University of Southern California Dissertations and Theses

The role of serotonergic receptor, HTR2B, on myeloid-derived suppressor cells in the brain metastatic environment

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The role of serotonergic receptor, HTR2B, on myeloid-derived suppressor cells in the brain metastatic environment

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Content THE ROLE OF SEROTONERGIC RECEPTOR, HTR2B, ON MYELOID-DERIVED
SUPPRESSOR CELLS IN THE BRAIN METASTATIC ENVIRONMENT
by
Mukund Iyer
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CANCER BIOLOGY AND GENOMICS)
May 2025
Copyright 2025 Mukund Iyer

ii
Dedication
To my wife, Kriti, my parents, Mohan and Uma, and my brother, Vighnesh, for their
unconditional love and unwavering support in my academic pursuits.

iii
Acknowledgements
Mentor
Josh Neman (Program Director)
Thesis Committee Members
Evanthia Roussos Torres, MD PhD (Chair)
Min Yu, PhD
I want to first extend my deepest gratitude to Dr. Josh Neman for his invaluable
guidance and unwavering support throughout my six-year doctoral journey. His
mentorship has been instrumental in my growth as a scientist and individual. Dr. Neman
nurtured my scientific curiosity, fostering intellectual exploration and critical thinking. He
expertly guided my research, translating questions into testable hypotheses, and
generously shared his vast knowledge and resources. His commitment to
compassionate, high-quality research, focused on patients with breast-to-brain
metastasis, deeply resonated with me. Dr. Neman instilled the importance of aligning
scientific pursuits with human compassion. His mentorship has left an indelible mark on
my academic and personal development and as I transition into the next phase of my
career, I carry his invaluable lessons and steadfast support with me.
I also want to sincerely thank the members of my committee, Dr. Evanthia
Roussos Torres and Dr. Min Yu for their insightful feedback and constructive criticism,
consistently shaping the direction of my research. I extend a special thanks to Dr.
Torres for graciously introducing me to the fascinating world of Myeloid-Derived
Suppressor Cells (MDSCs) and sharing her lab’s expertise and resources, including
access to cutting-edge techniques and invaluable guidance in this area. Dr. Yu's

iv
insightful comments and critical evaluation consistently challenged me to think more
deeply about my research and strengthened the overall quality of my work. I am deeply
grateful for their dedication to my success.
Working alongside my lab mates in the Neman Lab has been one of the best
parts of my PhD. I especially want to thank Dr. Diganta Das, who started his
postdoctoral studies at the same time I joined the lab and lent an open ear to bounce
ideas off each other which led to full-fledged experiments generating valuable data. I
would like to thank Brooke Nakamura, who always has the answer for any scientific or
administrative question I could think of, and Dr. Saman Sedighi whose infectious
positivity brightens the entire mood of the lab. I would additionally like to thank my
undergraduate mentees, Max Reed and Priya Shah, for their assistance in generating
data shown in this dissertation.
I’d like to thank Aaron Baugh and Dr. Julie Jang of the Dr. Roussos Torres Lab,
for their invaluable contributions to my MDSC project. Their insightful knowledge and
technical expertise on immunology and MDSCs, including late-night experiments, were
instrumental in the development and successful execution of my project.
I also want to thank the core facilities and resources at USC that facilitated my
research. I am very grateful to Ms. Ivetta Vorobyova, who assisted with numerous
intracardiac injections and bioluminescent imaging over years to generate successful in
vivo data for my project. I thank Ms. Bernadette Masinsin at the FACS core who helped
me sort countless co-culture models to distinguish changes in gene expression in
certain cell populations.

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I am grateful for the camaraderie of my fellow graduate students, especially Dr.
Tuo Shi, Janielle Cuala, Oscar Alberto, Bowen Wang, and Sayuri Pacheco, for their
support and joyful presence.
Finally, I would like to express my deepest gratitude to my family for their
constant love and support throughout this challenging journey. My wife, Kriti, has been
my constant source of strength and encouragement. Her steadfast belief in me and her
selfless dedication, including accompanying me in the lab on many weekends, have
been instrumental to my success. I am eternally grateful to my parents, Uma and
Mohan, for their encouragement and love through my PhD journey. Their thoughtful
gestures, such as providing meals for me throughout the week when I was too busy with
research to cook, have been invaluable. Witnessing my brother, Vighnesh, navigate his
own challenging PhD journey has been a constant source of inspiration. Observing his
dedication, perseverance, and commitment to his research has fueled my own
determination and drive. Their love and support have made this challenging journey not
only possible but also incredibly rewarding.

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Table of Contents
Dedication.........................................................................................................................ii
Acknowledgements..........................................................................................................iii
List of Tables ...................................................................................................................ix
List of Figures ...................................................................................................................x
Abstract...........................................................................................................................xii
Chapter 1 – Introduction .................................................................................................. 1
1.1 Prevalence and Impact of Breast-to-Brain Metastasis ........................................... 1
1.2 Diagnosis and Current Therapeutic Options .......................................................... 1
1.3 Immunological Landscape of Brain Metastasis...................................................... 2
1.4 Role of MDSCs in Tumor Progression ................................................................... 3
1.5 The Influence of Neurotransmitters on the Tumor Microenvironment.................... 4
1.6 The Impact of Serotonin on Immune Cells............................................................. 5
1.7 The Mechanisms Underlying Serotonin Receptor, HTR2B.................................... 5
1.8 Overview of the NF-κB Signaling Cascade ............................................................ 6
1.9 The Role of MDSCs in Immunotherapy Resistance............................................... 7
1.10 Scope of the Current Dissertation Research........................................................ 7
1.11 Summary.............................................................................................................. 9
Chapter 2: MDSCs are Present in the Breast-to-Brain Metastatic
Microenvironment and Upregulate NF-κB Signaling...................................................... 11
2.1 Abstract ................................................................................................................ 11
2.2 Introduction .......................................................................................................... 12
2.3 Materials and Methods......................................................................................... 13
2.3.1 Cell Culture.................................................................................................... 13
2.3.2 Animals.......................................................................................................... 17
2.3.3 RNA Isolation and qPCR Analysis................................................................. 19
2.3.4 Histology........................................................................................................ 19
2.3.5 Microscopy and Imaging................................................................................ 20
2.3.6 Flow Cytometry.............................................................................................. 20
2.3.7 ELISA ............................................................................................................ 21
2.3.8 Bioinformatics ................................................................................................ 22
2.3.9 Statistics ........................................................................................................ 23
2.4 Results: ................................................................................................................ 24
2.4.1 Myeloid Infiltration and T cell Suppression in Brain Metastasis..................... 24
2.4.2 pNF-κB Signaling Upregulation Drives the Pro-Inflammatory Phenotype
of Brain Metastatic MDSCs..................................................................................... 27

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2.4.3 The Brain Microenvironmental Education of MDSCs Drives T cell
Suppression and Supports Tumor Proliferation...................................................... 32
2.5 Discussion:........................................................................................................... 33
Chapter 3: Upregulation of Serotonergic Receptor, HTR2B, Drives NF-κB
Signaling in Brain Metastatic MDSCs ............................................................................ 36
3.1 Abstract ................................................................................................................ 36
3.2 Introduction .......................................................................................................... 36
3.3 Results ................................................................................................................. 38
3.3.1 Serotonin Mediates NF-κB Activation in Brain-Adapted MDSCs................... 38
3.3.2 Brain Metastatic MDSC Express the Serotonergic Receptor, HTR2B .......... 44
3.3.3 HTR2B-NF-κB Signaling Axis Regulates MDSC Activity and Promotes
Tumor Growth......................................................................................................... 47
3.4 Discussion............................................................................................................ 51
Chapter 4: MDSCs Modulate Neuronal Properties of Breast Cancer Brain
Metastasis...................................................................................................................... 54
4.1 Abstract ................................................................................................................ 54
4.2 Introduction .......................................................................................................... 54
4.3 Results ................................................................................................................. 56
4.3.1 MDSCs Regulate Neuronal Acquisition in Breast Cancer Cells .................... 56
4.3.2 MDSC-HTR2B Modulation Mediates Tumor Cells’ Neuronal Acquisition
in the Brain Microenvironment................................................................................ 58
4.4 Discussion............................................................................................................ 59
Chapter 5: HTR2B Antagonism and Immunotherapy Synergistically Reduce
Breast-to-Brain Metastasis and Improve Survival.......................................................... 62
5.1 Abstract ................................................................................................................ 62
5.2 Introduction .......................................................................................................... 63
5.3 Results ................................................................................................................. 65
5.3.1 Small Molecule HTR2B Inhibition Modulates NF-κB Activity in MDSCs........ 65
5.3.2 Targeting HTR2B Augments Immunotherapy in Breast-to-Brain
Metastasis............................................................................................................... 67
5.3.3 Combined HTR2B Blockade and Immunotherapy Promotes T Cell
Infiltration Within Intracranial Tumors ..................................................................... 70
5.4 Discussion............................................................................................................ 72
Chapter 6: Conclusions and Future Directions .............................................................. 75
6.1 Recap................................................................................................................... 75

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6.2 Limitations and Future Directions......................................................................... 75
6.3 Therapeutic Avenues ........................................................................................... 77
References .................................................................................................................... 79
Appendix........................................................................................................................ 89

ix
List of Tables
Table 1: Genes and Primer Sequences......................................................................... 89
Table 2: Antibodies ........................................................................................................ 92
Table 3: Allen Brain Atlas Donor Information................................................................. 93

x
List of Figures
Figure 2. 1: Infiltrating Myeloid Derived Suppressor Cells (MDSCs) are present in
brain metastases ........................................................................................................... 26
Figure 2. 2: High myeloid infiltration accompanies reduced T cell infiltration in brain
metastasis of triple-negative breast cancer. .................................................................. 27
Figure 2. 3: In-vitro models to study human and mouse MDSC function in the brain
microenvironment. ......................................................................................................... 28
Figure 2. 4: MDSCs upregulate inflammatory pathways in the brain
microenvironment. ......................................................................................................... 29
Figure 2. 5: Brain metastatic MDSCs exhibit elevated pNF-κB signaling and
increased immunosuppressive capacity. ....................................................................... 31
Figure 2. 6: Brain metastatic MDSCs suppress T cell responses and promote tumor
growth. ........................................................................................................................... 32
Figure 3. 1: MDSCs upregulate neuronal markers in the brain microenvironment........ 39
Figure 3. 2: Serotonin regulates MDSC activation of NF-κB and its downstream
markers.......................................................................................................................... 41
Figure 3. 3: Neurons are a key driver of serotonin synthesis in brain metastasis. ........ 42
Figure 3. 4: Brain Metastases Exhibit Increased Serotonin Compared to Other
Breast Metastatic Sites .................................................................................................. 43
Figure 3. 5: MDSC-mediated serotonin upregulation in neurons modulates
expression of NF-κB downstream markers in MDSCs. ................................................. 44
Figure 3. 6: MDSCs upregulate serotonergic receptor, HTR2B, in our mMDSC- and
h-MDSC-brain models. .................................................................................................. 45
Figure 3. 7: MDSC-HTR2B expression is detected in human and mouse brain
metastatic tissues. ......................................................................................................... 46
Figure 3. 8: Establishment of MDSC-HTR2B knockdown (J774M Htr2bKD) cell lines. .. 47
Figure 3. 9: HTR2B regulates NF-κB signaling pathway in MDSCs. ............................. 48
Figure 3. 10: Inhibition of NF-κB signaling identifies specific markers influenced by
HTR2B-NF-κB signaling cascade. ................................................................................. 50
Figure 3. 11: MDSC-HTR2B influences breast tumor proliferation................................ 51
Figure 4. 1: MDSCs Downregulate Neuronal Genes in Tumor Cells Within the Brain
Microenvironment. ......................................................................................................... 57
Figure 4. 2: MDSC-HTR2B regulates neuronal acquisition in breast cancer cells. ....... 59
Figure 5. 1: HTR2B antagonists inhibit NF-κB signaling cascade in MDSCs. ............... 67
Figure 5. 2: HTR2B antagonism and immunotherapy synergistically delay breast-tobrain metastasis and improve survival........................................................................... 70

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Figure 5. 3: HTR2B inhibition and immunotherapy promotes T cell infiltration into
intracranial tumor........................................................................................................... 71
Figure 5. 4: Graphical Conclusion. ................................................................................ 72

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Abstract
Brain metastases (BM) represent the most prevalent form of intracranial neoplasm
and pose a significant clinical challenge due to limited treatment options and a dismal
patient prognosis. Within the complex BM microenvironment, myeloid-derived suppressor
cells (MDSCs) are abundantly recruited and contribute substantially to tumor progression
and immunosuppression. This study delves into the critical role of the serotonin receptor,
HTR2B, in modulating MDSC function within the unique context of the BM
microenvironment. Utilizing in vitro co-culture models that mimic the intricate interplay
between tumor cells, neurons, and immune cells, alongside analyses of patient tissue
microarrays and single cell RNA sequencing, we demonstrate that MDSCs residing within
BM exhibit a marked upregulation of HTR2B. This heightened HTR2B expression drives
the activation of NF-κB signaling and its downstream inflammatory cascade within
MDSCs. We further establish that this HTR2B-NF-κB axis plays a crucial role in
augmenting MDSC-mediated suppression of T cell activity, fostering breast cancer cell
proliferation, and influencing the complex process of tumor cell integration into the central
nervous system (CNS) microenvironment. Importantly, we found that HTR2B inhibition,
achieved through the use of the FDA-approved drug clozapine, effectively disrupts this
pro-tumorigenic signaling pathway. In in vivo models of metastatic and intracranial breast
tumors, the combination of clozapine with anti-PD-1 immunotherapy resulted in a
significant reduction in overall tumor burden, a marked increase in survival rates, and a
notable enhancement of T cell infiltration into the brain metastatic lesions. These
compelling findings not only illuminate a novel HTR2B-NF-κB signaling pathway within
MDSCs as a driver of breast-to-brain metastasis but also suggest that targeting this axis

xiii
with HTR2B antagonists, such as clozapine, holds considerable therapeutic promise. This
approach, particularly in conjunction with immune checkpoint inhibitors, may offer a novel
and translatable strategy to improve clinical outcomes for patients grappling with the
devastating consequences of brain metastases. Moreover, this work underscores the
importance of considering the neuro-immune axis in cancer progression and opens
avenues for exploring the repurposing of neurological drugs to optimize immunotherapy
efficacy.

1
Chapter 1 – Introduction
1.1 Prevalence and Impact of Breast-to-Brain Metastasis
Breast cancer, the most prevalent malignancy among women worldwide, can be
categorized into four primary molecular subtypes. These subtypes are classified based
on the expression of three specific receptors: estrogen receptor (ER), progesterone
receptor (PR), and human epidermal growth factor receptor 2 (HER2). The distinct
expression patterns of these receptors define the following subtypes: Luminal A
(ER+/PR+/HER2-; 73% of cases), Luminal B (ER+/PR+/HER2+; 11% of cases), HER2+
(ER-, PR-, HER2+; 4% of cases), and triple-negative (ER-, PR-, HER2-; 12% of cases)
[1]. While early-stage breast cancer often has favorable outcomes with treatments like
surgery, chemotherapy, and hormone therapy, metastatic breast cancer remains
incurable, with a 5-year survival rate of 29%.
Brain metastases (br-mets), with an incidence ranging from 8.3 to 14.3 per 100,000
individuals, are the most prevalent type of intracranial neoplasm [2, 3]. Breast cancer
metastasizes to the brain in approximately 40% of patients with HER2+ or triple negative
subtypes [4]. 90% of patients with these breast cancer subtypes will die of metastasis to
the brain. Despite recent therapy advancements targeting immune checkpoint inhibitors
and oncogenic kinases, the median survival for patients with breast-to-brain metastases
remains a bleak 3-19 months [5].
1.2 Diagnosis and Current Therapeutic Options
Breast-to-brain metastasis is often detected through neuroimaging techniques like
MRI or CT scans, which can reveal abnormal growths within the brain. Initial symptoms,

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such as vision, hearing, balance, and reflex imbalances can be assessed through a
neurological exam to identify potential areas of brain involvement. A biopsy is then
preformed to determine the nature of the tumor, whether it is malignant or benign, and its
origin.
Prognosis for patients with breast-to-brain metastasis remains challenging due to
the blood-brain barrier (BBB), which hinders drug delivery to the central nervous system.
Consequently, local treatments such as surgery, stereotactic radiation therapy, and
whole-brain radiation therapy are considered standard approaches. For patients with 1-4
lesions, a combination of surgery and radiotherapy is recommended, while radiotherapy
alone is utilized for patients with greater than 4 lesions [6].
Ongoing clinical trials are exploring the effectiveness of systemic treatments like
endocrine therapy and chemotherapy for breast-to-brain metastasis, focusing on their
ability to cross the blood-brain barrier and improve survival. Additionally, targeting the
tumor immune microenvironment (TIME) with immune checkpoint inhibitors, a strategy
proven effective in other tumor types, remains an understudied area of research.
1.3 Immunological Landscape of Brain Metastasis
While the brain was long heralded as an immunological sanctuary site where the
presence of the BBB and blood-cerebrospinal fluid barrier (BCSFB) restricted the entry
of peripheral immune cells, recent evidence indicates brain metastatic cells recruit bone
marrow derived myeloids (BMDMs) which can account for 30-50% of the total brain tumor
mass [7, 8]. Clinical evidence suggests a correlation between BMDMs infiltrates and
melanoma brain metastasis survival [9]. Further, activated microglia and infiltrating

3
monocytes in metastatic brain tumors are skewed towards the pro-tumor “M2” phenotype
by secreted cytokines such as IL-4, IL-10, and TGF-β [10]. However, the functional
contributions and mechanisms guiding BMDMs in the breast-to-brain metastatic tumor
microenvironment have yet to be resolved.
1.4 Role of MDSCs in Tumor Progression
A subset of BMDMs are myeloid-derived suppressor cells (MDSCs), a
heterogeneous population of immature myeloid cells known for their remarkable
immunosuppressive and tumorigenic activities [11-14]. MDSCs are broadly categorized
into two main subtypes: monocytic (M-MDSCs) and granulocytic (G-MDSCs) [15]. Human
M-MDSCs are typically CD11b+, CD14+, HLA-DRlow/-, and CD15-, while G-MDSCs are
CD11b+, CD15+, and CD14-/low; in mice, M-MDSCs are generally CD11b+, Ly6C+, and
Ly6G-, whereas G-MDSCs are CD11b+, Ly6G+, and Ly6C- [16]. Exploiting tumor cells
secrete inflammatory mediators which result in abnormal myelopoiesis in the bone
marrow, generating and recruiting MDSCs which confer an immunosuppressive tumor
niche and assist in tumor immune evasion [17]. MDSCs exert their suppressive activity
via the production of reactive oxygen species, arginase, nitric oxide synthase, and
cytokines among others to inhibit the activity of T cell receptors, NK cell function while
promoting regulatory T cell and tumor associated macrophage functions [18]. Recent
insights have demonstrated that these tumor-educated MDSCs sustain tumor growth
though a myriad of non-immune processes as well such as promotion of tumor
neovascularization, stemness, invasion, proliferation, and metastatic colonization [19].
Thus, in this context, MDSCs dual role has shown to be a great partner for tumor cells

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since circulating MDSCs can nurture tumor cells along each step of the metastatic
cascade. In metastatic breast cancer, higher levels of MDSCs are associated with worse
prognoses and survival [20]. Currently, there is scant understanding of the role of MDSCs
in brain metastases although they have been detected in the blood of mouse and human
models [21, 22].
1.5 The Influence of Neurotransmitters on the Tumor Microenvironment
The brain metastatic environment presents a distinct challenge because of the
ability of central nervous system (CNS)-resident cells to act as local mediators,
influencing the plasticity and differentiation of invading immune cells [23]. Notably,
neurons play a role in cellular signaling through the secretion of classical
neurotransmitters such as GABA, glutamate, acetylcholine, dopamine, and serotonin.
CNS infiltrating tumor cells overexpress neurotransmitter receptors, allowing them to
hijack these signaling pathways and facilitate their adaption and metastatic growth [24,
25]. Tumor-infiltrating immune cells have also exhibited the capacity to upregulate
neurotransmitter receptors in extracranial tumors to drive cell migration and
immunosuppressive function [26]. While there are no published papers on the role of the
classic brain mediators on MDSCs, studies have shown that MDSCs utilize non-CNS
derived acetylcholine and norepinephrine to enhance their immunosuppressive
capabilities [27, 28].

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1.6 The Impact of Serotonin on Immune Cells
The role of serotonin in neuroimmune circuits regulating inflammation and
immunity is an emerging area of research [29-31]. Serotonin (5-HT) is a monoamine
produced from the essential amino acid tryptophan. Within the CNS, serotonin is
synthesized and stored in presynaptic neurons [32]. There are at least 15 distinct
subtypes of serotonin receptors (5-HTR1-7), all of which belong to the G protein-coupled
receptor superfamily (GPCRs) except for 5-HT3R [33]. Tissue microarray in 102 ductal
carcinoma and invasive lobular carcinoma breast cancer patients showed that they
express many serotonin receptor subtypes [34]. Several types of immune cells express
serotonergic receptors/transporters to modulate their regulatory functions. While there
has been no research into the role of serotonin on MDSCs, macrophages express 5-
HTR2B, 5-HTR7, and SERT [35-37] to promote M2 macrophage polarization.
1.7 The Mechanisms Underlying Serotonin Receptor, HTR2B
One serotonin receptor of interest is HTR2B. Activation of HTR2B is mitogenic in
pulmonary vessels, heart, interstitial cells of Cajal, mouse and human tumors [38-40].
Interestingly, in lung vasculature and cardiomyocytes HTR2B activates transcription
factor NF-κB, which regulates a number of tumor-promoting biological processes,
including: cell growth and survival, inflammation, and immune response [38, 41, 42].
While serotonin receptor signaling through HTR2B in macrophages regulates the
transcription of several genes (AP1, c/EBP, and SRF), which contributes to macrophage
activation and polarization through phosphorylation of ERK1/2 molecules, no studies
have examined the effect of HTR2B mediated NF-κB activation on immune cells [33, 37].

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If this HTR2B-NF-κB signaling axis is activated in MDSCs residing within the brain
metastatic microenvironment, it could have significant implications for both tumor
progression and immunosuppression.
1.8 Overview of the NF-κB Signaling Cascade
NF-κB signaling, a crucial regulator of inflammation and immunity, is typically
activated by a diverse array of receptors, including TNF receptors, Toll-like receptors
(TLRs), and antigen receptors on immune cells [43, 44] . The pathway's core involves a
family of transcription factors, including p65 (RelA), p50, RelB, c-Rel, and their inhibitors,
the IκB proteins [45]. In the canonical pathway, activation of upstream receptors triggers
a cascade involving the IκB kinase (IKK) complex, which phosphorylates IκBα, leading to
its ubiquitination and subsequent degradation by the proteasome [46]. This degradation
releases NF-κB dimers, most commonly p65/p50, from their cytoplasmic sequestration
by IκB. These dimers then translocate to the nucleus, where they bind to specific κB DNA
motifs in the promoter regions of target genes, acting as transcription factors to induce
the expression of a vast array of downstream genes. These genes encode proinflammatory cytokines (e.g., TNFα, IL-1β, IL-6), chemokines (e.g., CXCL8, CCL2),
adhesion molecules (e.g., ICAM-1, VCAM-1), and other proteins involved in immune
responses and cell survival [47]. The dysregulation of NF-κB signaling is implicated in a
wide range of diseases, including cancer, autoimmune disorders, and chronic
inflammatory conditions, highlighting its importance in cellular homeostasis.

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1.9 The Role of MDSCs in Immunotherapy Resistance
Immunotherapy harnesses to body’s immune system to attack tumor cells. For
example, anti-PD-1 therapy blocks the PD-1 receptor on T cells to enhance their antitumor activity. However, the effectiveness of immunotherapy can be significantly hindered
by MDSCs, which suppress T cell activity within the tumor microenvironment [48]. By
targeting and reducing the immunosuppressive effects of MDSCs, we can free T cells to
effectively utilize immunotherapy treatments like anti-PD-1 to recognize and eliminate
tumors [49]. This combined approach holds the promise of enhancing the efficacy of
cancer immunotherapy and improving patient outcomes.
1.10 Scope of the Current Dissertation Research
Through the research presented in this dissertation, I aimed to understand the
dynamic interplay between MDSCs, breast tumor cells, and the brain microenvironment.
To this effect, the research was conducted in four parts, examining different aspects of
brain-metastatic colonization and growth:
In the second chapter of the dissertation, I investigated the presence and function
of MDSCs within the brain microenvironment. I examined MDSC populations in human
brain metastatic samples and in vivo models of breast tumor intracranial metastasis. IHC
staining of breast-to-brain tumor patient tissues showed triple negative breast cancer
subtype has the highest rates of infiltrating myeloids and the lowest rates of T cells,
underscoring the potential importance of understanding MDSC function in this
microenvironmental context. Employing in vitro mouse models of MDSCs, I conducted
bulk RNA sequencing to identify differentially expressed pathways. This analysis revealed

8
an upregulation of inflammatory signaling, driven by increased NF-κB activation. I
subsequently assessed downstream effectors of NF-κB in both human and mouse
MDSCs. Additionally, I explored the impact of the brain microenvironment on MDSC
immunosuppressive capabilities through T cell suppression assays. Chapter three will
delve into the mechanisms underlying these phenotypic changes in MDSCs within the
brain microenvironment.
In the third chapter of the dissertation, I examined a novel mechanism that drives
NF-κB activation in MDSCs within the brain microenvironment. I identified that MDSCs
have the capacity to upregulate neuronal acclimation and functional markers in the brain
microenvironment. Further analysis demonstrated that serotonin upregulates NF-κB
activation in MDSCs, specifically through the serotonergic receptor HTR2B. This receptor
was found to be expressed in MDSC populations in human brain metastatic samples. We
demonstrate that MDSCs induce increased serotonin expression in neurons, and
inhibiting their serotonin production leads to a decrease in expression of NF-κB
downstream markers in MDSCs. Further, utilizing lentiviral-mediated HTR2B knockdown
MDSC cell lines, I observed a decrease in NF-κB activation, as well as in the expression
of its downstream and pro-inflammatory markers. HTR2B knockdown in MDSCs
alleviated T cell suppression, highlighting the receptors role in MDSC function in the brain.
In the fourth chapter of the dissertation, I examined the role of MDSCs in the brain
metastatic environment on tumor colonization and growth. Utilizing in vitro co-culture
models with breast tumor cells, mouse MDSCs, and neural cells, I found that MDSCs
promote tumor cell proliferation while downregulating CNS-acclimatizing markers in
tumor cells. However, knockdown of HTR2B in MDSCs leads to a reduction in tumor

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proliferation and an increase in CNS-acclimating genes, suggesting a strong tumorpromoting role of MDSC-HTR2B. Consequently, in chapter 5 we examine the effects of
small-molecule inhibition of HTR2B on MDSC function and tumor growth.
In the fifth chapter of this dissertation, I investigated the effects of HTR2B inhibition
using the FDA-approved drug clozapine, in combination with anti-PD-1 immunotherapy,
to sensitize the tumor immune microenvironment and reduce tumor burden. In both
intracardiac and intracranial in vivo models of breast cancer, I observed that selective
targeting of HTR2B with clozapine, coupled with anti-PD-1 treatment led to decreased
brain metastasis formation, reduced tumor burden, and improved survival. Specifically,
this dual combination promoted increased infiltration of T cells into the brain
microenvironment, facilitating the destruction of tumor cells. Collectively, my research
demonstrates that upregulated NF-κB activation via HTR2B in MDSCs augments tumor
growth and reduces survival in brain metastatic breast cancer.
1.11 Summary
Despite significant advances in brain metastasis management, the development
of more effective treatments targeting the TIME remains a pressing clinical need. Limited
knowledge about the immunological landscape of BMs, a lack of actionable clinical
targets, and the challenges of crossing the blood brain barrier contribute to the complexity
of treating these tumors.
In recent years, myeloid derived suppressor cells have emerged as key
immunosuppressive mediators within the tumor microenvironment, inhibiting targeted T
cell removal of cancer cells and advancing tumor progression. Effectively targeting these

10
cells and releasing their grip on T cell inhibition is essential for unlocking effective
immunotherapy treatments. Sensitizing the immune microenvironment to immunotherapy
requires a deeper understanding of the mechanisms underlying MDSC pro-tumorigenic
functions.
Through the research conducted in this dissertation, I aim to advance our
understanding of MDSCs in the metastatic brain microenvironment and develop targeted
therapeutic strategies that can be translated into clinical trials for BM patients.

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Chapter 2: MDSCs are Present in the Breast-to-Brain Metastatic
Microenvironment and Upregulate NF-κB Signaling
2.1 Abstract
Myeloid Derived Suppressor Cells (MDSCs) support breast cancer growth via
immune suppression and non-immunological mechanisms. Although 15% of breast
cancer patients develop brain metastasis, the presence and role of MDSCs within this
environment remain largely unexplored. Utilizing patient tissue staining and scRNAsequencing paired with syngeneic in vivo mouse models, we demonstrated that MDSCs
infiltrate brain metastases. Further, by establishing novel mouse and human MDSCneural models, we identified that MDSCs are influenced by CNS-derived neural cues to
adopt a pro-inflammatory phenotype. Specifically, MDSCs upregulate NF-κB signaling, a
master transcription factor involved in inflammation and tumor progression. This
activation promotes the expression of various NF-κB downstream inflammatory
cytokines, including IL-6, IL-1β, IL-10, and TNF, as well as immunosuppressive enzymes
such as COX2, MMP9, IDO1, NOS2, and ARG1. Consequently, these neural-educated
MDSCs exhibit enhanced immunosuppressive capabilities and promote tumor
proliferation, highlighting their detrimental role in cancer progression. Our findings
emphasize the significance of MDSCs in the progression of brain metastases and
underscore the need for further research to develop targeted therapies that can effectively
counteract their immunosuppressive and pro-tumorigenic effects.

12
2.2 Introduction
Breast cancer is the second most common origin of brain metastasis after lung
cancer. This disease is associated with poor prognosis and a lack of effective therapeutics
due to the blood brain barrier preventing drug delivery to the central nervous system.
Current therapeutics have primarily focused on targeting the tumor itself, neglecting to
reprogram the tumor microenvironment, specifically MDSCs.
MDSCs are characterized by their plasticity and can drastically alter their
immunosuppressive and pro-tumorigenic pathways in response to the local
microenvironment [50]. The BBB creates a unique niche within the CNS, where neurons
influence the cellular landscape of invading tumor cells [24]. While the presence of
MDSCs in the brain metastatic microenvironment has yet to be demonstrated, research
has shown that peripherally derived MDSCs constitute 30-50% of the tumor mass in
primary brain cancer (gliomas) [51, 52]. Further, while MDSCs have shown to exert an
immunosuppressive niche in gliomas, their ability to adapt to CNS environmental cues
remains largely unexplored [53].
In this chapter, we utilized in vivo mouse models and patient-derived human brain
metastatic tissues to validate MDSCs presence in brain metastasis. We employed both
mouse and human MDSC models to investigate MDSC-neural interactions. This
approach allowed us to characterize MDSC phenotypic shifts and upregulated pathways
in response to the neural microenvironment. Additionally, we assessed the effects of
neural-educated MDSCs on immunosuppressive function and breast tumor proliferation.
Our findings provide novel insights into the role of neural input in facilitating immunological
and non-immunological adaptations of MDSCs. A deeper understanding of this

13
adaptation may reveal novel mechanisms of immunosuppression and tumor progression,
opening new avenues for drug targeting.
2.3 Materials and Methods
2.3.1 Cell Culture
The following commercially available cell lines were used: 4T1 triple negative
breast cancer cells (ATCC), MDA-MB-231 triple negative breast cancer cells (ATCC), and
E0771 Luminal B breast cancer cells (ATCC). MDSC-like J774Ms were kindly provided
from Dr. Kebin Liu’s lab at Augusta University. All cell cultures were maintained at 37°C,
5% CO2 in humidified incubators. These commercial cell lines, differentiated Human
MDSCs, and T cells were cultured in RPMI1640 (Thermo Fisher, catalog no. 11875119)
supplemented with 10% FBS (Omega Scientific, catalog no. FB-12), 1.5% HEPES buffer
(Gibco, catalog no. 25-060-Cl), 1x GlutaMAX (Thermo Fisher, catalog no. 35050061), 1%
MEM nonessential amino acids (Corning, catalog no. 25-025-Cl), 1x AntibioticAntimycotic (Thermo Fisher, catalog no. 15240062), 1% sodium pyruvate (Gibco, catalog
no. 11360-070), 0.0004% beta-mercaptoethanol (Sigma, catalog no. M3148) and will be
referred to as complete RPMI media. Primary mouse neurons were maintained in
Neurobasal-A Media (Thermo Fisher, catalog no. 10888022) supplemented with 1x B-27
(Thermo Fisher, catalog no. 17504044), 1x Antibiotic-Antimycotic (Thermo Fisher,
catalog no. 15240062), and 1x GlutaMAX (Thermo Fisher, catalog no. 35050061) and will
be referred to as complete Neurobasal-A media. All cell lines were stored in liquid nitrogen
and frozen down between 5 to 10 passages from original cell line. All cells thawed from
liquid nitrogen were passaged once before experiment setup and no cell lines were

14
utilized past 15 passages. Cell lines were negative for mycoplasma and were frequently
tested utilizing MycoAlert Kits (VWR, catalog no. 75860-360).
Lentiviral Particle Production, Transduction, and Selection
HTR2B knockdown and Scrambled (GeneCopoeia, Cat# MSH027376-LVRU6P)
and pHIV-Luc-ZsGreen (Addgene, plasmid no. 39196) lentiviral particles were produced
using the 293T cell system as previously described [54]. J774M cells were transduced to
stably express knockdown (Htr2bKD) or Scrambled. These cells were treated with 4 μg/ml
puromycin to ensure proper cell selection. Six J774M Htr2bKD variants were tested and
three variants with significant knockdown were chosen to be utilized in future
experiments. 4T1, MDA-MB-231, and E0771 cells were transduced to stably express
Firefly Luciferase/GFP (FF/GFP). These cells were FACS sorted for the GFP+ population.
Primary Mouse Brain Cells
Mouse brain cells were isolated from whole brain tissue of 0 - 4 day postnatal mice
and cultured in vitro as previously described [55]. Media collected from these cultures
every 72 hours serve as mouse brain conditioned media (mBCM) for subsequent
experiments.
Human iPSC-derived Forebrain Neurons
Human PGP-1 induced pluripotent stem cells (iPSCs) were kindly provided by Dr.
Giorgia Quadrato. Neural progenitor cells (NPCs) were generated from PGP-1s utilizing
the STEMdiffTM SMADi Neural Induction Kit (STEMCELL Technologies, cat# 08581) as
per manufacturers instructions. Mature forebrain neurons were generated from NPCs
utilizing the STEMdiffTM Forebrain Neuron Differentiation Kit (STEMCELL Technologies,
cat# 08600) and STEMdiffTM Forebrain Neuron Maturation Kit (STEMCELL Technologies,

15
cat# 08605) as per manufacturer’s instructions. Conditioned media was collected from
mature forebrain neurons between 8-20 days after incubation in neuron maturation
medium.
Human MDSC Derivation
Blood from healthy donors was obtained at the University of Southern California
under approved consent and IRB. Peripheral blood mononuclear cells (PBMCs) were
separated via a Ficoll gradient and isolated for CD33+ cells via the EasySep Human
CD33+ Selection Kit II (Stem Cell, catalog no. 17876) per manufacturer’s instructions.
These cells were plated at 5 x 105 cells/mL in complete RPMI media for 7 days in the
presence of recombinant human GM-CSF (20 ng/mL; Stem Cell, catalog no. 78140) and
IL-6 (20 ng/mL; Stem Cell, catalog no. 78050.1). GM-CSF was added on days 1, 3, and
5 whereas IL-6 was only added on day 5. After 7 days, the cells were utilized for
downstream experiments and T cell suppression assays were preformed to validate the
suppressive capability of these Human MDSCs.
Tumor/MDSC-Neuron Co-cultures
Tumor cells and MDSCs were stained with cell permeable Far Red dye (Thermo
Fisher, catalog no. C34564) at 10 uM or CMFDA dye (Thermo Fisher, catalog no. C2925)
at 5 uM as per manufacturers recommendation to allow for future separation of cell
populations. Cells were resuspended in complete Neurobasal-A media and seeded onto
neuronal cultures (1:1:125 tumor to MDSC to neuron ratio) to model Tumor-MDSCNeuron interactions. 72 hours post seeding, half of the media was replaced. 96 hours
post seeding, tumor cells/MDSCs were either separated via FACS for qPCR, stained and

16
processed for flow cytometry, or fixed with 4% formaldehyde for immunofluorescence
studies.
In vitro Suppression Assay
CD8+ T cells were isolated from spleens of healthy 8-12 week female BALB/c mice
via the EasySep Mouse CD8+ Isolation Kit (Stem Cell, catalog no. 19853) per
manufacturer’s instruction. Isolated CD8+ T cells were stained with CFSE at 3 uM. 5x104
CFSE-labeled CD8+ T cells were co-cultured with J774M cells (Control, Scrambled,
HTR2BKD) at a 4:1 ratio (T cell:J774M) and anti-CD3/CD28 beads (Thermo Fisher
Scientific, catalog no. 11453D) as per manufacturer’s instructions in complete RPMI
media. Media was changed after 24 hours to mBCM. T cells were allowed to proliferate
for a total of 60 hours. Afterwards, the samples were collected, stained with Live/Dead
Fixable Aqua (Thermo Fisher, catalog no. L34965) and analyzed via flow cytometric
analysis.
Exogenous Compound Treatments
J774M were seeded at 7.5 x 104 cells onto a 6 well plate in complete RPMI media.
For exogenous neurotransmitter assays, the following day the media was replaced with
complete Neurobasal-A Media and concurrently treated with Serotonin (Sigma-Aldrich,
catalog no. H9523), Dopamine (Sigma-Aldrich, catalog no. H8502), Acetylcholine
(Sigma-Aldrich, catalog no. A6625), Norepinephrine (Sigma-Aldrich, catalog no. A7256),
or GABA (Sigma-Aldrich, catalog no. A2129). For HTR2B antagonist treatment assays,
the following day the media was replaced with mouse BCM and concurrently treated with
SB-204741 (Cayman Chemical, catalog no. 32965), clozapine (TCI Chemicals, catalog
no. 2547), RS-127445 (Adooq Bioscience, catalog no. A11165) or aripiprazole (TCI

17
Chemicals, catalog no. A2496). For MLN120B treatment assays, the following day the
media was replaced with mouse BCM and concurrently treated with MLN120B
(Medchemexpress, catalog no. HY-15473). For LX-1031 treatment assays, previously
isolated mouse neural cells as described above were treated with LX-1031
(Medchemexpress, catalog no. HY-13041) and concurrently seeded with J774M (1:125
MDSC to neuron ratio). Samples were collected for downstream RNA isolation/qPCR,
flow cytometry, or ELISA.
Proliferation Assay
4T1 FF/GFP and J774M (Scrambled, HTR2BKD) cells were resuspended in
complete Neurobasal-A media and seeded onto neuronal cultures (1:1:125 tumor to
MDSC to neuron ratio) on a 96 well plate. MDA-MB-231 FF/GFP and J774M (Scrambled,
HTR2BKD) cells were resuspended in complete RPMI media and seeded (2:1 tumor to
MDSC ratio) on a 96 well plate. Media was changed the following day to mouse brain
conditioned media (mBCM). Fluorescent GFP signal is indicative of the number of tumor
cells in the well. Fluorescence was measured daily on a Varioskan™ Lux multimode
microplate reader (ThermoFisher Scientific) at 485/520 (Ex/Em).
2.3.2 Animals
9–12-week-old adult female BALB/c mice (strain#000651) or C57BL/6
(strain#000664) were purchased from Jackson Laboratories and used for either in vivo
intracardiac or intracranial injection experiments to model metastatic disease. Animal
procedures were performed under approved IACUC protocols and guidelines. All animals

18
were humanely euthanized upon signs of morbidity, including development of tumor
symptoms (paralysis, hydrocephalus, weight loss, severely hunched).
Tumor Model, Treatment Dosing Scheme, and Tumor Resection
To model metastatic disease, 5 x 103 triple-negative breast cancer 4T1 FF/GFP or
Luminal B breast cancer E0771 FF/GFP cells resuspended in serum-free RPMI1640 were
injected intra-cardiac into the heart’s left ventricle with a 25-gauge needle. Clozapine was
dosed daily by intraperitoneal injection at 1 mg/kg body weight. Clozapine was diluted in
0.1N HCl and neutralized with 4.0N NaOH and diluted in PBS. Anti-PD-1 (BioXCell;
RMP1-14) and IgG2a isotype control (BioXCell, Lym-1) was diluted in PBS and dosed
2x/week by intraperitoneal injection at 100 ug/mouse. Development of brain metastasis
and other distant metastasis was measured by optical imaging of animals twice per week.
Luciferin (1µL/gram bodyweight of animal) was injected into the peritoneal cavity of each
mouse and bioluminescence was imaged (dorsal and ventral) 10 minutes after injection.
We measured bioluminescent signal from brain metastases utilizing the same brain ROI
for all experimental animals on all imaging days. To model brain metastatic disease, 5 x
103 4T1 resuspended in serum-free RPMI1640 were injected into the brain parenchyma
utilizing a stereotaxic frame. After locating the intersection point of bregma and midline (x
= 0, y = 0, z = 0), tumor cells were injected with a 25-guage needle at (x = +1.0, y = +1.0,
z = -1.0). Clozapine and anti-PD-1 were dosed as described above. Tumors were seeded
for 3 days prior to beginning treatment. Mice for all experiments were monitored and
weighed daily for presentation of tumor burden related symptoms and were humanely
euthanized. Immediately post-euthanasia, whole brains were resected and placed into

19
formalin before being sent to the histology core at USC for paraffin embedding and
processed into 10 uM-thick sections for subsequent staining.
2.3.3 RNA Isolation and qPCR Analysis
Cells were harvested by scraping when appropriate or trypsinization for 3-5
minutes at 37°C, followed by neutralization in media containing 10% FBS, and
centrifugation at 360 rcf for 5 minutes. The resulting cell pellet was either processed for
RNA extraction immediately or frozen for subsequent use in qPCR (preformed in triplicate
per sample) as previously described [24]. All primers utilized for qPCR analysis were
purchased from IDT (Appendix Table 1).
2.3.4 Histology
Mouse brain tissues were fixed, paraffin embedded and processed as described
above. Tissues were stained on the Leica Bond III Autostainer (Leica, Cat# 21.2201).
Antigen unmasking was conducted with BOND Epitope Retrieval Solution 1 & 2 (Leica,
Cat# AR9961/AR9640) for 30 minutes as per manufacturer's instructions. Primary
antibodies were incubated for 60 minutes. Subsequent use of a DAB chromogen and
hematoxylin & eosin stain were conducted utilizing the BOND Polymer Refine Detection
DAB (Leica, Cat# DS9800) and Modified Mayer’s Hematoxylin (American Mastertech,
Cat# HXMMHGAL) as per manufacturer’s instructions.

20
2.3.5 Microscopy and Imaging
Confocal and widefield imaging were performed and quantified as previously
described [25].
Immunofluorescence
Brain-metastatic tumor tissues from patients were acquired via USC Neurosurgery
under approved consent and IRB. These tissues were formalin fixed, paraffin embedded,
and processed into 10 uM-thick sections. Selected regions of interest were punch-holed
and embedded into a tissue microarray with 50+ samples. Immunofluorescence protocol
on tissue and cells were performed as previously described [24]. Primary antibodies and
secondary antibodies list can be found in Appendix Table 2.
2.3.6 Flow Cytometry
To detect intracellular markers, The Foxp3/Transcription Factor Staining Buffer Set
(Invitrogen, catalog no. 00-5523-00) was utilized to fix and permeabilize cells as per
manufacturers recommendation. Cells were subsequently blocked in CD16/CD32 Fc
block (BD Biosciences; catalog no.553142) at a 1:100 dilution overnight. The next day,
samples were incubated with primary antibody and secondary antibodies (Appendix
Table 2) and ran on the Attune NxT Flow Cytometer at the USC Stem Cell Flow Cytometry
Facility. To detect extracellular markers, cells were not fixed or permeabilized but rather
directly stained with primary and secondary antibodies and resuspended in FACS buffer
(5% FBS, 0.1% NaN3, PBS) prior to running on the Attune NxT. For Fluorescent Activated
Cell Sorting (FACS), previously stained CMFDA or Far-Red cells were harvested,

21
resuspended in FACS buffer, and analyzed on the BD FACSAria Cell Sorter. Resulting
data was analyzed utilizing FlowJo v10.10 software.
2.3.7 ELISA
In this study, we used Luminex xMAP technology for multiplexed quantification of
45 Mouse cytokines, chemokines and growth factors. The multiplexing analysis was
performed using the Luminex™ 200 system (Luminex, Austin, TX, USA) by Eve
Technologies Corp. (Calgary, Alberta). Forty-five markers were simultaneously measured
in the samples using Eve Technologies' Mouse Cytokine 45-Plex Discovery Assay®
which consists of two separate kits: one 32-plex and one 13-plex (MilliporeSigma,
Burlington, Massachusetts, USA). The assay was run according to the manufacturer's
protocol. The 32-plex consisted of Eotaxin, G-CSF, GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17, IP-10, KC,
LIF, LIX, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNFα, and VEGF. The
13-plex consisted of 6Ckine/Exodus2, Erythropoietin, Fractalkine, IFNβ-1, IL-11, IL-16,
IL-20, MCP-5, MDC, MIP-3α, MIP-3β, TARC, and TIMP-1. Assay sensitivities of these
markers range from 0.3 – 30.6 pg/mL for the 45-plex. A cubic spline and 5-paramter
logistic regression were used when formatting and optimizing the standard. Regression
analysis was performed utilizing the Bio-Plex Manager™ software. Individual analyte
sensitivity values are available in the MilliporeSigma MILLIPLEX® MAP protocol.

22
2.3.8 Bioinformatics
scRNA-seq of Human Brain Metastasis Samples
Publicly available single-cell RNA sequencing (scRNA-seq) data from GSE186344
(PMID: 35063085) were utilized for this study. This dataset profiled 15 human
parenchymal brain metastases from 15 patient with diagnosis of melanoma (n=3), breast
cancer (n=3), lung cancer (n=3), ovarian cancer (n=2), CRC (n=1), renal cell carcinoma
(n=1), unknown primary carcinoma (n=1) and a case of adult rhabdomyosarcoma that
was a non-carcinoma, non-melanoma sample used for comparison. Raw sequencing
data (FASTQ files) were downloaded from the NCBI Gene Expression Omnibus (GEO).
PCA and UMAP were performed to reduced dimensionality and colors based on
log(CPM+1) as the measure of expression. Clustering was performed using the Louvain
algorithm with multilevel refinement, as implemented in Seurat. Cell types were annotated
based on the expression of canonical marker genes.
Bulk RNA-seq of J774M cells
J774M cells were cultured independently or in a MDSC-Neuron co-culture as
described above and J774M were separated via FACS in singlet. Transcriptomic
sequencing was performed at the USC Norris Molecular Genomics Core. Samples were
simultaneously library prepped using the Kapa mRNA HyperPrep kit following
manufacturer's protocol (Roche, KK8580). Prepared libraries were sequenced on the
Illumina Nextseq500 at single end 75 cycles. RNA integrity number values of the samples
ranged from 8.5 to 10. Samples were read at 25 million reads per sample at a read length
of 1x75. Processing of the data was conducted on Partek Flow via DESeq2 to generate
a differentially expressed gene list amongst samples. These differentially expressed

23
genes were inputted into Ingenuity Pathway Analysis and analyzed for significantly
enriched canonical signaling pathways.
Allen Brain Atlas
Map of serotonin synthesis enzymes at the gene expression levels was adapted
from the Allen Human Brain Atlas, which is an open-source database of quantitative genelevel transcriptome from DNA microarrays (Allen Institute for Brain Science. Allen Human
Brain Atlas. 2010. Available from: https://human.brain-map.org/microarray/search). TPH1
probe name: CUST_16083_PI416261804; NCBI Accession: NM_004179.1. TPH2 probe
name: A_24_P401787; NCBI Accession: NM_173353.2. DDC probe name:
CUST_16986_PI416261804; NCBI Accession: CUST_16986_PI416261804. MRI
images acquired at (0, -18, 18). Details on patients analyzed are available in Appendix
Table 3.
2.3.9 Statistics
Statistics were preformed using GraphPad Prism 8 software. To assess statistical
significance, Student’s t test, one-way ANOVA, and log-rank statistical analyses were
utilized. For one-way ANOVA, post hoc analysis was performed using Tukey’s multiple
comparison test where necessary. All histogram data show individual data points with the
mean ± SEM (Standard Error of the Mean) and p < 0.05 was considered to be statistically
significant. Statistical significance and Hazard Ratio for survival data from in vivo
experiments were calculated using Log-Rank Test.

24
2.4 Results:
2.4.1 Myeloid Infiltration and T cell Suppression in Brain Metastasis
While Myeloid Derived Suppressor Cells (MDSCs) are well-established
contributors to the tumor microenvironment and are known to be recruited to tumor sites
from the bone marrow, there is a paucity of literature discussing their presence and role
within the brain microenvironment [56]. To address this, we performed single-cell RNA
sequencing (scRNA-seq) on patient BM tissues. We identified myeloid cluster enriched
in PTPRC, CD33 and ITGAM, and within this cluster, identified MDSCs based on
ITGAM+, CD14+/-, FUT4+/-, CD3-, HLA-DR low/-, CD19-, MS4A1- expression (Figure
2.1A-D). To further investigate MDSCs infiltration in human BM, we collected human brain
metastatic tissues and generated a tissue microarray containing breast and lung, among
others, to brain metastasis samples. This microarray was stained for M-MDSCs (CD11b+
CD14+ CD15- HLA-DR-) and G-MDSCs (CD11b+ CD14- CD15+ HLA-DR-).
Immunofluorescence analysis revealed the presence of both M-MDSCs and G-MDSCs
in both breast and lung to brain metastases (Figure 2.1E-F). To examine MDSC
infiltration in mouse brain metastatic tumors, we stained resected tumors in a syngeneic
model of 4T1 brain metastases. Flow cytometric analysis revealed infiltration of both MMDSCs (CD45+, CD11b+, Ly6c+, Ly6g-) and G-MDSCs (CD45+, CD11b+, Ly6c-, Ly6g+)
(Figure 2.1G-H). A slightly higher, but not statistically significant, infiltration of G-MDSCs
compared to M-MDSCs was observed, suggesting an environment with increased
reactive oxygen species [12].

25

26
Figure 2. 1: Infiltrating Myeloid Derived Suppressor Cells (MDSCs) are present in brain metastases.
(A) UMAP visualization of single-cell RNA sequencing data of human breast to brain metastasis patient
tissues (n=15). Cells are color-coated by major cell type cluster. (B) Expression of representative myeloid
marker genes (PTPRC, CD33, ITGAM) is shown. (C) UMAP visualization of myeloid cells, color by
classification as MDSCs or other myeloid cells. (D) Expression of MDSC marker genes is shown.
Immunofluorescence (IF) and H&E of breast-to-brain (E) and lung-to-brain (F) metastatic patient tissues.
M-MDSCs (CD11b+ CD14+ CD15- HLA-DR-) and G-MDSCs (CD11b+ CD14- CD15+ HLA-DR-). Arrows
point to MDSCs. Scale bar = 50 μm. Flow cytometric gating strategy (G) and bar graph (H) of M- (CD45+
CD11b+ Ly6c+Ly6g-) and G-MDSCs (CD45+ CD11b+ Ly6c-Ly6g+) from 4T1 brain metastases 14 days
post-transplantation (n=3). Error bars represent ± SEM.
To investigate the immune crosstalk between infiltrating myeloid cells and T cells
in breast-to-brain metastasis, we analyzed human brain metastasis tissue originating from
the four primary breast cancer subtypes (Luminal A, Luminal B, HER2+, and Triple
Negative (TNBC)) [57]. Immunohistochemical (IHC) analysis revealed a significantly
higher ratio of CD33+ myeloid cells to CD3+ T cells, CD4+ helper T cells, and CD8+
cytotoxic T cells specifically in brain metastasis from TNBC (Figure 2.2A).This finding is
particularly significant given that triple-negative breast cancer is the most aggressive
subtype, constitutes the highest percentage of breast-to-brain metastasis cases, and is
associated with the worst patient survival [58, 59]. Therefore, we will focus on
characterizing the dynamic interplay between myeloid cells and the brain
microenvironment in the context of triple-negative breast-to-brain metastasis.

27
Figure 2. 2: High myeloid infiltration accompanies reduced T cell infiltration in brain metastasis of
triple-negative breast cancer.
(A) IHC images and analysis of breast to brain tumor patient tissue sections from four primary subtypes
(Luminal A, B, HER2+ and TN) stained for CD33+ myeloids, CD3+ T cells, CD4+ Helper T cells and CD8+
Cytotoxic T cells. H&E staining is included. Arrows point to positively stained cells. n=4-7 patients per
subtype, analyzed 5-6 images per tissue. Scale bar = 50 μm. Error bars represent ± SEM.
2.4.2 pNF-κB Signaling Upregulation Drives the Pro-Inflammatory Phenotype of
Brain Metastatic MDSCs
The immunologic landscape and tumor microenvironments between different
organs distinctly influence MDSC plasticity and differentiation [60]. To understand MDSC
adaptation and phenotypic plasticity within the brain microenvironment, we created
murine and human in vitro models where we co-cultured mouse MDSC-like J774M cells
with isolated mouse neural cells (mMDSC-brain model) and cultured human peripheral
blood mononuclear cell- (PBMC-) derived MDSCs with human iPSC-derived forebrain
neuron conditioned media (hMDSC-brain model; Figure 2.3A) [61, 62]. To validate the
establishment of the hMDSC-brain model, we observed iPSC forebrain neurons were

28
positively stained for TUJ1, a neuron-specific marker (Figure 2.3B). Further, a defining
characteristic of MDSCs is their ability to suppress the proliferation and growth of T cells
[63]. Therefore, via a T cell suppression assay, we validated that our differentiated human
MDSCs maintained the ability to suppress T cell growth (Figure 2.3C). The models in
Figure 2A are utilized throughout the dissertation to model MDSC function in the brain
environment.
Figure 2. 3: In-vitro models to study human and mouse MDSC function in the brain
microenvironment.
(A) Schematic representation of MDSC-brain microenvironment models. Top: Conditioned media was
collected from induced pluripotent stem cell (iPSC)-derived human forebrain neurons and added to human
MDSCs (hMDSCs) differentiated from peripheral blood mononuclear cells (PBMCs) of healthy donors
(hMDSC-brain model). Bottom: Mouse MDSC J774M cells were co-cultured with neural cells isolated from
postnatal mouse brains (mMDSC-brain model). (B) IF staining of human mature forebrain neurons
differentiated from iPSCs generated using the PGP-1 cell line. Images captured 8 days post incubation in
neuron maturation media. Scale bar = 50 μm. (C) CFSE proliferation assay of human CD3+ T cells cocultured with hMDSCs at a 1:1 ratio. Data represent n=3 replicates per group. Error bars represent ± SEM.
To characterize the top upregulated pathways and upstream regulators in mouse
MDSCs within the brain microenvironment, we conducted bulkRNA-seq of isolated
MDSCs from our mMDSC-brain model. Differential gene expression analyses revealed
an upregulation of inflammatory and hypercytokinemia pathways (Figure 2.4A). To
ascertain the effectors influencing these pathways, we utilized Ingenuity Pathway
Analysis to identify the top upstream predicted regulators. This analysis implicated LPS,

29
TNF, IFNG, IL1B, and TLR, which are known activators of canonical NF-κB signaling
(Figure 2.4B) [45, 64, 65].
Figure 2. 4: MDSCs upregulate inflammatory pathways in the brain microenvironment.
Ingenuity Pathway Analysis of significantly upregulated pathways (A) and predicted upstream regulators
(B) in J774M in mMDSC-brain model by bulk RNA sequencing.
Inflammatory cytokine expression is frequently driven by activation of the NF-κB
transcription family, via nuclear translocation of cytoplasmic complexes [66, 67]. NF-κB
activation in MDSCs enhances immunosuppressive capacity and promotes tumor
survival and growth [62, 68]. We therefore hypothesized that MDSCs exhibit increased
activation of NF-κB within the brain microenvironment. Indeed, mouse MDSCs exposed
to mouse brain conditioned media (mBCM) upregulated pNF-κB, as demonstrated by flow
cytometric analysis (Figure 2.5A). Furthermore, we assessed whether NF-κB
downstream inflammatory signals were increased in MDSCs in the brain
microenvironment. In mouse MDSCs, a significant increase in interleukins (Il6, Il1b, Il10),
enzymes (Cox2, Mmp9, Ido1, Nos2, Arg1), chemokines (Cxcl1, Cxcl2), receptors (Tlr1,
Tlr8) and other cytokines (Tnf, NF-κBiz) mRNA expression was observed (Figure 2.5B).
In differentiated human MDSCs, we detected upregulated mRNA expression of Il6, Cxcl2,

30
Cox2, Il1b, and Nos2 (Figure 2.5C). Key upregulated immunosuppressive markers such
as Il10, Il1b, Cox2, Ido1, Nos2, and Arg1 have been demonstrated to be essential for
immunosuppressive MDSCs to impair lymphocyte function, induce free radical
production, and deplete critical metabolites required for T cell receptor function [69].
These markers can also have a marked non-immunological effect on advancing tumor
progression by promoting the metastatic cascade, cell proliferation, tumor cell survival,
angiogenesis, and inflammation [70].
To determine whether the changes detected at the mRNA expression level
translated to protein expression, we conducted a multiplex ELISA cytokine panel which
revealed an upregulation of 85% of the cytokines assayed in mouse MDSCs in the brain
microenvironment (Figure 2.5D). Many of the key upregulated cytokines are interleukins
(IL-6, IL-20, IL-1B) supporting inflammation and chemokines (CXCL10, CXCL1, CCL2,
CCL3, CXCL2) guiding mobility and migration [71]. While cytokine release was assayed
via analysis of the extracellular media, we examined the expression of the enzymes
COX2, IDO1, NF-ΚBIZ, and MMP9 using flow cytometric analysis. This revealed a similar
upregulation of these markers in mouse MDSCs in the brain microenvironment (Figure
2.5 E-H). Our results suggest that MDSCs in the brain microenvironment express an
inflammatory phenotypic profile, potentially driven by NF-κB activation.

31
Figure 2. 5: Brain metastatic MDSCs exhibit elevated pNF-κB signaling and increased
immunosuppressive capacity.
(A) Flow cytometric analysis of pNF-κB expression in J774M treated with mouse brain conditioned media
(mBCM). qPCR of NF-κB associated inflammatory markers in the mMDSC- (B) and hMDSC- (C) brain
models. (D) Heatmap depicting the results of an ELISA analysis comparing the levels of NF-κB associated
inflammatory markers in the mMDSC-brain model. (E-H) Flow cytometric analysis of NF-κB associated
inflammatory markers in the mMDSC-brain model. Signal measured either by percent positive or median
fluorescent intensity (MFI). Data represent n=3 replicates per group. Error bars represent ± SEM.

32
2.4.3 The Brain Microenvironmental Education of MDSCs Drives T cell Suppression
and Supports Tumor Proliferation
We next wanted to assess the immune and non-immune impact of these neuraleducated MDSCs on the brain metastatic environment. Considering MDSCs in the brain
microenvironment have shown to upregulate multiple immunosuppressive enzymes and
cytokines such as NOS2 and COX2, we first sought to ascertain whether these MDSCs
have an increased capacity to suppress T cells. Indeed, neural conditioned MDSCs
exhibited an increased capacity to suppress CD8+ T cell proliferation, enhancing their
immunosuppressive capabilities (Figure 2.6A). Beyond immunosuppression, previous
studies have demonstrated that tumor proliferation can be enhanced via paracrine
secretion of IL-6, IL-10, and TNF as well as through oxidative metabolism including the
generation of ROS and NO [19, 72, 73]. Given that MDSCs in the brain microenvironment
upregulate these key pro-proliferative cytokines and enzymes, we investigated whether
these MDSCs would have an increased capability to promote tumor proliferation. Indeed,
we observed an increase in breast tumor cell proliferation when in the presence of mouse
MDSCs in the brain microenvironment (Figure 2.6B). These findings suggest a novel,
pro-tumorigenic phenotype in MDSCs within the brain metastatic microenvironment.
Figure 2. 6: Brain metastatic MDSCs suppress T cell responses and promote tumor growth.
(A) CFSE proliferation assay of mouse CD8+ T cells co-cultured with J774M in control or mBCM conditions
at a ratio of 4:1 (T cell:J774M). Each peak represents a cell division as determined by CFSE dilution.
Percentage represents percent proliferating CD8+ T cells. (B) Proliferation assay of 4T1 triple negative

33
breast cancer cells co-cultured with mouse neural cells and J774M MDSCs. Data represent n=3-9 replicates
per group per timepoint. Error bars represent ± SEM.
2.5 Discussion:
Investigating MDSCs adaptation and phenotypic alterations within the CNS
microenvironmental niche is crucial to understanding the functions of MDSCs in breastto-brain metastasis. Given the long-held belief that the brain restricted the entry of
peripherally derived immune cells, most studies have focused on MDSC function in
extracranial tumors such as the lung, melanoma, breast, and colon [14]. Recently,
research has expanded to explore the functions of MDSCs in the primary brain cancer,
glioblastoma. While recent studies demonstrate that the density of MDSCs increases
during glioma progression and correlates with worse patient survival, the effects of the
unique brain mileu on MDSC mechanisms and function have yet to be adequately
examined [53].
Overall, our study provides novel evidence that MDSCs are present in breast-tobrain metastasis patient tissues and mouse models. M-MDSCs are more similar to
macrophages and exhibit immunosuppression via NO and ROS, while G-MDSCs are
similar to neutrophils and primarily use ROS as their mechanism of immune suppression
[74]. In our mouse model, we observed an elevated number of G-MDSCs as compared
to M-MDSCs (Figure 2.1A). While this finding isn’t further explored in this thesis, it may
guide our understanding of CNS-specific effects on MDSC plasticity and function.
Traditionally, mouse MDSCs are isolated from tumor-bearing mice or derived
through GM-CSF and IL-6 treatment of bone marrow derived cells [75]. However, these
methods have limitations, including difficulty in acquiring a large number of MDSCs,
minimal to no proliferative capacity, and challenges in genetic manipulation for assessing

34
the effects of certain driver genes. To address these limitations, we utilized J774M mouse
MDSC-like cells, which are isolated CD11b+ Gr1+ cells of Balb/c mouse origin that
demonstrate strong immunosuppressive properties [76]. To validate these in vitro
findings, we generated human MDSCs from healthy peripheral blood donors using an
adapted protocol published by Dr. Epstein of USC. This procedure involves isolating
CD33+ cells from peripheral blood mononuclear cells and treating them with GM-CSF
and IL-6 over the course of the week to differentiate them into suppressive human MDSCs
[61]. The combination of mouse J774M MDSCs, which can proliferate and be genetically
manipulated, coupled with primary-derived human MDSCs expands the mechanistic
capabilities and translatability of our work.
The unique brain microenvironment can alter MDSC function in facets distinct from
other organs. The activation of different pathways in MDSCs can significantly influence
mechanisms of tumor metastasis and survival. Our RNA-seq data demonstrates an
upregulation of proinflammatory pathways and specifically the transcription factor, NF-κB
in MDSCs in the brain microenvironment. Recent literature has shown NF-κB to be a
versatile regulator of tumor-promoting mechanisms, ranging from inflammation,
immunosuppression, metastasis, to angiogenesis. However, NF-κB’s primary
downstream targets are inflammatory, leading to a dual role in tumor formation:
obstructing the immune system from attacking tumor cells and inducing cell proliferation
through mitogenic pathways [77]. Furthermore, NF-κB activation can activate a lethal
positive feedback loop, where NF-κB downstream effectors such as TNF, TLRs, and IL1B
can lead to the reactivation of the transcription factor [78]. Therefore, as shown in our

35
data in Figure 2.6A, NF-κB activation correlates with strong immunosuppressive
capabilities and the ability to increase tumor proliferation.
While the analysis from this chapter implicates NF-κB as the key modulator of
MDSC activity in the brain microenvironment, further mechanistic studies are required to
identify which neural cues are influencing MDSC phenotypic changes. This examination
is the focus of Chapter 3.

36
Chapter 3: Upregulation of Serotonergic Receptor, HTR2B, Drives NFκB Signaling in Brain Metastatic MDSCs
3.1 Abstract
While MDSCs exhibit plasticity and adapt to their local niche, their ability to
respond to neural cues in the CNS-metastatic environment remains poorly understood.
Our study demonstrates that MDSCs in the brain express neural receptors and markers
and, when treated with serotonin, upregulate NF-κB signaling. Further, MDSCs cause
neurons to upregulate their serotonin production, fueling a potential positive feedback
loop. Through patient tissue arrays, scRNA-seq, and in vitro MDSC models, we show that
MDSCs express HTR2B, a receptor previously shown to activate NF-κB signaling in
cardiomyocytes. Knockdown of HTR2B expression in MDSCs reduced NF-κB activation
and the expression of NF-κB downstream inflammatory cytokines, including IL-6, TNF,
and COX2. Consequently, these HTR2BKD MDSCs exhibited diminished
immunosuppressive capabilities and a reduced ability to promote tumor cell proliferation,
highlighting the functional effects of the HTR2B-NF-κB signaling axis. Our findings
suggest that targeting the HTR2B-NF-κB axis may represent a promising therapeutic
strategy to reduce the immunosuppressive effects of MDSCs and improve outcomes in
brain metastases.
3.2 Introduction
In the preceding section, we demonstrated that both mouse and human MDSCs
upregulate NF-κB signaling and downstream inflammatory pathways within the brain
microenvironment. While these MDSCs adapt to the neural cues of the CNS

37
microenvironment to drive their phenotypic changes, the specific signals they respond to
remain unclear.
Neurons play a key role in cellular signaling through the secretion of classical
neurotransmitters such as GABA, glutamate, acetylcholine, dopamine, and serotonin.
Recent research has focused on the neuro-immune axis, investigating functional
interactions between the neuronal and immune systems in homeostasis, multiple
sclerosis, autism, chronic inflammatory disorders, and cancer [79]. Studies suggest that
tumor-infiltrating immune cells can upregulate neurotransmitter receptors in extracranial
tumors to drive cell migration and immunosuppressive function [26]. For example,
macrophages in hepatocellular sarcoma and fibrosarcoma utilize glutamate signaling to
activate NMDAR-mediated calcium influx and subsequent ROS production, driving
immunosuppressive function [80]. While there are no published studies on the role of
classic brain mediators on MDSCs in the context of cancer, research has shown that
MDSCs utilize non-CNS derived acetylcholine and norepinephrine to enhance their
immunosuppressive capabilities through ROS production and IL-10 secretion,
respectively [27, 28].
Serotonin has been attributed to various immunoregulatory functions on immune
cells. Nearly all immune cells express at least one of the 15 distinct subtypes of serotonin
receptors (5-HTR1-7), which belong to the G protein-coupled receptor (GPCR)
superfamily except for 5-HT3R [33]. Serotonin has shown to stimulate monocytes and
lymphocytes, influencing the secretion of pro-inflammatory cytokines [81, 82].
Specifically, serotonin can increase the production of IL-1B, IL-6, and TNF in
macrophages via 5-HT2 receptor stimulation [83, 84]. These same cytokines were

38
significantly upregulated in our MDSCs within the brain microenvironment, as described
in Chapter 2. However, there have been no studies examining the effects of serotonin on
MDSC phenotype or expression of serotonin receptor machinery.
One serotonin receptor of interest is HTR2B, as it has been shown to activate the
transcription factor NF-κB in lung vasculature and cardiomyocytes [38, 41]. Given our
findings in Chapter 2 that MDSCs exposed to the brain microenvironment upregulate NFκB and its downstream effectors, we hypothesized that MDSCs might utilize the HTR2BNF-κB signaling axis to exert an immunosuppressive and pro-tumorigenic niche.
In this study, we first investigated whether MDSCs in the brain microenvironment
upregulate classical neurotransmitter receptors and whether exogenous treatment with
classical neurotransmitters upregulate NF-κB signaling. We examined the source of
serotonin in brain metastasis and whether HTR2B expression in both human patient
tissues and in vitro MDSC models is present. Furthermore, we generated HTR2B
knockdown MDSC cell lines to examine changes in NF-κB activation and its downstream
regulators. Finally, we assessed the effect of MDSC-HTR2B on immunosuppression and
the promotion of breast tumor proliferation. This study provides key insights into the role
of neuronal input in facilitating activation of the HTR2B-NF-κB axis, which fuels a protumorigenic niche.
3.3 Results
3.3.1 Serotonin Mediates NF-κB Activation in Brain-Adapted MDSCs
Previous studies have demonstrated a gain of neurotransmitter receptors in
immune cells, signaling through these receptors to modulate immune function [85].

39
However, this phenomenon has yet to be studied in MDSCs within the metastatic brain
environment. Utilizing bulkRNA-seq data of isolated MDSCs from our mMDSC-brain
model, we assessed whether neural-exposed MDSCs upregulated key neurotransmitter
and neural markers within key neurotransmitter subclasses (serotonin, acetylcholine,
dopamine, GABA, reelin, glial factors, and neuronal factors). This analysis revealed that
neural-exposed MDSCs upregulated 49 out of 59 assessed markers (Figure 3.1A).
Notably, key neuronal factors such as BDNF and its receptor NTRK2 were highly
expressed, which are crucial neurotrophic factors regulating neuronal growth, synapse
formation, and structural plasticity [86]. When examining classical neurotransmitters, we
observed a distinct upregulation of multiple GABAergic, dopaminergic, and serotonergic
transporters, receptors, and catabolic enzymes. Collectively, this data suggests that
MDSCs uniquely adapt to CNS neural cues by upregulating neuronal characteristics.
Figure 3. 1: MDSCs upregulate neuronal markers in the brain microenvironment.
(A) Selected neuronal gene expression of key neurotransmitter pathways in J774Ms in mMDSC-brain
model by bulk RNA sequencing.

40
To identify which of these neurotransmitters might be guiding MDSC shifts to a
pro-inflammatory phenotype in the brain microenvironment, we treated our mouse
MDSCs with serotonin, acetylcholine, dopamine, GABA, and norepinephrine and
assessed pNF-κB expression. Immunofluorescence staining revealed that MDSCs
treated with exogenous serotonin exhibited a significant increase in pNF-κB expression
compared to acetylcholine, dopamine, GABA, or norepinephrine (Figure 3.2A). The
immunomodulatory function of serotonin on MDSCs remains largely unexplored. To
assess the effects of serotonin on NF-κB downstream effector gene expression, we
treated mouse MDSCs with exogenous serotonin and evaluated the gene expression of

41
Figure 3. 2: Serotonin regulates MDSC activation of NF-κB and its downstream markers.
(A) IF staining and quantification of pNF-κB expression in J774M after treatment with serotonin,
acetylcholine, dopamine, GABA, or norepinephrine. 3 images per group, 7-10 cells per image. Scale bar =
50 μm. (B) qPCR of NF-κB associated inflammatory markers in J774Ms treated with 50 nM exogenous
serotonin (5-HT) over multiple timepoints. (C) Flow cytometric representative image and analysis of pNFκB in J774M treated with 50 nM 5-HT. Data represent n=3 replicates per group. Error bars represent ±
SEM.
Il6, Cxcl2, Il1b, Cox2, and Mmp9 over various timepoints. We observed a significant
increase in NF-κB downstream inflammatory gene expression at earlier timepoints, but a
significant decrease at later timepoints (Figure 3.2B). This suggests serotonin activates
an inflammatory cycle with pro-inflammatory signals followed by anti-inflammatory
signals, reflecting a dynamic balance [87]. Together, this data strongly implicates
serotonin as a key immunomodulatory regulator of MDSC function in the brain metastatic
environment.
To investigate the source of serotonin in brain metastases, we analyzed the
expression of serotonin precursor enzymes TPH1, TPH2, and DDC in brain metastatic
tissue samples. scRNA-sequencing revealed that neurons were the primary cell type
expressing these serotonin-synthesizing enzymes (Figure 3.3A-B). Consistent with this
observation, gene microarray analysis of healthy brains demonstrated that all brain lobes
and regions express the necessary machinery for serotonin synthesis (Figure 3.3C).
Furthermore, in mice bearing 4T1 intracardiac tumors, high serotonin levels were
observed in infiltrating MDSCs, brain tumor tissue, and adjacent brain tissue, specifically
within the brain microenvironment, but not in other common breast cancer metastatic sites
(liver and lung; Figure 3.4A-B).

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Figure 3. 3: Neurons are a key driver of serotonin synthesis in brain metastasis.
(A) UMAP visualization of single-cell RNA sequencing data of human breast to brain metastasis patient
tissues (n=15). Cells are color-coated by major cell type cluster. (B) Expression of serotonin synthesis
enzymes (TPH1, TPH2, DDC) is shown. (C) Gene microarray analysis of serotonin synthesis genes TPH1,
TPH2, and DDC by anatomical segmentations of healthy human brain tissue (n=6). Data obtained from the
Human Brain Atlas of Allen Institute (available from: human.brain-map.org/microarray/search). MRI crosssections included. Abbreviations: FL, frontal lobe. Ins, insula. cc, corpus callosum. OL, occipital lobe. PL,
parietal lobe. TL, temporal lobe. BG, basal ganglia. BF, basal forebrain. Amg, amygdala. HiF, hippocampal
formation. ET, epithalamus. TH, thalamus. Hy, hypothalamus. MES, mesencephalon. MET,
metencephalon. MY, myelencephalon.

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Figure 3. 4: Brain Metastases Exhibit Increased Serotonin Compared to Other Breast Metastatic
Sites
(A) IF of MDSCs (GR-1) and 5-HT from differing metastatic organs (Brain, Liver, Lung) after 4T1
intracardiac injection. Orthogonal images demonstrate colocalization of 5-HT and GR-1. Arrows point to
MDSCs. (B) IF of 5-HT in tumor and tumor-adjacent tissue from differing metastatic organs (Brain, Liver,
Lung) after 4T1 breast cancer intracardiac injection. B = Brain, LU = Lung, LI = Liver, T = Tumor. Scale bar
= 50 μm. Data represent n=3 replicates per group.
To explore the potential interplay between MDSCs and neurons on serotonin
expression, we performed co-culture experiments that revealed MDSCs induced
serotonin upregulation in neurons, whereas neurons did not reciprocally upregulate
serotonin production in MDSCs (Figure 3.5A-B). These findings strongly suggest that
neurons are the primary source of serotonin in the brain metastatic environment.
Importantly, inhibiting neuronal serotonin production with LX-1031 resulted in
downregulation of key NF-κB downstream genes in MDSCs, highlighting the critical role
of serotonin availability for MDSC adaptation and function within the neural niche (Figure
3.5C). While further investigation is needed, these results suggest that MDSCs in the
brain microenvironment may be particularly sensitive to serotonin modulation compared
to other metastatic sites, potentially due to the brain’s unique cellular composition and
neurochemical signaling pathways.

44
Figure 3. 5: MDSC-mediated serotonin upregulation in neurons modulates expression of NF-κB
downstream markers in MDSCs.
IF staining and quantification of 5-HT in Neurons alone or co-cultured with J774Ms (A) or in J774Ms alone
or co-cultured with brain cells (B). J774Ms were stained with a Far Red dye immediately prior to co-culture
seeding to distinguish from other cell populations. Neurons stained with TUJ1. 5-HT measured by mean
intensity per cell. 4-5 images per group, 5-7 cells per image. (C) qPCR analysis of NF-κB associated
inflammatory markers in the mMDSC-brain model treated with 5 μm TPH inhibitor LX1031. Data represent
n=3 replicates per group. Error bars represent ± SEM.
3.3.2 Brain Metastatic MDSC Express the Serotonergic Receptor, HTR2B
Given the importance of serotonin for MDSC function in the brain, we sought to
identify the specific serotonin receptors involved in mediating NF-κB signaling. We
analyzed the expression of various serotonin receptors in MDSCs isolated from the brain
microenvironment and found that HTR2B was the most highly upregulated in MDSCs
(Figure 3.6A). Additional In vitro analysis our mMDSC- and hMDSC-brain models further
revealed a significant upregulation of HTR2B gene and protein expression (Figure 3.6BD).

45
Figure 3. 6: MDSCs upregulate serotonergic receptor, HTR2B, in our mMDSC- and h-MDSC-brain
models.
(A) qPCR analysis of serotonergic receptors in the mMDSC-brain model. (B) Flow cytometric analysis of
HTR2B in the mMDSC-brain model. qPCR analysis (C) and flow cytometric analysis (D) of HTR2B in the
hMDSC-brain model. Flow cytometric signal measured by percent positive. Data represent n=2-3 replicates
per group. Error bars represent ± SEM.
HTR2B, a serotonergic receptor previously shown to promote NF-κB activation in
lung vasculature and cardiomyocytes was next investigated for its expression in MDSCs
within the brain metastatic microenvironment (15, 16). Single-cell RNA-sequencing
(scRNA-seq) analysis confirmed HTR2B expression in MDSC subpopulations from brain
metastasis patients (Chapter 2), across differing primary tumor origins (Figure 3.7A-B).
Immunofluorescence staining of our brain metastatic tissue microarray (Chapter 2) further
revealed HTR2B expression in both M-MDSCs (CD11b+ CD14+ CD15- HLA-DRHTR2B+) and G-MDSCs (CD11b+ CD14- CD15+ HLA-DR- HTR2B+) in human breast
and lung-to-brain metastatic tissues (Figure 3.7C-D). Consistent with these findings,
MDSCs in the brain metastatic niche of mice bearing 4T1 intracardiac tumors expressed
HTR2B, whereas MDSCs in other common metastatic sites (liver and lung) did not
(Figure 3.7E). These results suggest that MDSC-HTR2B expression may be a brain-

46
specific phenomenon, potentially driven by the unique microenvironmental factors
present in the brain.
Figure 3. 7: MDSC-HTR2B expression is detected in human and mouse brain metastatic tissues.
(A) UMAP visualization of single-cell RNA sequencing data of human breast to brain metastasis patient
tissues (n=15). UMAP visualization of myeloid cells, color by classification as MDSCs or other myeloid cells.
(B) Expression of HTR2B is shown. IF and H&E of MDSCs and HTR2B in breast-to-brain (C) and lung-tobrain (D) metastatic patient tissues. Scale bar = 50 μm. (E) IF of MDSCs (GR-1) and HTR2B from differing
metastatic organs (Brain, Liver, Lung) after 4T1 intracardiac injection. Orthogonal images demonstrate
colocalization of HTR2B and GR-1. Arrows point to MDSCs. Data represent n=3 replicates per group. Scale
bar = 50 μm.

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3.3.3 HTR2B-NF-κB Signaling Axis Regulates MDSC Activity and Promotes Tumor
Growth
These findings led us to hypothesize that MDSCs upregulate pNF-κB via an
HTR2B-mediated mechanism. To test this hypothesis, we generated and validated
shRNA-HTR2B knockdown (HTR2BKD) and shRNA-Control (Scrambled) mouse MDSC
cells by assessing their gene and protein expression of HTR2B (Figure 3.8A-B).
Figure 3. 8: Establishment of MDSC-HTR2B knockdown (J774M Htr2bKD) cell lines.
qPCR analysis (A) and flow cytometric analysis (B) of HTR2B in J774M transduced with either shRNA
control (Scrambled) or shRNA targeting HTR2B (Htr2bKD) after treatment with mBCM. Data represent n=3
replicates per group. Error bars represent ± SEM.
Further, HTR2BKD MDSCs in neural conditioned media displayed a significant
downregulation of pNF-κB expression (Figure 3.9A). Additionally, HTR2BKD MDSCs in
our mMDSC-brain model exhibited a significant downregulation of NF-κB downstream
gene expression, including Il6, Cxcl2, Cox2, Nos2, Tnf, Mmp9, and Tlr8 (Figure 3.9B).
To determine whether the changes detected at the mRNA expression level translated to
protein expression, we conducted a multiplex ELISA cytokine panel, which revealed a
downregulation of 67% of the cytokines assayed in our HTR2BKD MDSCs compared to
Scrambled MDSCs in the brain microenvironment (Figure 3.9C). Many of the key
downregulated cytokines were IL-6, TNFa, CXCL2, CX3CL1, IL10, and CXCL9. While
cytokine release was assayed via analysis of the extracellular media, we examined the
expression of enzymes COX2 and MMP9 using flow cytometric analysis. This revealed a

48
similar downregulation of these markers in HTR2BKD MDSCs compared to Scrambled
MDSCs in the brain microenvironment (Figure 3.9D-E).
Given the known roles of NF-κB activation and its downstream effectors in
regulating immune suppression, we investigated the impact of the HTR2B-NF-κB
signaling axis on MDSC immunosuppressive function [69, 88]. Neural conditioned
HTR2BKD MDSCs demonstrated a decreased capacity to suppress CD8+ T cell
proliferation, reducing their immunosuppressive capabilities (Figure 3.9F). These
findings suggest that HTR2B regulates NF-κB activation and suppressive signaling within
MDSCs residing in the brain microenvironment.
Figure 3. 9: HTR2B regulates NF-κB signaling pathway in MDSCs.
(A) Flow cytometric analysis of pNF-κB in J774M Scrambled or Htr2bKD after treatment with mBCM. (B)
qPCR analysis of NF-κB associated inflammatory markers in J774M Scrambled or Htr2bKD in the mMDSC-

49
brain model. (C) Heatmap depicting the results of ELISA analysis comparing the levels of NF-κB associated
inflammatory markers in the media of J774M Scrambled or Htr2bKD in the mMDSC-brain model. (D-E) Flow
cytometric analysis of NF-κB associated inflammatory markers in J774M Scrambled or Htr2bKD in the
mMDSC-brain model. Signal measured either by percent positive or MFI. (F) CFSE proliferation assay of
CD8+ T cells co-cultured with J774M Scrambled or Htr2bKD at a ratio of 4:1 (T cell:J774M) after treatment
with mBCM. Data represent n=3 replicates per group. Error bars represent ± SEM.
To distinguish between inflammatory markers directly regulated by the HTR2BNF-κB signaling axis and those influenced by other transcription factors, we used
MLN120B, an IκB kinase β inhibitor that blocks canonical NF-κB activation. BCM-treated
MDSCs exposed to MLN120B exhibited a significant, dose-dependent decrease in NFκB activation (Figure 3.10A). Comparing gene expression profiles of Scrambled control
and HTR2BKD MDSCs following MLN120B treatment allowed us to identify genes
specifically regulated by the HTR2B-NF-κB pathway. Markers downregulated in the
scrambled control but not the HTR2B-knockdown condition were considered direct
targets of this cascade. This analysis revealed Il6, Cxcl2, Il1b, Nos2, and Tlr8 as key
transcriptional targets of the HTR2B-NF-κB signaling axis (Figure 3.10B).

50
Figure 3. 10: Inhibition of NF-κB signaling identifies specific markers influenced by HTR2B-NF-κB
signaling cascade.
(A) Flow cytometric analysis of pNF-κB in J774M after treatment with IKKβ Inhibitor, MLN120B, at
increasing concentrations in mBCM. (B) qPCR analysis of NF-κB associated inflammatory markers in
J774M Scrambled and J774M Htr2bKD after treatment with MLN120B in mBCM. Data represent n=3
replicates per group. Error bars represent ± SEM.
Beyond immunosuppression, MDSCs can enhance tumor cell proliferation [19].
Moreover, NF-κB downstream cytokines, IL-6 and TNF, have been shown to stimulate
cancer cell growth [89, 90]. Therefore, we hypothesized that MDSC-HTR2B signaling may
promote breast tumor proliferation. In the brain microenvironment, mouse 4T1 and human
MDA-MB-231 breast cancer cells co-cultured with HTR2BKD MDSCs exhibited
significantly decreased proliferative capacity (Figure 3.11A-B). Exogenous serotonin
treatment alone increased the proliferative capacity of 4T1 cells in the absence of brain
microenvironmental components (Figure 3.11C). However, co-culturing 4T1 cells with
HTR2BKD MDSCs, compared to Scrambled MDSCs, did not significantly decrease
proliferation, even when supplemented with serotonin (Figure 3.11D). This suggests that
the impact of HTR2BKD MDSCs on tumor cell proliferation is dependent on factors specific
to the brain microenvironment. Collectively, these results support a critical mechanistic
role for the HTR2B-NF-κB signaling axis in shaping both the immunological and nonimmunological functions of MDSCs within the breast-to-brain metastatic
microenvironment.

51
Figure 3. 11: MDSC-HTR2B influences breast tumor proliferation.
(A) Proliferation assay of 4T1 breast cancer cells co-cultured with mouse neural cells and either J774M
Scrambled or Htr2bKD. Data represent n=3-9 replicates per group per timepoint. (B) Proliferation assay of
MDA-MB-231 breast cancer cells co-cultured with either J774M Scrambled or Htr2bKD in mBCM. Data
represent n=5-10 replicates per group per timepoint. (C) Relative cell count of 4T1 treated with 50 nM 5-
HT at 96 hours. Data represent n=6 replicates per group. (D) Relative cell count of 4T1 co-cultured with
J774M Scrambled or Htr2bKD treated with 50 nM 5-HT at 96 hours. Data represent n=6 replicates per group.
Error bars represent ± SEM.
3.4 Discussion
Effectively targeting MDSCs in the brain metastatic microenvironment requires a
thorough understanding of how the local niche influences MDSC adaptation and function.
Previous research has been limited in examining how immune cells, particularly MDSCs,
adapt to the CNS niche to promote the metastatic cascade. Our study aims to
characterize the underlying mechanisms guiding the activation of NF-κB in MDSCs within
the brain metastatic, as observed in Chapter 2.
We discovered that MDSCs in the brain environment upregulate regulators of CNS
competence and neural acclimatation, as previously described in tumor cells [24, 25].
Specifically, exogenous serotonin treatment led to the upregulation of pNF-κB signaling
in MDSCs. There are 13 characterized unique serotonergic receptors, all but one
belonging to the G protein receptor superfamily with distinct signal-transduction

52
mechanisms. The 5-HT2 receptor family stimulates phospholipase C (PLC) and
specifically, HTR2B has shown to utilize this pathway to activate NF-κB in
cardiomyocytes, which is essential for regulating mitochondrial function [38]. Although
HTR2B expression and function have primarily been studied in cardiomyocytes and tumor
cells, our patient-derived tissues and in-vitro analysis with our MDSC models reveal the
upregulation and expression of HTR2B in MDSCs within the brain microenvironment. This
data implicates serotonin and HTR2B signaling as potential activators of the NF-κB
pathway in MDSCs.
Manipulating individual molecules and pathways within the complex networks of
immune responses is essential for understanding immune cell function [91]. Examining
MDSC function has been restricted by their rarity, short lifespan, heterogeneity, poor
viability, and most importantly, difficulty in genetic manipulation. RNA interference
technology, which utilizes short hairpin RNAs (shRNA), offers an alternative strategy for
manipulating gene expression. Lentiviral-mediated transduction using this method of
targeted gene knockdown enables a durable, long-lasting effect. We successfully
employed this technology to knockdown HTR2B expression in our mouse MDSC J774M
cells. However, it is important to note that low levels of HTR2B expression persisted in
mouse MDSCs in the brain environment, as this technology provides a sustained
knockdown of gene expression rather than a complete knockout.
We found that HTR2B knockdown in our mouse MDSCs downregulated pNF-κB
expression and its downstream effectors, suggesting a link between the two. However,
this observation doesn't preclude the involvement of other transcription factors in HTR2Bmediated gene regulation. To dissect the specific contribution of the HTR2B-NF-κB axis,

53
we employed the NF-κB inhibitor MLN120B in conjunction with both scrambled control
and HTR2B knockdown cell lines. This approach allowed us to distinguish genes directly
regulated by the HTR2B-NF-κB pathway from those solely influenced by HTR2B,
potentially through other transcription factors.
Among the HTR2BKD downregulated effector cytokines and enzymes such as IL6, COX2, and TNF, they have been shown to significantly augment the
immunosuppressive abilities of MDSCs while also accelerating tumor proliferation [92-
94]. Correspondingly, we observed HTR2BKD MDSCs exhibited a significantly decreased
capacity to suppress CD8+ T cells and a reduced ability to promote both mouse and
human breast cancer proliferation. This data indicates that HTR2BKD MDSCs in the brain
microenvironment have a subdued MDSC phenotype and exert a weaker pro-tumorigenic
niche.
This study identifies a novel HTR2B-NF-κB signaling axis in MDSCs that supports
tumor progression and anti-tumor immunity. Developing methods to effectively inhibit this
signaling cascade could have significant implications for breast-to-brain metastatic
patients.

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Chapter 4: MDSCs Modulate Neuronal Properties of Breast Cancer
Brain Metastasis
4.1 Abstract
This thesis investigated the role of MDSCs and their serotonin receptor HTR2B in
breast-to-brain metastasis. While previous research has demonstrated that breast cancer
cells adapt to the neural microenvironment by upregulating neuronal genes, the influence
of immune cells and specifically MDSCs on this adaptation remains unclear. Using a
novel in vitro co-culture model incorporating tumor cells, neurons, and MDSCs, we found
that the presence of MDSCs attenuated the upregulation of neuronal markers (SRRM4,
ABAT, and Reelin) in breast cancer cells. Conversely, disruption of MDSC-HTR2B
signaling partially rescued the expression of these neural markers. These findings
suggest that MDSCs, via HTR2B signaling, may establish a permissive microenvironment
that reduces the selective pressure for tumor cells to fully acquire a neural phenotype for
survival in the brain. This work highlights the complex interplay between tumor cells,
neurons, and immune cells within the brain metastatic niche and suggests a novel role
for MDSC-HTR2B signaling in modulating tumor cell adaptation, potentially opening new
avenues for therapeutic intervention.
4.2 Introduction
Breast cancer is the most prevalent malignancy in women worldwide and the
second leading cause of cancer-related deaths. While early-stage breast cancer is
considered curable, distant metastasis drastically reduces the median survival reduces
to a dismal 30 months [95]. Breast cancer brain metastasis accounts for 10-30% of all

55
metastatic breast cancer cases and is associated with poor quality of life and limited
therapeutic options due to the blood-brain barrier [96]. Understanding the intricate
mechanisms underlying the formation and progression of these metastases is crucial for
enhancing treatment efficacy and improving patient prognoses.
The "seed and soil" hypothesis emphasizes the importance of both tumor cell
characteristics and the unique microenvironment of the central nervous system (CNS) in
the development of brain metastases [97]. Unlike extracranial sites, the brain’s distinct
cellular composition, anatomical structure, and metabolic constraints drive the adaptation
and survival of breast tumor cells, forcing them to co-opt a neural niche [98, 99]. Recent
findings from our lab demonstrates that neuronal exposure can induce the expression of
classical neurotransmitter receptor genes and synaptic mediators in breast cancer cells,
facilitating their adaptation and survival within the CNS [24]. Specifically, our studies have
identified the activation of ABAT, Reelin, and SRRM4 signaling, key neuronal signatures
previously implicated in the neural acquisition of tumors.
Breast cancer cells can upregulate GABA transaminase (ABAT) to confer a
proliferative advantage by utilizing CNS-derived GABA as an oncometabolite. [100].
Additionally, Reelin, an extracellular matrix glycoprotein essential for neuronal synaptic
communication, has been demonstrated to regulate tumor migration and invasiveness in
breast-to-brain metastasis and its expression is associated with worse prognoses for
patients [24]. Moreover, Serine/Arginine Repetitive Matrix Protein 4 (SRRM4), a neuralspecific RNA splicing factor critical for development and function, has been shown to play
a crucial role in driving CNS acclimation, dormancy, and brain colonization in breast
cancer cells [25].

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While previous studies have focused on the role of neurons in tumor neuronal
adaptation, this examination has overlooked the contributions of immune cells, primarily
relying on neuron-tumor co-cultures and immunodeficient mouse models. To address this
gap, in this chapter, we utilized co-culture models that mimic a tumor-neuron-immune
microenvironment to investigate the effects of MDSCs on tumor cell neural acquisition.
Furthermore, based on the diminished pro-tumorigenic functions of HTR2BKD MDSCs
observed in Chapter 3, we sought to determine whether HTR2BKD MDSCs differentially
influence colonization and adaptation of tumors within the brain microenvironment. These
findings provide novel insights into the role of immune cells, particularly those expressing
the serotonergic receptor HTR2B, in regulating tumor adaptation and survival within the
CNS.
4.3 Results
4.3.1 MDSCs Regulate Neuronal Acquisition in Breast Cancer Cells
While breast tumor cells have demonstrated adaptation to the neural niche and
exhibited CNS competency for their survival, the role of MDSCs on tumor-CNS
acclimatization remains unexplored. To better understand the effects of MDSCs on tumor
cells, we established a MDSC-tumor-neural in vitro co-culture model and assessed the
gene expression of neurotransmitter receptor and synaptic plasticity markers in tumor
cells. As expected, our gene expression array analysis revealed a strong upregulation of
neurotransmitter and synaptic plasticity mediators in breast cancer cells co-cultured with
neural cells. However, the addition of MDSCs to the brain microenvironment weakened
this neuronal acquisition in breast tumor cells (Figure 4.1A). To further corroborate these

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findings, we investigated the gene expression of three key neural markers: SRRM4,
master mRNA splicing regulator of neuronal gene expression and breast-to-brain
metastasis colonization; ABAT, a critical enzyme in GABA metabolism promoting breast
tumor proliferation; and Reelin, a key extracellular protein regulating neural synapse
formation, migration, and breast-to-brain metastatic proliferation (Figure 4.1B) [25, 100-
102]. Notably, Srrm4 and Abat, but not Reelin gene expression, were significantly
downregulated in 4T1 breast cancer cells within the brain microenvironment when cocultured with MDSCs as compared to when MDSCs were absent. This suggests that
MDSCs play a critical role in regulating tumor cell neural assimilation and adaptation in
the brain environment.
Figure 4. 1: MDSCs Downregulate Neuronal Genes in Tumor Cells Within the Brain
Microenvironment.
(A) Clustergram depicting mRNA expression of mouse neurotransmitter receptors and synaptic mediators
in 4T1 under three conditions: 4T1 alone, Brain co-culture, Brain-J774M co-culture. (B) qPCR analysis of
key neuronal adaptation markers in the three conditions listed in (A).

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4.3.2 MDSC-HTR2B Modulation Mediates Tumor Cells’ Neuronal Acquisition in the
Brain Microenvironment
Given the effects we observed in Chapter 3 of disrupting the MDSC-HTR2B
signaling cascade on immunosuppression and pro-tumorigenic function, we hypothesized
that MDSC-HTR2B could influence breast tumor cell neural adaptation in the brain
microenvironment. Gene expression of neurotransmitter and synaptic plasticity markers
was further downregulated in breast tumor cells in the brain microenvironment when cocultured with Scrambled MDSCs as compared to HTR2BKD MDSCs (Figure 4.2A).
Validation of key neural markers – SRRM4, ABAT, and Reelin – corroborated these
findings. We observed reduced nuclear colocalization of SRRM4 and decreased protein
expression of ABAT and Reelin in 4T1 breast tumor cells within the brain
microenvironment co-cultured with Scrambled MDSCs as compared to HTR2BKD MDSCs
(Figure 4.2B). These results suggest a novel role of MDSC-HTR2B in regulating the
neural acquisition and colonization of breast tumor cells seeking to exploit the CNS
microenvironment.

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Figure 4. 2: MDSC-HTR2B regulates neuronal acquisition in breast cancer cells.
(A) Clustergram depicting mRNA expression of mouse neurotransmitter receptors and synaptic mediators
in 4T1 under three conditions: Brain co-culture, Brain-J774M Scrambled co-culture, Brain-J774M Htr2bKD
co-culture. (B) IF staining and quantification of SRRM4, ABAT, and Reelin protein expression in 4T1 in the
three conditions listed in (A). Tumor cells were stained with CMFDA dye immediately prior to co-culture
seeding to distinguish from other cell populations. SRRM4 signal is measured by nuclear colocalization
rate, whereas ABAT and Reelin are measured by mean intensity per cell. 4-5 images per group, 10-12 cells
per image. Error bars represent ± SEM.
4.4 Discussion
During the metastatic cascade, primary tumor cells acquire cellular and phenotypic
plasticity to survive and adapt to their novel metastatic site [103]. Tumor growth and
metastasis involve various microenvironmental components, including resident and
infiltrating lymphocytes cells, neuronal cells, and glial cells. As metastatic cancer cells

60
establish in target organs that have microenvironmental factors markedly different from
the primary site, the interactions between the tumor and its stromal components influence
metastatic growth. In the context of breast-to-brain metastases, the interactions between
neurons and brain-seeking tumor cells induce CNS adaptation and facilitate the
establishment of brain metastases [24]. However, this gain of CNS-specific attributes has
been studied only in models modeling tumor-neuron interactions, disregarding the effects
of other stromal cells in this brain metastatic niche.
Therefore, our study aims to characterize the effects of MDSCs and MDSC-HTR2B
on tumor cells’ ability to adapt to the neural niche. We observed that while tumor cells
upregulated neural markers when co-cultured with neural cells, the addition of MDSCs to
this co-culture weakened this neural acquisition in breast tumor cells. This finding is
particularly interesting because tumor cells have shown to upregulate neuronal genes to
survive within the brain microenvironment, and we hypothesized that MDSCs would
further upregulate neuronal genes to promote the breast-to-brain metastatic cascade.
However, we observed in Chapter 2 that MDSCs promote tumor proliferation in the brain
microenvironment and advance a pro-tumorigenic niche. This leads us to believe that by
fostering a pro-tumorigenic niche in the brain microenvironment, MDSCs allow tumor cells
to thrive without requiring a drastic change in their phenotype to mimic neural cells.
To further ascertain whether MDSC’s ability to foster a pro-tumorigenic niche
affects tumor cells’ acquisition of neural traits, we compared the effects of MDSC
HTR2BKD against MDSC Scrambled. We observed that MDSC Scrambled further
downregulated neural markers as compared to MDSC HTR2BKD in breast tumor cells in
the brain microenvironment. Further, as observed in Chapter 3, MDSC HTR2BKD

61
exhibited a diminished ability to accelerate breast tumor cell growth compared to MDSC
Scrambled. These findings potentially demonstrate that when the pro-tumorigenic
functions of MDSCs are diminished via HTR2BKD, tumor cells may require more drastic
phenotypic shift to adopt neural features and survive in the brain microenvironment, as
they receive less metastatic assistance from MDSCs.
Cells require a significant amount of energy to alter gene expression, making it
more energy-efficient to turn on genes only when necessary [104]. This is particularly
critical for cancer cells seeking to optimize energy expenditure to accommodate their
metastatic aspirations. We therefore hypothesize that MDSC-HTR2B signaling in the
brain microenvironment creates a pro-tumorigenic niche, allowing tumor cells to thrive
without requiring drastic energetic demands to mimic neural cells. However, further
studies are warranted to elucidate the intricacies of this interplay between MDSCs,
HTR2B signaling, and tumor cell behavior within the brain environment. This study
underscores the importance of examining the effects of the broader tumor
microenvironment in shaping tumor plasticity and adaptation to different metastatic site
niches. This may allow us to uncover novel phenomena that more accurately recapitulate
disease physiology than more restrictive, isolated models.

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Chapter 5: HTR2B Antagonism and Immunotherapy Synergistically
Reduce Breast-to-Brain Metastasis and Improve Survival
5.1 Abstract
This study investigated the therapeutic potential of targeting the serotonin receptor
HTR2B in myeloid-derived suppressor cells (MDSCs) within the brain microenvironment
to enhance immunotherapy efficacy in brain metastases. MDSCs contribute significantly
to immune suppression in tumors, hindering effective cancer immunotherapy. We
hypothesized that HTR2B antagonism could reshape the immunosuppressive
microenvironment and synergize with immune checkpoint blockade. Our results
demonstrate that HTR2B antagonists, including clozapine, a clinically relevant
antipsychotic, effectively suppress the HTR2B-NF-κB signaling axis in MDSCs, reducing
the expression of pro-inflammatory and pro-tumorigenic cytokines. In syngeneic mouse
models of breast cancer brain metastasis (4T1 and E0771), the combination of clozapine
and anti-PD-1 immunotherapy significantly improved survival and reduced both brain
tumor and overall burden compared to either treatment alone. Furthermore, using an
intracranial metastasis model, we observed that the combined therapy increased T cell
infiltration into the brain tumor microenvironment. These findings highlight a novel
mechanism by which MDSCs utilize HTR2B signaling to promote immunosuppression in
the brain and suggest that targeting HTR2B with clozapine may be a promising strategy
to sensitize brain metastases to immunotherapy.

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5.2 Introduction
The T lymphocyte, with their capacity for antigen-directed cytotoxicity, have
emerged as pivotal targets for harnessing the immune system to combat cancer
progression [105]. In-depth mechanistic studies exploring the molecular and cellular
biology of T cells have paved the way for innovative approaches, including immune
checkpoint blockade. Several evolutionarily conserved negative regulators of T cell
activation act as "checkpoint molecules" to maintain immune homeostasis and prevent
excessive activation. PD-1, a key checkpoint molecule, primarily restrains immune
responses through inhibitory intracellular signaling in effector T cells and regulatory T
cells [106]. Malignant cancer cells can exploit this mechanism by upregulating PD-1
ligands, leading to T cell exhaustion and creating a tumor-supportive microenvironment
[107]. Neutralizing the PD-1 axis with monoclonal antibodies (mAbs) or secreted PD-1
extracellular domains has been shown to reverse these effects and enhance T cell
cytotoxicity against tumor cells [108, 109]. Building upon preclinical success, mAbs
targeting the PD-1 axis have been developed and demonstrated efficacy in clinical trials,
offering promising therapeutic options for cancer patients [110].
A substantial portion of patients exhibit minimal response to immunotherapy due
to the suppressive effects of MDSCs on T cell activity. MDSCs contribute to the immune
escape of malignant tumors, creating a challenging microenvironment for effective
immunotherapy. Recent findings suggest that targeting MDSCs could be a promising
alternative approach to reshaping the immunosuppressive microenvironment and
enhancing the efficacy of cancer immunotherapy. Consequently, eliminating these cells
and restoring a functional immune response has become a focal point of research in this

64
field. A preclinical study demonstrated that entinostat, a histone deacetylase inhibitor, can
increase the efficacy of immune checkpoint inhibitors (ICIs) in murine colorectal and
breast cancers [111]. Entinostat reprogrammed tumor-infiltrating MDSCs by significantly
inhibiting the expression of ARG1, iNOS, and COX2, suppressing their
immunosuppressive functions, and ultimately overcoming immune resistance [112, 113].
These findings highlight the potential of targeting MDSCs as a promising strategy to
improve immunotherapy outcomes.
The brain is a common site of metastasis for several solid tumors, including lung,
breast, and melanoma. Despite the availability of treatments like surgery and stereotactic
or whole-brain radiotherapy, immunotherapy remains largely unexplored for metastatic
brain cancer patients. While clinical trials have shown limited therapeutic benefits of
immunotherapy in brain metastatic patients, novel approaches are being investigated,
such as combining immunotherapy with radiosurgery in breast-to-brain metastasis
patients (NCT03449238). However, current research has largely overlooked the role of
immunosuppressive cells, like MDSCs, in modulating the immune response and
potentially enhancing the efficacy of immunotherapy.
Given our findings in Chapter 3 regarding the immunosuppressive and protumorigenic functions of MDSC-HTR2B, targeting this receptor could potentially reshape
the immunosuppressive microenvironment and enhance the efficacy of cancer
immunotherapy in brain metastases. A diverse array of small molecule HTR2B
antagonists exists, with some demonstrating hyper-selectivity for HTR2B over closely
related receptors such as SB204741 and RS127445 [114, 115]. Others, such as
clozapine and aripiprazole, exhibit strong selectivity for HTR2B, readily cross the blood-

65
brain barrier, and are FDA-approved antipsychotics for schizophrenia and bipolar disorder
[116]. Interestingly, clozapine is occasionally used to manage cancer-induced seizures in
brain metastatic patients, making it a promising candidate for clinical translation in this
context due to its FDA approval.
In this study, we investigated whether MDSCs within the brain microenvironment
respond to HTR2B antagonism, disrupting the HTR2B-NF-κB signaling cascade. We
hypothesized that targeting HTR2B could reshape the immunosuppressive
microenvironment and enhance the efficacy of immunotherapy in brain metastases. To
test this, we examined brain metastatic formation, overall tumor burden, and survival in
syngeneic breast tumor-bearing mice treated with immunotherapy (anti-PD-1) in
combination with the HTR2B antagonist, clozapine. Furthermore, using a syngeneic
intracranial breast-to-brain metastatic model, we investigated whether mice treated with
anti-PD-1 and clozapine exhibited improved survival and increased T cell infiltration into
the tumor. These findings provide valuable insights into the synergistic effects of
immunotherapy and HTR2B antagonism, highlighting the potential of targeting HTR2B to
sensitize the microenvironment to immunotherapy in brain metastases.
5.3 Results
5.3.1 Small Molecule HTR2B Inhibition Modulates NF-κB Activity in MDSCs
Recognizing the critical role of MDSC-HTR2B on breast tumor proliferation and T
cell immunosuppression, we investigated whether HTR2B antagonism could disrupt the
HTR2B-NF-κB signaling pathway in MDSCs. Treatment of MDSC J774Ms in neural
media with HTR2B antagonists resulted in significant downregulation of key NF-κB-

66
mediated downstream inflammatory cytokines (Il6, Cxcl2, Il1b) and enzymes (Cox2,
Mmp9) at the transcriptional level (Figure 5.1A). This pattern held true for protein
expression of IL6 and CXCL2, with reduced levels observed in MDSC J774Ms treated
with HTR2B antagonists SB204741 and clozapine (Figure 5.1B). We extended these
findings to human MDSCs in human neural media, where clozapine treatment
significantly reduced gene expression of Il6, Cxcl2, Cox2, and Tnf (Figure 5.1C).
Because clozapine can target both dopaminergic and serotonergic receptors, we
examined the effects of exogenous dopamine and HTR2B antagonism on 4T1 cell
proliferation in co-culture with MDSC J774Ms. We observed that the HTR2B antagonists
SB204741 and clozapine had similar effects on tumor cell proliferation, regardless of
dopamine presence (Figure 5.1D). These results suggest that HTR2B antagonists can
effectively mitigate the HTR2B-NF-κB signaling axis in MDSCs, leading to a reduction in
pro-inflammatory and pro-tumorigenic markers.

67
Figure 5. 1: HTR2B antagonists inhibit NF-κB signaling cascade in MDSCs.
(A) qPCR analysis of NF-κB associated inflammatory markers in J774M treated with either 1 uM SB204741, 1 uM RS-127445, 1 uM aripiprazole, or 1 uM clozapine in mBCM. Data represent n=3 replicates
per group. (B) qPCR analysis of NF-κB associated inflammatory markers in hMDSCs treated with 10 uM
clozapine in hBCM. Data represent n=3 replicates per group. (C) ELISA analysis of NF-κB associated
inflammatory markers in J774M treated with either 10 uM SB-204741 or 10 uM clozapine in mBCM. Data
represent n=3 replicates per group. (D) Relative cell count of 4T1 co-cultured with J774M and treated with
10 μm Dopamine, 10 μm SB-204741 or 10 uM clozapine in mBCM at 96 hours. Data represent n=6
replicates per group. Error bars represent ± SEM.
5.3.2 Targeting HTR2B Augments Immunotherapy in Breast-to-Brain Metastasis
MDSC-HTR2B dysregulation reduces tumor growth and promotes T cell
proliferation within the brain microenvironment, creating a potentially favorable
environment for enhanced anti-tumor immunity. However, The PD-L1 ligand on tumor
cells suppresses the immune response by binding to the PD-1 receptor on T cells,
hindering T cell proliferation and cytokine production [117]. Blocking the PD-1 pathway

68
with an immune checkpoint inhibitor can reinvigorate T cells, enhancing their ability to
recognize and eliminate tumors. Therefore, we hypothesized combining HTR2B
antagonism (clozapine) with immune checkpoint blockade (anti-PD-1) could
synergistically augment the anti-tumor response leading to increased survival and
reduced metastatic brain tumor burden.
To test this hypothesis, we injected either Triple Negative 4T1-FF/GFP or Luminal
B E0771-FF/GFP metastatic breast tumor cells intracardially into immune-competent
female Balb/c mice, followed by concurrent treatment with clozapine + anti-PD-1 (Figure
5.2A). Mice receiving this dual treatment exhibited a significant increase in survival
compared to those receiving single-agent or no treatments (Figure 5.2B-C). For 4T1
bearing mice, median survival for vehicle-treated mice was 16 days post-tumor
implantation, whereas mice treated with both clozapine and anti-PD-1 survived for 25
days – a 56% increase. For E0771 bearing mice, median survival for vehicle-treated mice
was 22 days post-tumor implantation, whereas mice treated with both clozapine and antiPD-1 survived for 43 days – a 95% increase.
Bioluminescent imaging showed that dual treatment of clozapine and anti-PD-1
but not single-agent treatments significantly reduced brain tumor burden compared to
vehicle-treated in both 4T1 and E0771 tumor-bearing mice (Figure 5.2 D-G). In 4T1-
bearing mice, the combination treatment also delayed the median onset of brain
metastasis (14 days) compared to vehicle (7 days), clozapine (10 days), and anti-PD-1
(10 days) alone (Figure 5.2H). While anti-PD-1 monotherapy reduced overall tumor
burden in 4T1-bearing mice similar to the combination treatment, it did not significantly
affect brain tumor burden (Figure 5.2I). In E0771-bearing mice, only the combination

69
treatment significantly reduced overall tumor burden (Figure 5.2J). There is concern that
clozapine treatment can lead to agranulocytosis [118]. Analysis of spleens from the
different treatment groups of 4T1 bearing mice shows that agranulocytosis was not
observed in any treatment group (Figure 5.2K). Overall, these findings suggest a
synergistic effect of HTR2B antagonism with anti-PD-1 specifically in reducing brain tumor
growth.

70
Figure 5. 2: HTR2B antagonism and immunotherapy synergistically delay breast-to-brain
metastasis and improve survival.
(A) In vivo mouse breast metastatic model. Female mice were injected intracardiac with Triple Negative
4T1 or Luminal B E0771 breast cancer cells and subsequently treated with either vehicle control, clozapine
(administered daily), anti-PD-1 (administered twice per week), or a combination of clozapine and anti-PD1. (B) Kaplan-Meier survival curve of mice described in (A). 4T1 injected mice in the clozapine + anti-PD-1
group exhibited improved survival compared to vehicle (p=0.0086, hazard ratio [HR]=11.25), clozapine
alone (p=0.0244, HR=6.715), and anti-PD-1 alone (p=0.0136, HR=8.601) (log-rank test). n=5 per group.
(C) E0771 injected mice in the clozapine + anti-PD-1 group exhibited improved survival compared to vehicle
(p=0.0123, HR=9.401) (log-rank test). n=5-6 per group. (D-E) 4T1 injected mice in vivo bioluminescence
imaging (BLI) at day 10 and 14 and bar graph of mouse head BLI signal between treatment groups at day
10. (F-G) E0771 injected mice in vivo BLI at day 9 and 13 and bar graph of mouse head BLI signal between
treatment groups at day 13. BLI intensity scale was normalized (4T1: Min: 2.5e5, Max:5.0e6; E0771: Min:
5e3, Max:2.5e4). (H) Graphical comparison of brain metastasis (BM) incidence over time in 4T1 injected
mice (n=5 per group). BM incidence determined through BLI signal. Mice in the clozapine + anti-PD-1 group
displayed a delayed onset of BM compared to vehicle (p=0.0116, HR=12.81) and clozapine alone
(p=0.0080, HR=14.38) (log-rank test). Bar graph represents total BLI signal at day 10 (4T1) (I) and day 13
(E0771) (J) post-tumor implantation. (K) H&E images of spleen from treatment groups of 4T1 intracardiac
injected mice. Arrows point to granulocytes. Scale bar = 20 μm. Error bars represent ± SEM.
5.3.3 Combined HTR2B Blockade and Immunotherapy Promotes T Cell Infiltration
Within Intracranial Tumors
Because dual treatment of clozapine and anti-PD-1 in a breast metastatic model
led to increased survival and reduced brain tumor burden, we sought to better understand
the mechanisms underlying this synergy within the brain microenvironment. To model
this, we injected 4T1 metastatic breast tumor cells into the brain of female Balb/c mice,
followed by treatment with clozapine and anti-PD-1 (Figure 5.3A). We observed a
significant increase in survival only in mice treated with the combination of clozapine +
anti-PD-1 but not with single-agent treatments (Figure 5.3B). Median survival of vehicletreated mice was 20 days post-tumor implantation, whereas mice treated with both
clozapine and anti-PD-1 survived for 26 days – a 30% increase. Given that HTR2B
inhibition has shown to increase T cell proliferation and anti-PD-1 blockade can generate
a robust T cell mediated anti-tumor response, we hypothesized that this dual treatment
regimen could promote increased T cell infiltration within the brain tumor

71
microenvironment. Indeed, immunohistochemical analysis of metastatic brain tumor
tissues revealed a significant increase in CD3+ T cell infiltration in mice treated with the
combination of clozapine and anti-PD-1 as compared to vehicle or single agent
treatments (Figure 5.3C). This suggests that the combined strategy of HTR2B
antagonism and immunotherapy has the potential to transform a breast-to-brain tumor
microenvironment from immunologically “cold” to “hot”, leading to improved survival
outcomes.
Figure 5. 3: HTR2B inhibition and immunotherapy promotes T cell infiltration into intracranial tumor.
(A) Mouse breast-to-brain metastatic model. Female mice were injected intracranially with 4T1 breast
cancer cells and subsequently treated with either vehicle control, clozapine (administered daily), anti-PD-1
(administered twice per week), or a combination of clozapine and anti-PD-1. (B) Kaplan-Meier survival
curve of mice described in (A). Mice in the clozapine + anti-PD-1 group demonstrated improved survival
compared to vehicle (p=0.019, HR=4.001), clozapine alone (p=0.0009, HR=7.849), and anti-PD-1 alone
(p=0.0193, HR=3.727) (log-rank test). n=9-10 per group. (C) IHC images and analysis of brain tumor tissue
sections stained for CD3+ T cells across the groups outlined in (A). H&E staining is included. Arrows
indicate CD3+ cells. 3 mice per group, 5-6 images per tissue. Scale bar = 50 μm. Error bars represent ±
SEM.
Our findings collectively suggest a novel mechanism by which MDSCs in the brain
microenvironment leverage extracellular serotonin to activate a pro-tumorigenic signaling
cascade through HTR2B. This HTR2B-mediated activation upregulates NF-κB, leading

72
to enhanced T cell suppression and promotion of tumor proliferation. However, HTR2B
antagonism with clozapine disrupts this suppressive pathway, resulting in restoration of
T cell activation and reduction of tumor proliferation (Figure 5.4C).
Figure 5. 4: Graphical Conclusion.
(A) Schematic illustration of the HTR2B-NF-κB signaling axis in MDSCs within the metastatic brain
microenvironment.
5.4 Discussion
This study investigated the therapeutic potential of targeting the serotonin
receptor HTR2B in MDSCs within the brain metastatic microenvironment, hypothesizing
that HTR2B antagonism could reshape the immunosuppressive landscape and enhance
the efficacy of immunotherapy. Our findings demonstrate that HTR2B antagonism with
clozapine effectively attenuates the HTR2B-NF-κB signaling axis in MDSCs, leading to
a reduction in pro-inflammatory and pro-tumorigenic markers. This effect was observed
both in murine and human MDSCs, highlighting the translational relevance of our
findings. Critically, we observed a synergistic effect of combining clozapine with anti-

73
PD-1 immunotherapy in reducing brain tumor burden and improving survival in
syngeneic breast cancer models (Fig. 3B-E). This synergy was evident in both triplenegative (4T1) and luminal B (E0771) breast cancer models, suggesting a broader
applicability of this therapeutic strategy.
The observed reduction in brain tumor burden and increased survival in the
combination treatment group can be attributed, at least in part, to the increased
infiltration of CD3+ T cells into the brain tumor microenvironment. This suggests that the
combined treatment effectively converts the immunologically "cold" tumor
microenvironment to a "hot" one, rendering it more susceptible to immune attack.
Clozapine disrupts the HTR2B-NF-κB pathway in MDSCs, diminishing their
immunosuppressive function, allowing for enhanced T cell activation and infiltration.
This effect is further amplified by the concurrent administration of anti-PD-1, which
reinvigorates exhausted T cells and enhances their cytotoxic potential.
Our findings build upon previous work demonstrating the importance of MDSCs
in mediating immune suppression in the tumor microenvironment [53]. While previous
studies have explored targeting MDSCs through various mechanisms, including
inhibition of ARG1, iNOS, and COX2 [111-113], our study introduces a novel
therapeutic approach by targeting the serotonin signaling pathway via HTR2B
antagonism. The use of clozapine, an FDA-approved drug that readily crosses the
blood-brain barrier [116], offers a significant advantage for clinical translation. Its
established safety profile and existing clinical use for other indications make it an
attractive candidate for repurposing in the context of brain metastases. Its occasional

74
use for cancer-induced seizures in brain metastasis patients further strengthens the
rationale for its investigation in this context.

75
Chapter 6: Conclusions and Future Directions
6.1 Recap
This dissertation has explored the critical role of MDSCs in the complex
microenvironment of breast-to-brain metastasis. Our findings provide compelling
evidence for a novel mechanism by which MDSCs contribute to tumor progression and
immune evasion within the central nervous system. We have demonstrated, for the first
time, the presence and functional significance of MDSCs in human and murine models
of brain metastasis, revealing a distinct inflammatory phenotype driven by NF-κB
activation. This activation is modulated by the serotonergic receptor HTR2B, expressed
on MDSCs and activated by neuronally derived serotonin. Crucially, we have shown
that targeting the HTR2B-NF-κB axis with the FDA-approved drug clozapine can
effectively reduce MDSC activity and synergize with anti-PD-1 immunotherapy to
improve survival and reduce tumor burden in preclinical models.
6.2 Limitations and Future Directions
While this study provides significant insights into the role of MDSCs and HTR2B
signaling in brain metastasis, it is important to acknowledge certain limitations. Although
we observed a significant increase in T cell infiltration in the combined treatment group,
a more detailed characterization of the infiltrating immune cell populations is warranted.
Future studies should investigate the specific T cell subsets (e.g., CD4+ vs. CD8+ vs
FOXP3+) and their functional status, as well as the presence and activity of other
immune cell types, such as macrophages, natural killer cells, and microglia. This will

76
provide a more comprehensive understanding of the immunomodulatory effects of
HTR2B antagonism.
While our in vitro and in vivo data strongly suggest a link between HTR2B
signaling and NF-κB activation in MDSCs, further mechanistic investigations are needed
to fully elucidate the underlying molecular mechanisms. For example, examining the
specific signaling pathways downstream of HTR2B that lead to NF-κB activation would
be valuable. Furthermore, investigating potential crosstalk between HTR2B signaling
and other pathways involved in MDSC activation and function could reveal additional
therapeutic targets. Exploring changes in the expression of other immunosuppressive
molecules, such as PD-L1 on tumor cells or other immune checkpoint molecules on T
cells, is also an important area for future research.
Our study primarily utilized preclinical mouse models, and the translatability of
our findings to human patients needs to be validated in clinical trials. Future studies
should investigate the expression of HTR2B in MDSCs from patients with brain
metastases to determine its potential as a biomarker for patient selection and response
to therapy. Furthermore, clinical trials evaluating the safety and efficacy of clozapine in
combination with immunotherapy in patients with brain metastases are warranted. Such
trials should carefully assess the impact of this combination therapy on tumor burden,
survival, and immune responses within the brain microenvironment.
Finally, our findings suggest a potential interplay between MDSCs and tumor cell
adaptation to the brain microenvironment. Future studies should further explore the
mechanisms by which MDSCs influence the expression of neuronal markers in tumor
cells and how this interaction contributes to tumor progression and colonization within

77
the brain. Investigating the role of other cell types within the brain metastatic niche, such
as astrocytes and microglia, would also provide a more complete picture of the complex
interactions shaping the tumor microenvironment.
6.3 Therapeutic Avenues
The results presented in this dissertation suggest that targeting the HTR2B-NFκB signaling axis in MDSCs may represent a promising therapeutic strategy for
improving outcomes in patients with brain metastases, particularly when combined with
immune checkpoint blockade. The use of clozapine, an FDA-approved drug with
established safety and blood-brain barrier penetration, offers an advantage for rapid
clinical translation. The potential of repurposing clozapine for this new indication
warrants further investigation in clinical trials.
Beyond clozapine, exploring other HTR2B antagonists with potentially improved
selectivity and pharmacokinetic properties could lead to the development of even more
effective therapies. Furthermore, investigating the potential of combining HTR2B
antagonism with other immunotherapeutic strategies, such as CAR T cell therapy or
other immune checkpoint inhibitors, warrants additional exploration. Given the complex
interplay between MDSCs, tumor cells, and the brain microenvironment, combination
therapies targeting multiple pathways are likely to be more effective than single-agent
approaches.
In conclusion, this dissertation has shed light on the critical role of MDSCs and
HTR2B signaling in the progression of breast-to-brain metastasis. By identifying a novel
therapeutic vulnerability in this challenging microenvironment, our findings pave the way

78
for the development of more effective therapies for patients with brain metastases.
While further research is needed to fully understand the intricate mechanisms involved
and to validate our findings in clinical trials, the results presented here offer a promising
new direction in the fight against this devastating disease.

79
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Appendix
Table 1: Genes and Primer Sequences
Gene (Mouse) Primer Sequence
Il6 Primer 1: 5’-AGCCAGAGTCCTTCAGAGA-3’
Primer 1: 5’-TCCTTAGCCACTCCTTCTGT-3’
Il1b Primer 1: 5'-GACCTGTTCTTTGAAGTTGACG-3’
Primer 2: 5'-CTCTTGTTGATGTGCTGCTG-3’
Il10 Primer 1: 5’-ATGGCCTTGTAGACACCTTG-3’
Primer 2: 5’-GTCATCGATTTCTCCCCTGTG-3’
Tnf Primer 1: 5’-TCTTTGAGATCCATGCCGTTG-3’
Primer 2: 5’-AGACCCTCACACTCAGATCA-3’
Cox2 Primer 1: 5'-CAAGACAGATCATAAGCGAGGA-3'
Primer 2: 5'-GCGCAGTTTATGTTGTCTGTC-3'
Nos2 Primer 1: 5'-GACTGAGCTGTTAGAGACACTT-3’
Primer 2: 5’-CACTTGCTCCAAATCCAAC-3’
Cxcl2 Primer 1: 5'-CAGAAGTCATAGCCACTCCAAG-3'
Primer 2: 5'-CTTTCCAGGTCAGTTAGCCTT-3'
Htr2b Primer 1: 5'-AGATTTGCTGGTTGGATTGTTTG-3’
Primer 2: 5'-GATGGAGGCAGTTGAAAAGAGA-3 '
Mmp9 Primer 1: 5’-GTGGGAGGTATAGTGGGACA-3’
Primer 2: 5’-GACATAGACGGCATCCAGTATC-3’
Srrm4 Primer 1: 5’-TCTGAAGGTCCATCCTGATCT-3’
Primer 2: 5’-CATCATCGTCGCCAGTATCAC-3’
Abat Primer 1: 5’-GGACTTCCGTCTTCATGAGTC-3’
Primer 2: 5’-ACCTCCACCTCTTCATACCT-3’
Reln Primer 1: 5’-AGCACTCTCTCCTCCTATCTG-3’
Primer 2: 5’-GTCGTGTCTTCTGGATCTTCTC-3’
Arg1 Primer 1: 5'-GAATGGAAGAGTCAGTGTGGT-3'
Primer 2: 5'-AGTGTTGATGTCAGTGTGAGC-3'
Rplpo Primer 1: 5’-GCACAGTGACCTCACACG-3’
Primer 2: 5’-AGAAACTGCTGCCTCACATC-3’

90
Gapdh Primer 1: 5’-AATGGTGAAGGTCGGTGTG-3’
Primer 2: 5’-GTGGAGTCATACTGGAACATGTAG-3’
Ido1 Primer 1: 5'-GTAGAGCGTCAAGACCTGAAAG-3'
Primer 2: 5’-GATATATGCGGAGAACGTGGAA-3'
Nfkbiz Primer 1: 5'-TGCAGGACCCTTACTAAGGA-3'
Primer 2: 5'-GTTCCAGATTTGCTTCTTCCG-3’
Tlr8 Primer 1: 5'-CGTTTTACCTTCCTTTGTCTATAGAAC-3’
Primer 2: 5'-TCTGGAATAGTTCGCTTTATGGA-3’
Tlr1 Primer 1: 5'-GAGCAGAACTAGTGTTGTGAATG-3'
Primer 2: 5'-TCTTCAGAGCATTGCCACAT-3'
Htr1a Primer 1: 5’-ACTCACCTCTCACAGTATCCA-3’
Primer 2: 5’-CTTTTGCTCCTTACCTCCTCTAC-3’
Htr1b Primer 1: 5’-CCAAGTCAAAGTGCGAGTCT-3’
Primer 2: 5’-CCAACACACAATAAATGCTCCT -3’
Htr1d Primer 1: 5’-GTGACCAAGACTCAAAGAATGC-3’
Primer 2: 5’-GGATGCTGGTGATAACAAGACA-3’
HTR1f Primer 1: 5’-ACCAGATCAGGAGGTGAAGT-3’
Primer 2: 5’AAGTTTTGGTCTGATGCGTTT-3’
Htr2a Primer 1: 5’-TCAACTCCAGAACCAAAGCC-3’
Primer 2: 5’CCTTCGAATCATCCTGTAGCC-3’
Htr2b Primer 1: 5’-GATGGAGGCAGTTGAAAAGAGA-3’
Primer 2: 5’-AGATTTGCTGGTTGGATTGTTTG-3’
Htr2c Primer 1: 5’-TTTGCTTTCGTCCCTCAGTC-3’
Primer 2: 5’-ACGTAATCCTATTGAGCATAGCC-3’
Htr3a Primer 1: 5’-GAAGCCTACTACTGTCTCCATTG-3’
Primer 2: 5’-AGTCCACTGCAGAAACTCATC-3’
Slc6a4 Primer 1: 5’-CGTTGGTGTTTCAGGAGTGAT-3’
Primer 2: 5’-CATCGTCTGTCATCTGCATCC-3’
Htr4 Primer 1: 5’-CGATCTTTCACCTGTGCTGTA-3’
Primer 2: 5’-CTCCCAACATTAATGCGATGC-3’
Htr5a Primer 1: 5’-CAGGAAGACCAACAGCGT-3’
Primer 2: 5’-TCCACGTATCCCCTTCTGT-3’
Htr5b Primer 1: 5’-GGAAAATATACAAAGCCGCCAA-3’

91
Primer 2: 5’-TGTGAACACCATCTCAGACTC-3’
Htr6 Primer 1: 5’-GCCTTGGAAACCTTGCAG-3’
Primer 2: 5’-GTGACAAAGAACATGCTCAGC-3’
Htr7 Primer 1: 5’-TCTGCAACGTCTTCATCGC-3’
Primer 2: 5’-TACATTTCCCATTCTGCCTCA-3’
Gene (Human) Primer Sequence
IL6 Primer 1: 5’-ATTCGTTCTGAAGAGGTGAGTG-3’
Primer 2: 5’-CCTTCCCTGCCCCAGTA-3’
IL1B Primer 1: 5'-CAGCCAATCTCATTGCTCAAG-3'
Primer 2: 5'-GAACAAGTCATCCTCATTGCC-3’
TNF Primer 1: 5’-TCAGCTTGAGGGTTTGCTAC-3’
Primer 2: 5’-TGCACTTTGGAGTGATCGG-3’
COX2 Primer 1: 5’-TGTTTGGAGTGGGTTTCAGA-3’
Primer 2: 5’-GAGTGTGGGATTTGACCAGTA-3’
NOS2 Primer 1: 5'-CACCATCCTTTGCGACA-3'
Primer 2: 5'-GCAGCTCAGCCTGTACT-3'
CXCL2 Primer 1: 5'-TTCACAGTGTGTGGTCAACAT-3'
Primer 2: 5’-TCTCGCTCTAACACAGAGGGA-3'
HTR2B Primer 1: 5'-AGAAGAAGCTGAGTATTAC-3'
Primer 2: 5’-AGGCAGGACATAGAACAAGTG-3 '
RPLPO Primer 1: 5’-TGTCTGCTCCCACAATGAAAC-3’
Primer 2: 5’-TCGTCTTTAAACCCTGCGTG-3’

92
Table 2: Antibodies
Primary Antibodies
Antibody Company Catalog # Host Concentration
used
CD11b (tissue) Invitrogen 53-0196-82 Mouse 1:50
HLA-DR (tissue) Novus Biologicals NB100-
2707PCP Mouse 1:50
CD14 (tissue) Novus Biologicals NBP2-
89259AF405 Rabbit 1:100
CD15 (tissue) Abcam ab281744 Rabbit 1:100
CD45 (flow) Biolegend 103151 Rat 1:200
CD11b (flow) ThermoFisher 48-0112-82 Rat 1:240
Ly6g (flow) Biolegend 127605 Rat 1:300
Ly6c (flow) Biolegend 128012 Rat 1:100
pNF-κB Cell Signaling 3033S Rabbit 1:500
COX2 Cell Signaling 12282S Rabbit 1:200
HTR2B MyBioSource MBS9608087 Rabbit 1:100
CD3 (tissue) Abcam ab16669 Rabbit 1:150
ABAT Novus Biologicals NBP-2-21598 Rabbit 1:250
SRRM4 biorbyt orb2296 Rabbit 1:200
Reelin Genetex GTX37552 Rabbit 1:200
TUJ1 R&D Systems MAB1195 Mouse 1:50
IDO1 Cell Signaling 51851S Rabbit 1:250
NFKBIZ Cell Signaling 93726S Rabbit 1:250
MMP9 Abcam ab38898 Rabbit 1:200
Serotonin Abcam ab66047 Goat 1:500
Serotonin (tissue) Bioss BS-1126R Rabbit 1:100
GR-1 R&D Systems MAB1037 Rat 1:50
TUJ1 ThermoFisher MA5-32035 Rabbit 1:100
Secondary Antibodies
Antibody Company Catalog # Host Concentration
used
Anti-Rabbit
Alexa Fluor 647 Jackson Immuno 111-6050144 Goat 1:300
Anti-Goat
Alexa Fluor 488 Jackson Immuno 705-545-147 Donkey 1:300
Anti-Mouse
Alexa Fluor 647 Jackson Immuno 715-605-151 Donkey 1:300
Anti-Rat
Alexa Fluor 647 Jackson Immuno 712-605-153 Donkey 1:300
Anti-Rabbit
Alexa Fluor 488 Jackson Immuno 711-545-152 Donkey 1:300
Anti-Rabbit
Alexa Fluor 594 Jackson Immuno 711-585-152 Donkey 1:300

93
Table 3: Allen Brain Atlas Donor Information
Donor Age Sex Ethnicity
H0351.1009 57 years Male White or Caucasian
H0351.1012 31 years Male White or Caucasian
H0351.1015 49 years Female Hispanic
H0351.1016 55 years Male White or Caucasian
H0351.2001 24 years Male Black or African American
H0351.2002 39 years Male Black or African American

Abstract (if available)

Abstract Myeloid-Derived Suppressor Cells (MDSCs) support breast cancer growth via immune suppression and non-immunological mechanisms. Although 15% of patients with breast cancer will develop brain metastasis, there is scant understanding of MDSCs’ contribution within the breast-to-brain metastatic microenvironment. Utilizing co-culture models mimicking a tumor-neuron-immune microenvironment and patient tissue arrays, we identified serotonergic receptor, HTR2B, on MDSCs to upregulate pNF-κB and suppress T cell proliferation, resulting in enhanced tumor growth. In vivo murine models of metastatic and intracranial breast tumors treated with FDA-approved, anti-psychotic HTR2B antagonist, clozapine, combined with immunotherapy anti-PD-1 demonstrated a significant increase in survival and increased T cell infiltration. Collectively, these findings reveal a previously unknown role of MDSC-HTR2B in breast-to-brain metastasis, suggesting a novel and immediate therapeutic approach using neurological drugs to treat patients with metastatic breast cancer.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Iyer, Mukund (author)

Core Title The role of serotonergic receptor, HTR2B, on myeloid-derived suppressor cells in the brain metastatic environment

Contributor Electronically uploaded by the author (provenance)

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Cancer Biology and Genomics

Degree Conferral Date 2025-05

Publication Date 04/02/2025

Defense Date 02/26/2025

Publisher University of Southern California (original), Los Angeles, California (original), University of Southern California. Libraries (digital)

Tag brain metastasis,breast cancer,HTR2B,MDSC,myeloid cells,OAI-PMH Harvest,serotonin,tumor microenvironment

Format theses (aat)

Language English

Advisor Roussos Torres, Evanthia (committee chair), Neman, Josh (committee member), Yu, Min (committee member)

Creator Email mmiyer@usc.edu,mukundiyer1@gmail.com

Unique identifier UC11399K54N

Identifier etd-IyerMukund-13884.pdf (filename)

Legacy Identifier etd-IyerMukund-13884

Document Type Dissertation

Format theses (aat)

Rights Iyer, Mukund

Internet Media Type application/pdf

Type texts

Source 20250403-usctheses-batch-1248 (batch), University of Southern California Dissertations and Theses (collection), University of Southern California (contributing entity)

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