Close
About
FAQ
Home
Collections
Login
USC Login
Register
0
Selected
Invert selection
Deselect all
Deselect all
Click here to refresh results
Click here to refresh results
USC
/
Digital Library
/
University of Southern California Dissertations and Theses
/
Characterization of a new HER2 positive mouse model of metastatic breast cancer
(USC Thesis Other)
Characterization of a new HER2 positive mouse model of metastatic breast cancer
PDF
Download
Share
Open document
Flip pages
Contact Us
Contact Us
Copy asset link
Request this asset
Transcript (if available)
Content
Characterization of a New HER2 Positive Mouse Model of Metastatic
Breast Cancer
By
Valerie Haruka Narumi
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR MEDICINE)
August 2023
ii
ACKNOWLEDGEMENTS
I would like to express my deepest gratitude and appreciation towards my mentor and PI, Dr.
Evanthia Roussos-Torres who has been here to support me since the summer of 2021. The
guidance and confidence she has offered in my scientific knowledge is something I can carry on
forever. I sincerely appreciate everything she has done to support my educational journey.
I would also like to express appreciation towards my two committee members, Drs. Alan Epstein
and Crystal Marconett for their time and efforts into improving my thesis project.
Finally, I would like to thank all members of the Roussos-Torres Lab. Although we have many
members I’d like to thank, I’d like to thank Aaron Baugh, Edgar Gonzales, and Sofi Castanon in
particular for always being there for questions and long hours in experiments!
iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ............................................................................................................ ii
LIST OF TABLES .......................................................................................................................... v
LIST OF FIGURES ....................................................................................................................... vi
LIST OF ABBREVIATIONS ....................................................................................................... vii
ABSTRACT ................................................................................................................................. viii
CHAPTER 1: INTRODUCTION ................................................................................................... 1
CHAPTER 2: MATERIALS AND METHODS ............................................................................ 6
2.1 NT2.5 and NT2.5LM Cell Lines ....................................................................................... 6
2.2 Utilization of NeuN Mice .................................................................................................. 8
2.4 Tumor Dissociation of Breast Tumors from NeuN Mice ................................................. 9
2.5 Flow Cytometry............................................................................................................... 10
2.6 Immunohistochemistry .................................................................................................... 10
2.7 Quantification of Lung Metastases ................................................................................. 11
2.8 Necropsy.......................................................................................................................... 11
2.9 Identification of Survival and Tumor Growth................................................................. 12
2.10 Single-Cell RNA Sequencing, Quality Control, and Data Analysis ............................. 12
2.11 Whole Exome Sequencing (WES) ................................................................................ 14
CHAPTER 3: RESULTS .............................................................................................................. 16
3.1 Derivation of NT2.5LM Syngeneic Model of HER2+ Breast Cancer ............................ 16
3.2 Breast tumors derived from NT2.5LM demonstrate faster growth rate and
increased development of lung metastases............................................................................ 19
3.3 NT2.5-LM expresses similar levels of HER2 but is more proliferative and
demonstrates basal cell morphology ..................................................................................... 21
3.4 Treatment of anti-Her2 therapy improves survival in a subset of mice .......................... 25
3.5 Whole exome seq of NT2.5 and NT2.5LM showed no significant differences
in mutations ........................................................................................................................... 27
3.6 ScRNA-seq of NT2.5LM tumors reveals increased cellular proliferation and
metastatic potential ................................................................................................................ 30
iv
CHAPTER 4: DISCUSSION ........................................................................................................ 34
CHAPTER 5: FUTURE DIRECTIONS ....................................................................................... 38
REFERENCES ............................................................................................................................. 39
v
LIST OF TABLES
Table 1. Subtypes of Breast Cancer ................................................................................................ 2
Table 2. Genes used to annotate and identify cancer cell clusters after Louvain clustering ........ 13
Table 3. Common breast cancer specific mutations ..................................................................... 15
vi
LIST OF FIGURES
Figure 1. Derivation of NT2.5LM Cell Line .................................................................................. 7
Figure 2. Necropsy of the NT2.5LM Spontaneous Model ........................................................... 16
Figure 3. Schematic of the three mouse models utilized .............................................................. 18
Figure 4. Survival experiment to compare NT2.5 and NT2.5LM ................................................ 20
Figure 5. Immunohistochemistry of NT2.5 breast tumor and NT2.5LM lung metastasis............ 22
Figure 6. H&E staining to identify tumor metastasis formation ................................................... 23
Figure 7. Flow cytometry experiment comparing HER2 expression ........................................... 24
Figure 8. Immunohistochemistry Quantification of Ki67............................................................. 25
Figure 9. anti-Her2 treatment in the NT2.5LM spontaneous mouse model ................................. 26
Figure 10. WES Analysis of common breast cancer specific mutations ...................................... 28
Figure 11. Overview of types of mutations between NT2.5 and NT2.5LM ................................. 29
Figure 12. Determining mutations in ErbB2 mRNA sequence .................................................... 30
Figure 13. scRNA seq comparing NT2.5 and NT2.5LM ............................................................. 32
vii
LIST OF ABBREVIATIONS
HER2 Human Epidermal Growth Factor Receptor 2
ER Estrogen Receptor
PR Progesterone Receptor
ICI Immune Checkpoint Inhibitors
FDA Food and Drug Administration
TME Tumor Microenvironment
H&E Hematoxylin and Eosin
scRNA seq Single-Cell RNA Sequencing
WES Whole Exome Sequencing
MFI Median Fluorescence Intensity
mRNA Messenger Ribonucleic Acid
viii
ABSTRACT
Animal models are essential tools for preclinical studies evaluating primary tumor development
and metastatic progression in breast cancer. Murine models with a fully intact immune system are
essential for studies investigating anti-cancer immune responses and reactions to
immunotherapies. In this thesis, characterization of a novel syngeneic murine tumor cell line that
provides a model of spontaneously metastatic neu-expressing breast cancer mammary tumor
implantation of a novel cell line, NT2.5LM, into FVB/NeuN mice was conducted. Mice growing
NT2.5-LM tumors in their mammary fat pads develop spontaneous metastasis to distant organs
including the lungs and bones, which mimics metastatic spread in patients. Development of this
syngeneic mouse model is instrumental in pre-clinical studies of novel therapeutic combinations
in a system that mimics aggressive metastatic breast cancer progression in patients targeted for
therapy.
1
CHAPTER 1
INTRODUCTION
In a study conducted in 2020, breast cancer was identified as one of the leading causes of
cancer death, surpassing lung cancer, with 2.3 million new cases, accounting for 11.7% of all
cancer cases globally
1
. Globally, it is the leading type of cancer incidence in women, and the
metastasis of breast cancer is the most common cause of cancer death, contributing to more than
685,000 deaths annually.
1
The survival rates of those with triple negative breast cancer and human
epidermal growth factor receptor (HER2) overexpressing breast cancer are seen to be the lowest.
Furthermore, there are subtypes in breast cancer that are used to stratify patient treatment
options based on the expression of either estrogen receptor (ER), progesterone receptor (PR), or
HER2 . Luminal A breast cancers are described to be ER positive, PR positive, and HER2 negative.
Luminal B breast cancers are ER positive, PR negative, and HER2 negative. HER2 positive breast
cancers are both ER and PR negative but HER2 positive. Triple negative breast cancers are ER,
PR, and HER2 negative (Table 1). We chose to focus on HER2 positive (HER2+) breast cancers
because they are described to be the one of the most aggressive breast cancers and have been
shown to be most promising with immunotherapy.
2
Table 1. Subtypes of Breast Cancer. Shows the four prevalent subtypes of breast cancer and their
expression of ER, PR, or HER2.
The increase in breast cancer cases is prevalent despite the availability of pre-screening
scans and treatments such as immune checkpoint inhibitors (ICI) for managing cancer and
improving patient quality of life.
2
Today, there is only one U.S. Food and Drug Administration
(FDA)-approved ICI treatment available for patients with breast cancer, however this treatment is
only for patients diagnosed with triple negative breast cancer and does not apply to patients with
HER2+ breast cancer that accounts for 20% of all breast cancers. HER2+ breast cancer
overexpresses the HER2 protein, which is the main cause of its growth.
3,4
Although not FDA
approved, there are immunotherapy options available for patients with HER2+ breast cancer,
although many patients show primary resistance or become eventually resistant to the treatment.
5,6
Identifying new ways to treat HER2+ breast cancer would allow for another treatment option for
patient populations that can improve survival and quality of life for patients. Furthermore,
researching options for HER2+ breast cancer that target the immune suppressor cells within the
tumor microenvironment could benefit all patients with metastatic breast cancer as there are
3
suppressive cells involved in all subtypes of breast cancer.
7,8
In order to research these subtypes of
breast cancers in the most biologically relevant way, murine models that mimic the characteristics
of these cancers are needed to enable study of the immune response.
The classification of clinical subtypes in breast cancer increases the complexity of studying
them and necessitates a good model in order to study their characteristics and therapeutic options.
Hence, the utilization of mouse models to study breast cancer is important. Mouse models for
breast cancer provide an important approach to examine the mechanisms and pathways in cancer
progression and metastasis in order to create and understand clinical therapeutics. Furthermore,
mouse models are one of the closest ways to understand tumor biology within an immune system
similar to human physiology. Currently, examples of models are 4T1 for studying triple negative
breast and NT2.5 for HER2+. However, there are some limitations in the usage of these models to
study cancer spread and the tumor microenvironment of distant metastasis effectively and
efficiently in mouse models. For example, the 4T1 mouse model that represents triple negative
breast cancer (TNBC) metastasizes quickly upon injection in BALB/C mice, and this rapid
mammary tumor growth necessitates the need to euthanize mice prior to adequate growth of distant
spontaneous metastasis.
9
The NT2.5 model that represents HER2+ also has limitations to its
utilization as a mouse model able to study the tumor microenvironment, which will be discussed
in the next section.
The aim of this thesis is to properly characterize a more aggressive murine model of
metastatic HER2+ breast cancer and describe methods to guide its use in preclinical studies.
Currently, there are only a few murine models of HER2+ breast cancer that develop spontaneous
mammary tumors and only some that form distant metastasis into the lungs and lymph nodes. The
original HER2/Neu transgenic mouse model for breast cancer was developed by Guy et. al. The
4
Neu proto-oncogene is part of the epidermal growth family as a transmembrane protein. The
amplification and overexpression of the human homologue of Neu (ERBB2) is observed in a large
percentage of primary breast cancers, where patients exhibiting high levels are implicated to have
low survival. The murine model developed by Guy et. al is an immunotolerant MMTV-HER2/Neu
transgenic murine model in which FBV/N mice overexpress their version of the Neu oncogene and
result in spontaneous mammary tumors around days 125-300. HER2/Neu is a proto-oncogene that
is commonly overexpressed in HER2+ breast cancer and normally identified by the immune
system, which recognizes Neu expression on the tumor cells as foreign and eliminates it. However,
because the murine model characterized in this thesis is immunotolerant to Neu, the immune
system will not recognize it and therefore will not eliminate the tumor, allowing the Neu-
expressing tumor to continue growing. This murine model is characteristic of human HER2+
breast cancer and not only develops spontaneous mammary tumors but also eventually develops
metastasis primarily to the lung. Although this murine model represents human HER2+ breast
cancer and spontaneously develops mammary tumors, the time it takes to do this is very long and
the extent of metastasis is low, making it difficult to study the extent of metastasis or the tumor
microenvironment in a timely manner
10
. After this transgenic mouse model was created, Jaffee et.
al developed a set of NT cell lines that were based off of the transgenic mice developed by Guy et.
al. These cell lines develop tumors and demonstrate immunotolerance to Neu, similar to the
transgenic mouse model. These cell lines used in the mouse model has the capability to mimic the
process of spontaneous metastasis, it does so with low penetrance in which only 20% of mice
develop spontaneous metastasis over many months. This does now allow for timely
experimentation and is difficult to coordinate.
10–12
From these cell lines, Sgouros et. al developed
a mouse model from the NT2.5 cell lines where they injected these cells via cardiac injection to
5
gain metastasis to distant organs. Although metastasis was observed within 3 weeks of cardiac
injection, this model is limited by its inability to recapitulate the process of spontaneous metastasis
and can be seen as artificial in the method in which it develops this metastasis.
10
The novel orthotopic NT2.5LM model presented here captures the entire process of
metastasis starting from the primary breast tumor to the lung. As opposed to the
colonization/tropism models of breast cancer metastasis that involve seeding the cancer cells
directly into the metastatic site,
13
this orthotopic model simulates not only the growth of metastases
at their new location but also the initial migration of the cancer cells from the established primary
tumor. As a breast cancer metastasis model, this orthotopic model involves an injection of cancer
cells within the mammary fat pad allowing reflection of the stages involved in cancer progression
that are commonly seen in patients.
14
Such orthotopic models, including the previously mentioned
4T1 TNBC model, have been employed to study drivers of breast cancer metastasis,
15
to screen
potential therapeutic options, and to study the metastatic tumor microenvironment (TME). This
orthotopic breast cancer metastasis model represents a useful addition to the preclinical scientist’s
toolkit that can aid in the development of novel treatments for patients suffering from HER2+
breast cancer.
6
CHAPTER 2
MATERIALS AND METHODS
2.1 NT2.5 and NT2.5LM Cell Lines
NT2.5 cells were derived from spontaneous HER2 overexpressing mammary tumors
growing in female NeuN mice. Originally, five Neu-expressing mammary tumor cell lines (NT1-
5) were derived from isolation of five different spontaneous tumors growing in transgenic NeuN
mice. In vitro cell lines were established by digestion of tumors with dispase and collagenase as
previously described.
16
Tumor cells were separated from untransformed epithelial cells with
trypsin.
17
Two of these cell lines, the NT2 and NT5 tumor lines, were expanded with production
performed at the NIH cGMP facility in Frederick, MD. Neu and MHC I levels were tested by
fluorescence-activated cell sorting and confirmed to be stable before freezing and storage in liquid
nitrogen and are available through ATCC. NT2.5 cells were derived from a spontaneous mammary
tumor growing in NeuN mice and obtained from Dr. Elizabeth Jaffe’s lab at Johns Hopkins
University.
18,19
NT2.5 cells were cultured from our frozen stocks and passaged twice before
injection. Culture conditions for NT2.5 cells were as follows: 37°C, 5% CO2 in RPMI 1640 (Gibco,
cat. 11875-093) supplemented with 20% fetal bovine serum (Gemini, cat. 100-106), 1.2% HEPES
buffer (Gibco, cat. 15630-080), 1% L-glutamine (Gibco, cat. 25030-081), 1% MEM non-essential
amino acids (Gibco, cat. 11140-050), 0.5% penicillin streptomycin (Gibco, cat. 15140-122), 1%
sodium pyruvate (Sigma, cat. S8636), 0.2% insulin (NovoLog, cat. U-100), 0.02% gentamicin
(Sigma, cat. G1397).
7
Figure 1. Derivation of NT2.5LM Cell Line. This schematic describes the development of the
NT2.5LM cell line from the parental cell line, NT2.
The NT2.5LM cell line was derived following mammary fat pad injection of 1x10^5 NT2.5
cells into the tail vein of five, 8-week old NeuN mice. Three weeks after tail vein injection, lung
metastases were macro-dissected from the lungs of all five mice, minced on ice, filtered using a
100-um filter and pooled. 1x10^5 cells were injected back into the tail vein of an additional five
NeuN mice. This was repeated once more, then on the third round of harvest, lung metastases were
macro-dissected from all five mice on ice, filtered using a 100-um filter, pooled, then 1x10^5 cells
were injected back into the mammary fat pad of additional five, 8-week old NeuN mice. Lungs
were harvested from all five mice three weeks after mammary fat pad injection and evaluated for
formation of spontaneous lung metastasis by serial H&E stains. Spontaneous lung metastases were
observed, thus this cell line was propagated in cell culture under the same conditions used for
culture of NT2.5 cells and given the name NT2.5LM for “Lung Metastasis”. Cell lines are tested
for mycoplasma every three months, and expression of HER2 is tested and characterized below.
Culture conditions for NT2.5-LM cells were as follows: 37°C, 5% CO2 in RPMI 1640 (Gibco, cat.
8
11875-093) supplemented with 20% fetal bovine serum (Gemini, cat. 100-106), 1.2% HEPES
buffer (Gibco, cat. 15630-080), 1% L-glutamine (Gibco, cat. 25030-081), 1% MEM non-essential
amino acids (Gibco, cat. 11140-050), 0.5% penicillin/streptomycin (Gibco, cat. 15140-122), 1%
sodium pyruvate (Sigma, cat. S8636), 0.2% insulin (NovoLog, cat. U-100), 0.02% gentamicin
(Sigma, cat. G1397). MCF7 cells were cultured in media composed of high glucose DMEM (Life
Technologies Inc., cat.10564029) supplemented with 10% fetal bovine serum and 0.5%
penicillin/streptomycin.
2.2 Utilization of NeuN Mice
A syngeneic mouse model of HER2+ breast cancer using the NT2.5 cell line was derived
from the NeuN transgenic mouse developed by Guy et al as described above.
20
NeuN transgenic
mice overexpress non-transforming rat Neu cDNA under the control of a mammary specific
promoter and develop spontaneous focal mammary adenocarcinomas after a long latency of 125
days with the majority of mice developing tumors by 300 days. Injection of this cell line into NeuN
mice leads to development of tumors 100% of the time given mice are tolerized to Neu. NT2.5 or
NT2.5LM cells were injected into the mammary fat pad (1x10^5 cells) for the survival/tumor
growth and anti-HER2 treatment studies.
21
Tumors were allowed to seed for 3-7 days prior to
initiating treatment with anti-HER2 as described below. Untreated mice developed palpable
tumors within one week. NT2.5LM cells were injected into the lateral tail vein (1x10^5 cells in
150 uL PBS) for the survival/tumor growth study. Mice were kept in pathogen-free conditions and
were treated in accordance with institutional and American Association of Laboratory Animal
Committee policies. NeuN mice were originally from W. Muller McMaster University in
Hamilton, Ontario, Canada, and overexpressed HER2 via the mouse mammary tumor virus
9
(MMTV) promoter. Colonies were renewed yearly from Jackson Labs and bred in-house by
brother/sister mating.
2.3 Treatment of Mice
Mice were treated with anti-HER2 antibody to mimic treatment with and without
trastuzumab as is as standard therapy for patients with HER2+ breast cancer. Anti-HER2 antibody
was given once a week for three weeks as described.
21
Following the initial three weeks of
treatment, maintenance dosing was continued every other week. During maintenance therapy,
animals received one dose of anti-HER2 antibody every other week. Dosage of anti-HER2
antibody was100 µg/mouse, which were injected intraperitoneally. Monoclonal antibodies were
obtained from BioXcell anti-HER2 isotype (clone 7.16.4) and diluted to 0.5 mg/mL in PBS.
Isotypes were used to treat vehicle mice and were also obtained from BioXcell anti-HER2 isotype
mouse IgG2a (clone C1.18.4). All isotype antibodies were also diluted to 0.5 mg/mL in PBS.
Dosages of antibody were based off of prior studies.
21
2.4 Tumor Dissociation of Breast Tumors from NeuN Mice
To obtain single-cell suspensions from breast tumors, tumors were harvested after three
weeks, diced, then filtered using a 100 mm cell strainer and red blood cells lysed using ACK lysis
buffer (Quality Biological, cat. 118-156-721). The resulting single-cell suspensions were used for
subsequent injections for cell line derivation. Tumor or lung dissociation for flow cytometry and
sequencing were done with the following protocol: following harvest, tumors were dissociated
using a tumor dissociation kit (Miltenyi Biotec, cat. 130-096-730) and the OctoDissociator
(Miltenyi Biotec) per the manufacturer’s instructions. The 37C_m_TDK_2 program was used to
10
dissociate tumors per the manufacturer’s instructions. Lungs were dissociated using the lung
dissociation kit (Miltenyi Biotec, cat. 130-095-927). Samples were filtered using a 40 mm cell
strainer, and red blood cells were lysed using ACK lysis buffer (Quality Biological, cat. 118-156-
721). Isolation of specific immune cell types or flow cytometry is described below, with single
cell RNA sequencing (scRNAseq) followed by an additional step of dead cell removal using the
MACS Dead Cell Removal Kit (Miltenyi Biotec).
2.5 Flow Cytometry
NT2.5 and NT2.5LM cells were cultured for 10 days in vitro, then washed with PBS and
incubated for 30 minutes at 4°C with Live/Dead Fixable Aqua (ThermoFisher, cat. L10119)
according to the manufacturer’s protocol. Cells were incubated with Fc receptor block (BD
Pharmingen, cat. 553142) for 10 minutes at room temperature, followed by a 30-minute incubation
with 1:100 PE-anti-HER2/Neu (R&D Systems, cat. FAB6744P) at 4°C for extracellular marker
staining. Cells were fixed and permeabilized for 30 minutes at room temperature using the
Foxp3/Transcription Factor Staining Buffer Set (Life Technologies Corp., cat. 00-5523-00),
followed by a 10-minute incubation at room temperature with Fc receptor block. Intracellular
markers were stained with 1:200 Alexa Fluor 647 anti-ER-α (Santa Cruz Biotechnology, Inc. cat.
sc-53493) or 1:200 FITC-anti-PR (Santa Cruz Biotechnology, Inc. cat. sc-398898) for 30 minutes
at room temperature. Samples were run on an Attune NxT flow cytometer (Invitrogen) and
analyzed using Kaluza software and Graphpad Prism.
2.6 Immunohistochemistry
Immunostaining was performed at the Oncology Tissue Services Core of Johns Hopkins
University. Immunolabeling for ErbB2, Ki67, CK5, CK6, AE1/3, and EGFR was performed on
11
formalin‐fixed, paraffin embedded sections. Briefly, following dewaxing and rehydration, slides
were immersed in 1% Tween-20, then heat‐induced antigen retrieval was performed in a steamer
using Antigen Unmasking Solution (Vector Labs, cat. H-3300) for 25 minutes. Slides were rinsed
in PBST, endogenous peroxidase and phosphatase were blocked (Dako, cat. S2003), and sections
were then incubated with primary antibody; anti‐ErbB2 (1:400 dilution; ThermoFisherScientific,
cat. MA5-15050, SF23975824), anti‐ERbeta (1:100 dilution; Bioss, cat. bs-0116R; 909984W),
anti‐Ki67 (1:200 dilution; Abcam, cat. Ab16667), anti-EGFR (1:50 dilution; LSBio, cat. LS-
B2914-5), anti-CK5 (1:2000 dilution; BioLegend, catalogue #905501), anti-CK6 (1:200 dilution;
Novus Biologicals, cat. NBP2-34358), antiAE-1/AE-3 (1:200 dilution; Novus Biologicals, cat.
NBP2-29429), for 45 minutes at room temperature. The primary antibodies were detected by 30-
minute incubation with HRP-labeled anti-rabbit secondary antibody (Leica Microsystems, cat.
PV6119) followed by detection with 3,3′‐Diaminobenzidine (Sigma‐Aldrich, cat. D4293)
counterstaining with Mayer’s hematoxylin, dehydration, and mounting.
2.7 Quantification of Lung Metastases
Lungs were fixed in formalin and stained with Hematoxylin and Eosin (H&E) stains. Each
section was taken 40 um apart with three sections from each lung. The sections were scanned and
the software HALO or NDPview.2 were used to quantify the number of metastasis and identify
the surface area of lung metastasis and total lungs.
2.8 Necropsy
Tissue sections were stained with H&E and visualized using light microscopy (Johns
Hopkins Tissue Pathology Core).
12
2.9 Identification of Survival and Tumor Growth
Following injection of tumor cells, mice were treated for three weeks as depicted in Figure
3, then received maintenance therapy until reaching the survival endpoint (tumor volume
exceeding 1.5 cm
3
or morbidity symptoms due to lung tumor burden). In the primary tumor model,
mice were examined for palpable tumors starting one week post-tumor cell injection, and
subsequently measured by calipers (± 0.01 mm) three times a week. Weekly tumor growth was
determined by taking the average of the differences in tumor volumes per week for each mouse.
Lung surface metastases were counted by visual inspection following euthanasia. Survival
experiments were continued until mice were euthanized according to protocol due to tumor burden
or morbidity caused by metastatic lung tumors as assessed by breathing, coat condition, activity,
and posture. The log rank (Mantel-Cox) test was used to test for significant differences between
survival curves, the Mann Whitney Wilcoxon test was used to compare tumor growth rate and
tumor volumes, and the Kruskal Wallis test was used for lung surface metastases.
2.10 Single-Cell RNA Sequencing, Quality Control, and Data Analysis
For library preparation, 10× Genomics Chromium Single Cell 3′ RNA-seq kits v3 were
used. Gene expression libraries were prepared according to the manufacturer’s protocol. Four
biological replicates from each cell line totaling eight processed tumors were sequenced in two
batches: Run A. two NT2.5 tumors, two NT2.5LM tumors; Run B. two NT2.5 tumors, two
NT2.5LM tumors. Each batch had an equal assortment of samples from multiple treatment groups
to reduce technical bias, however we restricted our analysis to replicates under the vehicle
treatment conditions. Illumina HiSeqX Ten or NovaSeq were used to generate total reads. Paired-
end reads were processed using CellRanger v3.0.2 and mapped to the mm10 transcriptome with
default settings. ScanPy v1.8.1 and Python v3 were used for quality control and basic filtering. For
13
gene filtering, all genes expressed in fewer than three cells within a cell line (NT2.5 and NT2.5LM)
were removed. Cells expressing less than 200 genes or more than 8,000 genes or having more than
15% mitochondrial gene expression were also removed. Gene expression was total-count
normalized to 10,000 reads per cell and log transformed. Highly variable genes were identified
using default ScanPy parameters, and the total of counts per cell and the percent of mitochondrial
genes expressed were regressed out. Finally, gene expression was scaled to unit variance and
values exceeding 10 standard deviations were removed. Neighborhood graphs were constructed
using the 10 nearest neighbors and 30 principal components. Tumors were clustered together
within cell lines using Louvain clustering (with resolution parameter 0.09) and cancer cells were
identified as Lcn+, Wfd2c+, Cd24a+, Cd276+, Col9a1+, Erbb2+, Pecam1-, Ptprc-. All other cell
clusters were removed.
Table 2. Genes used to annotate and identify cancer cell clusters after Louvain clustering.
Representative gene markers used in scRNA sequencing to identify tumor clusters.
14
2.11 Whole Exome Sequencing (WES)
Cells were cultured as described above. WES was performed by the Johns Hopkins
Genomics Core. One microgram or more of mouse genomic DNA from each sample was analyzed
by WES using the SureSelectXT Mouse All Exon kit (Agilent) followed by next generation
sequencing using the NovaSeq 6000 S4 flow cell (Illumina) with a 2x150 bp paired-end read
configuration following the manufacturer’s protocols. bcl2fastq v2.15.0 (Illumina) was used to
convert BCL files to FASTQ files using default parameters. Running alignments against the mm10
genome was done by bwa v0.7.7 (mem) along with Piccard-tools1.119 to add read groups and
remove duplicate reads. GATK v3.6.0 base call recalibration steps were used to create a final
alignment file. MuTect2 v3.6.0 was used to call somatic variants against a panel of normals using
default parameters. snpEFF (v4.1) was used to annotate the passed variant calls and to create a
clean tab-separated table of variants. VCF2MAF was used to convert VCF files to MAF files.
Maftools was used to generate visual representations of data. IGV v2.13.2 was used to identify
breast cancer specific mutations (PTEN, RAD51C, RAD51D, STK11, TP53, BRCA1, BRCA2,
ATM, BARD1, CDH1, CHEK2, NF1, PALB2, Arid1a, Foxa1, Pik3ca, ESR1) from MuTect2 files.
15
Table 3. Common breast cancer specific mutations. List of common breast cancer specific
mutations adapted from Alcazar et. al.
SnapGene Viewer v.6.2 was used to visually align and determine the mutations between
the two cell lines against the mRNA sequence of the ERBB2 receptor tyrosine kinase 2 of Mus
musculus. Annotations were used in order to create a visual representation of the mutational
differences.
16
CHAPTER 3
RESULTS
3.1 Derivation of NT2.5LM Syngeneic Model of HER2+ Breast Cancer
In the NT2.5 syngeneic mouse model, NT2.5 cells are injected into the mammary fat pad
and grown out until they reach a size of 1.5cm after which the mice are sacrificed. The tumors
become palpable at 7-10 days post injection and grow the maximum allowable volume by 4-5
weeks at which time the mice are sacrificed (Figure 3A). In the NT2.5-LM spontaneous model,
the tumor cells are injected into the second mammary fat pad and left to grow and metastasize
(Figure 3B) into heart, lymph nodes, lungs, kidney, adrenal gland, stomach, colon, spleen, skull,
ear, body wall, teeth, ovaries, eyes, and pancreas spontaneously (Figure 2).
Figure 2. Necropsy of the NT2.5LM Spontaneous Model. Necropsy data from NT2.5LM tumor-
bearing mouse showing breast cancer metastases (indicated by scale bars) in different tissues
including heart (A), lymph nodes (B), lungs (C), kidney (D), adrenal gland (E), stomach (F), colon
17
(G), spleen (H), skull (I), ear (J), body wall (K), and teeth (L). Other metastatic sites include
ovaries, eyes, and pancreas (data not shown). Tissue sections were stained with hematoxylin and
eosin (H&E).
Surgery is performed to remove the primary tumor at day 12 or once the tumors become
palpable to ensure that the primary tumor does not reach maximum allowable volume too quickly
and can metastasize for an ample period of time. Primary tumors usually regrow, and mice are
sacrificed 6-7 weeks after injection (4-5 weeks after resection) or once the primary tumor regrows
to a size of 1.5 cm. In the NT2.5LM experimental model, 1x10
5
NT2.5LM cells are injected via
tail vein into a NeuN mouse (Figure 3C). While no primary tumor develops, metastases develop
in the lungs. Mice are monitored according to approved IACUC protocols and sacrificed when
they exhibit morbidity symptoms, often increased rate of breathing, poor appetite, lethargy, weight
loss, and withdrawal from touch or attention which occurs 4-6 weeks after injection.
18
Figure 3. Schematic of the three mouse models utilized. A. NT2.5 Syngenic Mouse Model
Schematic; B. NT2.5LM Spontaneous Mouse Model Schematic; C. NT2.5LM Experimental
Mouse Model Schematic
19
3.2 Breast tumors derived from NT2.5LM demonstrate faster growth rate and increased
development of lung metastases
To determine if the NT2.5LM cell line would be appropriate as an in vivo model of breast
cancer metastasis, we injected FVB-NeuN mice with the NT2.5LM spontaneous model and tail
vein model in order to compare the survival and tumor burden with FVB-NeuN mice injected with
NT2.5 syngeneic model.
22
All mice in the NT2.5LM experimental and spontaneous groups
succumbed to disease by day 45 and 46 post-injection, respectively, while the survival of mice in
the NT2.5 group extended until day 59 (Figure 4A). Despite surgical resection of primary tumors
on day 12 post-injection in the NT2.5LM spontaneous group, these tumors regrew starting day 24
post-injection and most mice reached the endpoint (15 mm tumor length or visible ulceration)
faster than those with NT2.5 primary tumors. Furthermore, the weekly tumor growth rate was
significantly higher for NT2.5LM primary tumors compared to NT2.5 primary tumors (Figure 4B).
20
Figure 4. Survival experiment to compare NT2.5 and NT2.5LM. A. Survival of FVB-NeuN mice
injected with 1x10
5
NT2.5 or NT2.5LM cells in the mammary fat pad (spontaneous model) or
injected with 1x10
5
NT2.5LM cells in the tail vein (experimental model). B. Primary tumor growth
rate in mm
3
per week in NT2.5 and NT2.5LM spontaneous groups. C. Ratio of lung metastases
area versus total lung tissue area calculated to determine metastatic invasion. D. Number of lung
surface metastatic nodules observable via visual inspection in mice euthanized during days 38-41
post-cancer cell injection. NT2.5LM primary tumors were resected day 12 post-injection and
allowed to regrow. The Mann-Whitney test was used to determine statistically significant
differences between groups. *p<0.05, **p<0.01.
To assess the applicability of the NT2.5LM cell line to breast cancer metastasis studies,
mice with NT2.5LM breast tumors were dissected and tissue sections representing various organs
were stained with H&E to visualize metastases using light microscopy. Metastases were observed
21
in the heart, lymph nodes, lungs, kidney, adrenal gland, stomach, colon, spleen, skull, ear, body
wall, and teeth (Figure 2). Due to the high metastatic burden observed in lungs, we focused on
lungs as a surrogate measure of total metastatic burden using the NT2.5/NT2.5LM models. Lungs
from mice in all three models that were euthanized between days 34 and 41 post-injection were
visually inspected for identification of surface metastases. Comparatively, the mice in the NT2.5
spontaneous group had a lower lung metastasis area to total lung tissue area ratio (Figure 4C).
Mice in the NT2.5LM experimental metastasis group demonstrated significantly higher numbers
of surface lung metastases compared to NT2.5 (Figure 4D). The majority of mice in the NT2.5LM
experimental group had overgrown lung metastases that had invaded most of the lung tissue, which
impeded the enumeration of total metastatic nodules (counts exceeded 25). Since mice in both the
NT2.5LM spontaneous and experimental models survived for 6+ weeks and developed numerous
lung metastases within that timeline, NT2.5LM represents a viable model for breast cancer
metastases studies involving treatments.
3.3 NT2.5-LM expresses similar levels of HER2 but is more proliferative and demonstrates
basal cell morphology
As we observed the more aggressive and faster growth rate in the NT2.5LM model, we
next moved to characterizing and comparing the histological features of the primary breast tumors
and lung metastases derived from the NT2.5 and NT2.5-LM breast cancer cell lines, respectively,
using immunohistochemistry staining against ERBB2, Ki67, Ck5, Ck6, AE1/3, and EGFR (Figure
5). ERBB2 is indicative of HER2+, Ki67 is indicative of proliferation, Ck5 and Ck6 indicate a
basal-like molecular phenotype and are associated with poor prognosis, AE1/3 is found positive
in cancers of epithelial origin, and EGFR is associated with HER2 overexpression. Overall, we
22
saw that the staining was positive for ERBB2, Ki67, and EGFR, intermediate for AE1/3, and
negative for Ck5 and Ck6. Lung metastases maintain similar histologic features to primary breast
tumors.
Figure 5. Immunohistochemistry of NT2.5 breast tumor and NT2.5LM lung metastasis.
Immunohistochemistry staining of ERBB2, Ki67, Ck5, Ck6, AE1/3, and EGFR was performed in
metastatic lung tumors collected at day 35 after injections in mice with the NT2.5LM cell line.
Images taken at x100 and x400; scale bars represent 280 mm and 60 mm.
23
To determine how early tumor cells seed distant organs we chose to investigate seeding of
the lungs and as such, sampled lungs from tumor bearing mice at days 7, 10, 22, 28, and 35 and
used H&E staining to visualize tumor cell growth. Lungs were evaluated by a pathologist, and we
note lung metastasis seeding as early as day 7 in mice injected with NT2.5LM (Figure 6).
Figure 6. H&E staining to identify tumor metastasis formation. H&E staining of lungs (pink) at
day 7, day 10, day 22, day 28 and day 35 post cell line injection showing tumor (purple) size
development. Images taken at x40 and x200; scale bars represent 600 mm and 150 mm.
Furthermore, we quantified the percentage of NT2.5 and NT2.5LM cells expressing
traditional biomarkers of breast cancer subtypes, including HER2, estrogen receptor, and
progesterone receptor via flow cytometry (Figure 7A). Representative images of gating used for
HER2 are shown in Figure 8B. A minority of the NT2.5 and NT2.5LM cells expressed ER and
PR in vitro (<7% total live cells for both markers). Nearly 100% of NT2.5 and NT2.5LM cells
expressed HER2/Neu, establishing these cells as HER2/Neu+ breast cancer cell lines. Of note, the
median fluorescence intensity (MFI) for HER2/Neu was higher in NT2.5LM (Figure 7A).
24
Figure 7. Flow cytometry experiment comparing HER2 expression. A. Flow Cytometry data
showing HER2+ cells by percent total live cells (left panel) and HER2 median fluorescence
intensity (MFI, right panel) in NT2.5, NT2.5LM, and NT4 cells cultured in vitro for 1 week (n=5
mice(?)/group). The Kruskall-Wallis test was used to determine statistically significant differences
between groups. *p<0.05, **p<0.01. B. Representative gating HER2 for NT2.5 (left columns) and
NT2.5LM (right columns). Representative samples of NT2.5/NT2.5LM stained with target
antibody (HER2) are shown on the left, while NT2.5/NT2.5LM samples stained with control
isotype antibody are shown on the right.
In Figure 6, Ki67 was identified to be a positive stain and identified as a potential option
for explaining the more proliferative potential of NT2.5LM. We conducted immunohistochemistry
quantification on the NT2.5LM lung and NT2.5 breast tumors. After quantification, there were
significantly more Ki67 positive cells identified ratio wise in NT2.5LM compared to the NT2.5
sample (Figure 8).
25
Figure 8. Immunohistochemistry Quantification of Ki67. 10 Regions of Interest were identified
for NT2.5 and NT2.5LM respectively. Ratio of Ki67 positive cells to all cells was calculated.
Welch’s t-test was used to determine statistically significant differences between groups.
****p<0.0001.
3.4 Treatment of anti-Her2 therapy improves survival in a subset of mice
In an experiment utilizing 10 mice per group, mice were injected using the NT2.5LM
spontaneous model and were treated with HER2 directed therapy. As the spontaneous model was
utilized, the mice were injected with NT2.5LM cells in the second mammary fat pad, and the
primary tumor was resected at day 12 after the tumors became palpable. Mice were started on
HER2 antibody treatment by IP injections two times a week starting 18 days after the tumor
injection, and the treatment was continued for the rest of the experiment (Figure 9A).
26
Figure 9. anti-Her2 treatment in the NT2.5LM spontaneous mouse model. A. Treatment timeline
schematic of NT2.5LM spontaneous mouse models undergoing anti-Her2 therapy. B. NT2.5LM
spontaneous mouse receiving anti-HER2 therapy. Results were plotted using a Kaplan-Meier
curve and statistical significance was determined via a log-rank test (n = 10 mice/group). C.
Treatment with aHER2 antibody shows no significant decrease in the formation in the number of
lung metastases. D. Treatment with aHER2 antibody shows a significant decrease in the ratio of
lung metastasis area to total lung tissue area. All experiments were repeated once. Statistically
significant p-values are abbreviated as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Mice treated with anti-HER2 antibody showed significantly improved survival compared
to vehicle and demonstrated about a 35% response rate to therapy (Figure 9B). Furthermore,
27
number of metastases in the lung and the tumor area to total lung tissue area were calculated.
(Figure 9D). The number of metastases in the lung did not change (Figure 9C), however the
metastases themselves shrank and were stunted in growth from their initial size.
3.5 Whole exome seq of NT2.5 and NT2.5LM showed no significant differences in
mutations
Whole exome sequencing was done on both the NT2.5 and NT2.5LM cell lines to identify
if there were any variations in mutations. The tumor mutational burden for NT2.5 was found to be
11.45 mutations/megabase, and for NT2.5LM, it was found to be 13.45 mutations/megabase, both
of which can be considered to have intermediate amounts of mutational counts.
23
In order to
identify if these cell lines expressed any specific mutations, we looked into genes that are
commonly mutated in breast cancer: PTEN, RAD51C, RAD51D, STK11, TP53, BRCA1, BRCA2,
ATM, BARD1, CDH1, CHEK2, NF1, PALB2, ARID1A, FOXA1, PIK3CA, and ESR1 (Table 3).
24
Of these, we found that RAD51C was exclusive to NT2.5 and BRCA1 was exclusive to NT2.5LM.
PTEN, BRCA2, ATM, CDH1, CHEK2, NF1, ARID1A, PIK3CA, and ESR1 were existent in both
cell lines (Figure 10).
28
Figure 10. WES Analysis of common breast cancer specific mutations. Venn-Diagram showing
similarities and differences in breast cancer specific mutations between NT2.5 and NT2.5LM.
Overall, we found that these cell lines had significant overlap in the common breast cancer
specific mutations as well as having the largest variant classification of missense mutations, variant
types of SNP’s, and SNV class of tyrosine to cytosine, or cytosine to tyrosine mutations (Figure
11).
29
Figure 11. Overview of types of mutations between NT2.5 and NT2.5LM. Maftools generation
of mutational comparison of NT2.5 vs NT2.5LM cell line.
However, we wanted to look closely at the mouse equivalent to the HER2 gene, ERBB2,
as we hypothesized that perhaps the difference between cell lines was related to HER2 expression.
In comparing the messenger ribonucleic acid (mRNA) sequence of ERBB2 in a NeuN mouse to
the NT2.5 and NT2.5LM cell lines, we found six total mutations within the coding sequence across
both cell lines (Figure 12).
30
Figure 12. Determining mutations in ErbB2 mRNA sequence. A. Comparisons between the mRNA
coding sequence of the ERBB2 gene in Mus Musculus with point mutations identified in both
NT2.5 and NT2.5LM. B. Flow cytometry data for NeuN shown for Percent Total Live Cell and
Median Fluorescence Intensity. To determine statistical significance, log-rank test was utilized.
Comparisons were found not to be statistically significant.
However, none of the SNP mutations appeared to cause a change in the amino acid
sequence. Because no difference was found, we wanted to see if there were any identifiable
differences at the protein level.
3.6 ScRNA-seq of NT2.5LM tumors reveals increased cellular proliferation and metastatic
potential
To study how cancer cell populations differ in tumors arising from NT2.5 and NT2.5LM
cells, four NT2.5 tumors and four NT2.5LM tumors were collected from syngeneic NeuN mice
and subjected to unsorted single-cell RNA sequencing, yielding approximately 9.6x108 total reads.
Tumor clustering within cell line-derived tumors by Louvain clustering and visualization by
31
Uniform Manifold Approximation and Projection (UMAP) genes in NT2.5LM tumors were
identified. Of those, genes associated with increased cellular proliferation (PDGFA, SOX9),
25–27
invasion and migration (LRP1, CD9, CXCL1, ANXA1, AREG, IFITM3),
28–34
epithelial-to-
mesenchymal transition (VIM, INHBA),
35,36
and stemness and metastatic potential (S100A4,
NRP2, ALDH2, JUNB)
37–42
were upregulated, whereas genes associated with decreased cellular
proliferation (CRIP1),
43
invasion (CLDN7),
44,45
and epithelial-to-mesenchymal transition
(EPCAM)
46
were downregulated (Figure 13B). We also identified Vimentin to be a potential
candidate to explain the metastatic potential of the NT2.5LM cell line and, through flow cytometry,
identified that NT2.5LM cells expressed more Vimentin staining compared to NT2.5.
32
Figure 13. scRNA seq comparing NT2.5 and NT2.5LM. A. UMAP of cancer cells
identified from Louvain clustering of NT2.5 and NT2.5LM tumors. B. Heatmap of top 25 up- and
down-regulated genes between NT2.5 and NT2.5LM cancer cell clusters. C. Percentage of total
live NT2.5 or NT2.5LM cells that express vimentin as determined via flow cytometry. NT2.5 and
NT2.5LM cells were cultured in vitro for one week prior to analysis. The Mann-Whitney U-test
was used to determine significant differences between NT2.5 and NT2.5LM. *p<0.05. D. Violin
plot of gene EPCAM is presented.
33
To gain further insight into differential pathway regulation between NT2.5 and NT2.5LM
tumors, the top 250 differentially expressed genes in the cancer clusters from each cell line were
identified and compared for overlap after removal of all other cell clusters showed distinct
clustering between the cancer cells of NT2.5 and NT2.5LM tumors, identified as LCN+,
WFD2C+, CD24A+, CD276+, COL9A1+, and ERBB2+ (Table 2).
47–52
In total, approximately
15,000 NT2.5 and 13,000 NT2.5LM cancer cells were identified. When a violin plot was created
to identify the expression of EPCAM in these cells, we found that EPCAM was significantly
downregulated in NT2.5LM tumors (Figure 13D).
34
CHAPTER 4
DISCUSSION
The NT2.5 and NT2.5LM cell lines and models are representative of HER2+ breast cancer.
In this study, we show that although these cell lines are similar, they show differences primarily
in the increased metastatic potential of NT2.5LM. To facilitate lung metastasis formation quickly
and consistently, we generated a more aggressively metastatic breast cancer cell line, NT2.5LM,
through multiple passages and intravenous injections of single-cell tumor suspensions isolated
from the lungs of a NeuN mouse initially injected with the syngeneic spontaneously metastatic
breast cancer cell line NT2.5. Spontaneously metastatic breast cancer cell lines are valuable tools
for studying how metastatic tumors differ from primary tissue tumors in mice, but the length of
time for spontaneous lung metastases to develop after injection of cancer cells into the primary
breast tissue site is immensely delayed and inconsistent. In order to study their potential, we
generated various models (NT2.5 spontaneous, NT2.5LM spontaneous and NT2.5LM
experimental) to test their characteristics. The spontaneous model allows us to study the effect of
our intervention on metastatic progression in the most biologically accurate setting whereas the
experimental model allows us to evaluate an intervention on treatment of already metastatic
disease without having to worry about primary tumor growth dictating the timing of sacrifice.
However, a caveat of the experimental model is that it does not test the effects of an intervention
on prevention of metastatic progression with as much biological accuracy as the spontaneous
model. In order to study the metastatic burden and intervention of these models, we had to identify
where the tumors metastasized to. According to our necropsy data, we identified that many organs
were subject to metastasis. Specifically, the lungs showed high metastatic burden and visible
metastasis. Out of the many options, we ultimately chose the lungs as the representative organ to
35
study the extent of metastasis on our models. In understanding the proliferative nature of the
NT2.5LM model, identifying Ki67, which is significantly upregulated in our
immunohistochemistry studies, as a potential gene of interest that lends an idea to this concept.
What drives the more metastatic potential of the cell line can help identify what to look for when
translationally using these items in patient and clinical populations.
In determining how similarly it can reflect what is characteristic for patients with HER2+
breast cancer who are treated with HER2 directed therapy, HER2 therapy was administered to
mice who underwent the NT2.5LM spontaneous mouse model as HER2+ models should reflect
what is characteristic for patients who have HER2+ breast cancer. Although 35% of mice treated
with anti-HER2 antibody had significantly better survival rates, the fact that only 35% of these
mice responded to HER2 therapy is considered much lower than expected as they should respond
with much higher efficacy.
53
Therefore, these mice undergoing the NT2.5LM spontaneous model
can be considered to be poor responders and represent the patient population as such. Furthermore,
from these anti-HER2 treatments, we identified that the statistical significance between the number
of metastases formed versus the lung metastasis area to total lung tissue area ratio was different
wherein the former showed no statistical significance but the latter did, where the anti-HER2
treatment group showed a decrease in surface area calculations. This potentially identifies that
anti-HER2 therapy did not prevent the formation of metastases but allowed for a decrease in the
size of the existing metastasis. The mechanism behind this may potentially be reasoning to a type
of resistance, and potentially combining another type of treatment with anti-HER2 therapy may
aid in better survival.
Within these models, we expected similarities between these two cell lines due to the
NT2.5LM cell line being derived from NT2.5, and therefore, we explored their characteristics to
36
identify what similarities and differences existed between them. From the WES analysis, we
observed no significant differences in mutation types between them. Both of the cell lines
displayed similar mutational data, showing more missense mutations, SNP variant types, as well
as sharing half of the top 10 mutated genes (OBSCN, SSPO, PKHD1, LRRC37A, AND ABCA13).
Upon further analysis, however, we discovered that there were some differences in common breast
cancer mutation types between the two cell lines. Within the list of genes we looked at, we
observed that a mutation in RAD51C existed only in NT2.5 and BRCA1 only in NT2.5LM. Upon
further analysis, we identified that RAD51C and BRCA1 mutations were in intron regions of the
exome and therefore, resulted in no coding and therefore no protein changes. Furthermore, WES
analysis on the HER2 gene revealed no differences between the two cell lines and resulted in only
silent mutations in the coding sequence of the mRNA sequence. This lends to our hypothesis that
the driving factor behind the more metastatic phenotype that NT2.5LM displays is likely not due
to the mutation of the HER2 gene.
In further scRNAseq, NT2.5LM demonstrated more up-regulation of genes that control
cell proliferation whereas NT2.5 demonstrated downregulation in genes that involve cell
proliferation and cancer initiation. We identified that genes involved in epithelial-to-mesenchymal
transition were differentially regulated in a way in which it affected the more proliferative nature
of the NT2.5LM cell line by making it more mesenchymal in nature. In particular, we identified
Vimentin to be upregulated within the NT2.5LM cell line compared to the NT2.5 cell line. After
conducting a flow cytometry experiment staining to identify the extent of Vimentin cell expression,
we saw that the NT2.5LM cells expressed Vimentin more significantly than NT2.5, which could
be a cause of greater NT2.5LM metastatic potential compared to the NT2.5 cell lines, which also
does not show as much metastatic potential in murine studies.
37
The NT2.5LM cell line and mouse model developed from the parental NT2.5 cell line is
representative of metastatic HER2+ breast cancer and represents a more aggressive and metastatic
tumor type. Increased proliferation as indicated by Ki67 as well as necropsy data revealed
widespread metastasis across multiple organs, showing causation on the tumor growth of NT2.5
and NT2.5LM. Our findings lend to the fact that epithelial-to-mesenchymal transition and the more
mesenchymal nature the NT2.5LM cell line is, as shown by scRNA seq, are identified as potential
reasons for why NT2.5LM becomes more proliferative and metastatic.
54,55
38
CHAPTER 5
FUTURE DIRECTIONS
The cell lines and tumor models that we characterize here are a good representation of
patients with HER2+ breast cancer and allow us to recapitulate the cancer progression in mouse
models. Patients with HER2+ breast cancer show both antibody and cytotoxic T lymphocyte
responses specific to HER2, however, these responses are not sufficient to prevent tumor
progression,
56,57,58
an observation that has also been seen in NeuN mice.
59
Furthermore, previous
studies investigating gene expression features of murine mammary carcinomas as compared to
breast tumors have identified spontaneous tumors that form in NeuN mice most closely mimic
luminal B breast cancers.
60
As shown in the anti-HER2 treatment study, we found that although
there was a decrease in tumor size, the number of tumors did not differ between the two groups.
In addition to the survival data that we presented here, there suggests a potential resistance to
therapy which is a future direction that can be explored. The application of this murine model can
be utilized to study various aspects of breast cancer, including studying the immune tumor
microenvironment in identifying how the metastasis is affected within its niche.
Further characterization of differences in immune tumor microenvironment as well as in-
depth analysis of the metastatic tumors of both models should be further pursued to understand the
effect of gene expression and changes that the tumor microenvironment causes. Finally, evaluating
how epigenetic modulation and immune checkpoint inhibition affect survival in both models can
help determine which patient subpopulation may benefit most in future clinical trials.
39
REFERENCES
1. Sung, H. et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and
Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71, 209–249
(2021).
2. Sung, H. et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and
Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71, 209–249
(2021).
3. Cortés, J. et al. HER2 and hormone receptor-positive breast cancer—blocking the right
target. Nature Reviews Clinical Oncology 2010 8:5 8, 307–311 (2010).
4. Rimawi, M. F., Schiff, R. & Osborne, C. K. Targeting HER2 for the treatment of breast
cancer. Annu Rev Med 66, 111–128 (2015).
5. Loibl, S. & Gianni, L. HER2-positive breast cancer. Lancet 389, 2415–2429 (2017).
6. Pohlmann, P. R., Mayer, I. A. & Mernaugh, R. Resistance to Trastuzumab in Breast
Cancer. Clin Cancer Res 15, 7479–7491 (2009).
7. Cha, Y. J. & Koo, J. S. Role of Tumor-Associated Myeloid Cells in Breast Cancer. Cells
2020, Vol. 9, Page 1785 9, 1785 (2020).
8. Koboldt, D. C. et al. Comprehensive molecular portraits of human breast tumours. Nature
2012 490:7418 490, 61–70 (2012).
9. Gregório, A. C. et al. Inoculated Cell Density as a Determinant Factor of the Growth
Dynamics and Metastatic Efficiency of a Breast Cancer Murine Model. PLoS One 11,
e0165817 (2016).
10. Song, H. et al. An immunotolerant HER-2/neu transgenic mouse model of metastatic
breast cancer. Clin Cancer Res 14, 6116–24 (2008).
11. Foote, J. B. et al. A STING Agonist Given with OX40 Receptor and PD-L1 Modulators
Primes Immunity and Reduces Tumor Growth in Tolerized Mice. Cancer Immunol Res 5,
468–479 (2017).
12. Paschall, A. v & Liu, K. An Orthotopic Mouse Model of Spontaneous Breast Cancer
Metastasis. J Vis Exp (2016) doi:10.3791/54040.
13. Eckhardt, B. L., Francis, P. A., Parker, B. S. & Anderson, R. L. Strategies for the
discovery and development of therapies for metastatic breast cancer. Nature Reviews Drug
Discovery vol. 11 479–497 Preprint at https://doi.org/10.1038/nrd2372 (2012).
40
14. Fantozzi, A. & Christofori, G. Mouse models of breast cancer metastasis. Breast Cancer
Res 8, 212 (2006).
15. John, T. & al, et. A novel orthotopic model of breast cancer metastasis to bone. Clin Exp
Metastasis 17, 163 (1999).
16. Jaffee, E. M. et al. Development and characterization of a cytokine-secreting pancreatic
adenocarcinoma vaccine from primary tumors for use in clinical trials. Cancer J Sci Am 4,
194–203.
17. Jaffee, E. M. et al. Development and characterization of a cytokine-secreting pancreatic
adenocarcinoma vaccine from primary tumors for use in clinical trials. Cancer J Sci Am 4,
194–203.
18. Reilly, R. T. et al. HER-2/neu is a tumor rejection target in tolerized HER-2/neu
transgenic mice. Cancer Res 60, 3569–76 (2000).
19. Machiels, J. P. et al. Cyclophosphamide, doxorubicin, and paclitaxel enhance the
antitumor immune response of granulocyte/macrophage-colony stimulating factor-
secreting whole-cell vaccines in HER-2/neu tolerized mice. Cancer Res 61, 3689–97
(2001).
20. Guy, C. T. et al. Expression of the neu protooncogene in the mammary epithelium of
transgenic mice induces metastatic disease. Proc Natl Acad Sci U S A 89, 10578–10582
(1992).
21. Christmas, B. J. et al. Entinostat converts immune-resistant breast and pancreatic cancers
into checkpoint-responsive tumors by reprogramming tumor-infiltrating MDSCs. Cancer
Immunol Res canimm.0070.2018 (2018) doi:10.1158/2326-6066.CIR-18-0070.
22. Eckhardt, B. L., Francis, P. A., Parker, B. S. & Anderson, R. L. Strategies for the
discovery and development of therapies for metastatic breast cancer. Nature Reviews Drug
Discovery 2012 11:6 11, 479–497 (2012).
23. Riviere, P. et al. High Tumor Mutational Burden Correlates with Longer Survival in
Immunotherapy-Naïve Patients with Diverse Cancers. Mol Cancer Ther 19, 2139–2145
(2020).
24. Gil Del Alcazar, C. R. et al. Insights into Immune Escape During Tumor Evolution and
Response to Immunotherapy Using a Rat Model of Breast Cancer. Cancer Immunol Res
10, 680–697 (2022).
25. Pinto, M. P., Dye, W. W., Jacobsen, B. M. & Horwitz, K. B. Malignant stroma increases
luminal breast cancer cell proliferation and angiogenesis through platelet-derived growth
factor signaling. BMC Cancer 14, 735 (2014).
41
26. Jansson, S. et al. The PDGF pathway in breast cancer is linked to tumour aggressiveness,
triple-negative subtype and early recurrence. Breast Cancer Res Treat 169, 231–241
(2018).
27. Ma, Y. et al. SOX9 Is Essential for Triple-Negative Breast Cancer Cell Survival and
Metastasis. Molecular Cancer Research 18, 1825–1838 (2020).
28. Xing, P. et al. Roles of low-density lipoprotein receptor-related protein 1 in tumors. Chin
J Cancer 35, 6 (2016).
29. Fayard, B. et al. The Serine Protease Inhibitor Protease Nexin-1 Controls Mammary
Cancer Metastasis through LRP-1–Mediated MMP-9 Expression. Cancer Res 69, 5690–
5698 (2009).
30. Rappa, G., Green, T. M., Karbanová, J., Corbeil, D. & Lorico, A. Tetraspanin CD9
determines invasiveness and tumorigenicity of human breast cancer cells. Oncotarget 6,
7970–7991 (2015).
31. Yang, C. et al. CXCL1 stimulates migration and invasion in ER-negative breast cancer
cells via activation of the ERK/MMP2/9 signaling axis. Int J Oncol (2019)
doi:10.3892/ijo.2019.4840.
32. Moraes, L. A., Ampomah, P. B. & Lim, L. H. K. Annexin A1 in inflammation and breast
cancer: a new axis in the tumor microenvironment. Cell Adh Migr 1–7 (2018)
doi:10.1080/19336918.2018.1486143.
33. Baillo, A., Giroux, C. & Ethier, S. P. Knock-down of amphiregulin inhibits cellular
invasion in inflammatory breast cancer. J Cell Physiol 226, 2691–2701 (2011).
34. YANG, M., GAO, H., CHEN, P., JIA, J. & WU, S. Knockdown of interferon-induced
transmembrane protein 3 expression suppresses breast cancer cell growth and colony
formation and affects the cell cycle. Oncol Rep 30, 171–178 (2013).
35. Paulin, D., Lilienbaum, A., Kardjian, S., Agbulut, O. & Li, Z. Vimentin: Regulation and
pathogenesis. Biochimie 197, 96–112 (2022).
36. Yu, Y., Wang, W., Lu, W., Chen, W. & Shang, A. Inhibin β-A (INHBA) induces
epithelial–mesenchymal transition and accelerates the motility of breast cancer cells by
activating the TGF-β signaling pathway. Bioengineered 12, 4681–4696 (2021).
37. Helfman, D. M., Kim, E. J., Lukanidin, E. & Grigorian, M. The metastasis associated
protein S100A4: role in tumour progression and metastasis. Br J Cancer 92, 1955–1958
(2005).
42
38. Elaimy, A. L. et al. VEGF–neuropilin-2 signaling promotes stem-like traits in breast
cancer cells by TAZ-mediated repression of the Rac GAP β2-chimaerin. Sci Signal 11,
(2018).
39. Yasuoka, H. et al. Neuropilin-2 expression in breast cancer: correlation with lymph node
metastasis, poor prognosis, and regulation of CXCR4 expression. BMC Cancer 9, 220
(2009).
40. Zhang, H. & Fu, L. The role of ALDH2 in tumorigenesis and tumor progression:
Targeting ALDH2 as a potential cancer treatment. Acta Pharm Sin B 11, 1400–1411
(2021).
41. Sundqvist, A. et al. JUNB governs a feed-forward network of TGFβ signaling that
aggravates breast cancer invasion. Nucleic Acids Res 46, 1180–1195 (2018).
42. Qiao, Y. et al. AP-1-mediated chromatin looping regulates ZEB2 transcription: new
insights into TNFα-induced epithelial-mesenchymal transition in triple-negative breast
cancer. Oncotarget 6, 7804–7814 (2015).
43. Ludyga, N. et al. The impact of Cysteine-Rich Intestinal Protein 1 (CRIP1) in human
breast cancer. Mol Cancer 12, 28 (2013).
44. Kominsky, S. L. et al. Loss of the tight junction protein claudin-7 correlates with
histological grade in both ductal carcinoma in situ and invasive ductal carcinoma of the
breast. Oncogene 22, 2021–2033 (2003).
45. Martin, T. A. & Jiang, W. G. Loss of tight junction barrier function and its role in cancer
metastasis. Biochimica et Biophysica Acta (BBA) - Biomembranes 1788, 872–891 (2009).
46. Hyun, K.-A. et al. Epithelial-to-mesenchymal transition leads to loss of EpCAM and
different physical properties in circulating tumor cells from metastatic breast cancer.
Oncotarget 7, 24677–24687 (2016).
47. Sidiropoulos, D. N. et al. Entinostat Decreases Immune Suppression to Promote
Antitumor Responses in a HER2+ Breast Tumor Microenvironment. Cancer Immunol Res
10, 656–669 (2022).
48. Gündüz, U. R. et al. A new marker for breast cancer diagnosis, human epididymis protein
4: A preliminary study. Mol Clin Oncol 5, 355–360 (2016).
49. Seaman, S. et al. Eradication of Tumors through Simultaneous Ablation of CD276/B7-
H3-Positive Tumor Cells and Tumor Vasculature. Cancer Cell 31, 501-515.e8 (2017).
50. Yang, J. et al. Lipocalin 2 promotes breast cancer progression. Proceedings of the
National Academy of Sciences 106, 3913–3918 (2009).
43
51. Berger, T., Cheung, C. C., Elia, A. J. & Mak, T. W. Disruption of the Lcn2 gene in mice
suppresses primary mammary tumor formation but does not decrease lung metastasis.
Proceedings of the National Academy of Sciences 107, 2995–3000 (2010).
52. Yeo, S. K. et al. Single-cell RNA-sequencing reveals distinct patterns of cell state
heterogeneity in mouse models of breast cancer. Elife 9, (2020).
53. Vogel, C. L. et al. First-line Herceptin monotherapy in metastatic breast cancer. Oncology
61, 37–42 (2001).
54. Pal, A. K. et al. Metabolomics and EMT Markers of Breast Cancer: A Crosstalk and
Future Perspective. Pathophysiology 29, 200–222 (2022).
55. Le Bras, G. F., Taubenslag, K. J. & Andl, C. D. The regulation of cell-cell adhesion
during epithelial-mesenchymal transition, motility and tumor progression. Cell Adh Migr
6, 365–373 (2012).
56. Disis, M. L. et al. Existent T-Cell and Antibody Immunity to HER-2/neu Protein in
Patients with Breast Cancer.
57. Disis, M. L. et al. High-titer HER-2/neu protein-specific antibody can be detected in
patients with early-stage breast cancer. Journal of Clinical Oncology 15, 3363–3367
(1997).
58. Disis, M. L., Grabstein, K. H., Sleath, P. R. & Cheever, M. A. Generation of Immunity to
the HER-2/neu Oncogenic Protein in Patients with Breast and Ovarian Cancer Using a
Peptide-based Vaccine 1.
59. Reilly, R. T. et al. HER-2/neu is a tumor rejection target in tolerized HER-2/neu
transgenic mice. Cancer Res 60, 3569–76 (2000).
60. Herschkowitz, J. I. et al. Identification of conserved gene expression features between
murine mammary carcinoma models and human breast tumors. Genome Biol 8, 1–17
(2007).
Abstract (if available)
Abstract
Animal models are essential tools for preclinical studies evaluating primary tumor development and metastatic progression in breast cancer. Murine models with a fully intact immune system are essential for studies investigating anti-cancer immune responses and reactions to immunotherapies. In this thesis, characterization of a novel syngeneic murine tumor cell line that provides a model of spontaneously metastatic neu-expressing breast cancer mammary tumor implantation of a novel cell line, NT2.5LM, into FVB/NeuN mice was conducted. Mice growing NT2.5-LM tumors in their mammary fat pads develop spontaneous metastasis to distant organs including the lungs and bones, which mimics metastatic spread in patients. Development of this syngeneic mouse model is instrumental in pre-clinical studies of novel therapeutic combinations in a system that mimics aggressive metastatic breast cancer progression in patients targeted for therapy.
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
PDF
Stable expression of human B7-H4 in a mouse mammary tumor model as a target for cancer immunotherapy
PDF
Immune signature of murine solid tumor models
PDF
DNA methylation markers for blood-based detection of small cell lung cancer in mouse models
PDF
Establishing a human EGFR expressing murine mammary carcinoma cell line-D2F2, as a syngeneic immunocompetent model
PDF
Distortions of HER2 status designation parameters using fluorescence in situ hybridization (FISH) in breast cancer: aneusomy and proliferative rates
PDF
Development of immunotherapy for small cell lung cancer using novel modified antigens
PDF
Calorie restriction reduces liver steatosis in liver specific Pten deleted mouse model
PDF
HER2 and co-amplified genes in breast and gastric cancer
PDF
Studies on the role of TMEM56 in tumorigenesis by using PTEN knockout mouse model
PDF
Optimizing an immortalized human alveolar epithelial cell line model system to recapitulate lung adenocarcinoma development in vitro
PDF
Mechanism of a new CK2 inhibitor triggering senescence in breast cancer cells
PDF
Roles of circadian clock genes in cancer
PDF
The effect of tumor-mediated immune suppression on prostate cancer immunotherapy
PDF
Neuronal master regulator SRRM4 in breast cancer cells facilitates CNS-acclimation and colonization leading to brain metastasis
PDF
Elucidating the cellular origins of lung adenocarcinoma
PDF
Conjugation of CpG oligodeoxynucleotides to tumor‐targeting antibodies for immunotherapy of solid tumors
PDF
Limit of detection analysis for cell-free DNA methylation using targeted bisulfite sequencing
PDF
Modeling lung adenocarcinoma progression in vitro using immortalized human alveolar epithelial cells
PDF
Tracking human acute lymphoblastic leukemia cell clones in xenograft mouse models
PDF
Characterization of a new chromobox protein 8 (CBX8) antagonist in a model of human colon cancer
Asset Metadata
Creator
Narumi, Valerie Haruka
(author)
Core Title
Characterization of a new HER2 positive mouse model of metastatic breast cancer
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Medicine
Degree Conferral Date
2023-08
Publication Date
07/12/2023
Defense Date
06/29/2023
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
Breast,cancer,HER2,Metastatic,mouse model,murine model,OAI-PMH Harvest
Format
theses
(aat)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Roussos-Torres, Evanthia (
committee chair
), Epstein, Alan (
committee member
), Marconett, Crystal (
committee member
)
Creator Email
narumi@usc.edu,valeriehnarumi@gmail.com
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-oUC113263927
Unique identifier
UC113263927
Identifier
etd-NarumiVale-12077.pdf (filename)
Legacy Identifier
etd-NarumiVale-12077.pdf
Document Type
Thesis
Format
theses (aat)
Rights
Narumi, Valerie Haruka
Internet Media Type
application/pdf
Type
texts
Source
20230713-usctheses-batch-1067
(batch),
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright.
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Repository Email
cisadmin@lib.usc.edu
Tags
HER2
mouse model
murine model