Stage-specific tumor microenvironment dynamics and cancer-associated fibroblast profiling in MBL-6 mouse models of breast cancer

It is well established that carcinoma-associated fibroblasts (CAFs) differ phenotypically from fibroblasts associated with normal tissue, but the mechanisms underlying these differences remain controversial. Because CAFs can be propagated in vitro for extended periods and still maintain their cancer promoting phenotype, it has been proposed that they might have acquired somatic genetic alterations analogous to those observed in malignant epithelium. Whereas some investigators have reported frequent and profound genomic alterations in CAFs, other groups have found no such evidence. One striking common trait of those studies reporting frequent clonal somatic alterations in CAFs is the use of tissues and techniques which are well known to be highly prone to generating artefacts, such as limiting and poor quality DNA followed by highly multiplexed PCR-based analysis. We conclude that reported frequent clonal somatic mutations in CAFs are likely to be artefacts and are not the biological basis of the cancer promoting attributes of CAFs. [corrected]

EphA2, a receptor tyrosine kinase, is elevated in many invasive human breast cancers, and the majority of EphA2 remains unphosphorylated. The successful attachment of ligand EphrinA1 present on the surface of adjacent cells to EphA2 initiates EphA2 phosphorylation leading to its turnover. In vivo efficacy of various approaches targeting EphA2 for breast cancer therapy is usually evaluated in nude mice bearing human breast cancer xenografts. In order to establish an immunocompetent mouse model of breast cancer for EphA2-targeted therapies, we evaluated a mouse breast cancer cell line (MT1A2) for EphA2 expression and phosphorylation. Overexpression of EphA2 was observed in MT1A2 cells and the majority of it remained unphosphorylated signifying that EphA2 in MT1A2 cells behaved similar to that of human breast cancer cells. Human adenovirus subtype 5 (HAd5) vectors expressing secretory forms of EphrinA1 were used for in vitro and in vivo targeting of MT1A2-derived EphA2. MT1A2 cells infected with HAd-EphrinA1-Fc (HAd expressing extracellular domain of human EphrinA1 attached to Fc portion of human IgG1 heavy chain) induced EphA2 activation and its turnover. This led to inhibition in MT1A2 cell colony formation in soft agar and cell viability in monolayer culture. In addition, MT1A2 cells-infected with HAd-EphrinA1-Fc failed to form tumors in syngeneic FVB/n mice at least 32 days postinoculation. Moreover, intratumoral inoculation of FVB/n mice-bearing MT1A2-induced tumors with HAd-EphrinA1-Fc slowed the tumor growth and also resulted in the development of vector-specific immune response. These results indicate that FVB/n mice-bearing MT1A2-induced tumors could serve as an immunocompetent model of breast cancer for EphA2-targeted therapeutic strategies.

The tumor microenvironment (TME) has been shown to play a vital role in tumor development and progression. Carcinoma associated fibroblasts (CAFs) form a very essential component of the TME. In fact, the TME in different types of carcinomas, particularly carcinomas of the breast, is comprised mainly of fibroblasts. CAFs in breast cancer TME secrete cytokines and growth factors that are known to activate a multitude of signaling pathways in breast cancer cells. One of these pathways is the MAPK pathway, a signaling pathway activated downstream of ERBB receptor tyrosine kinase family members. We have previously demonstrated that activation of the MAPK pathway represses estrogen receptor (ER) expression leading to an ER-negative phenotype in breast cancer. We have generated dissociated CAF cell cultures from ER+, ER-/Her2+, and triple negative (TN) primary breast tumors. We have characterized these CAFs on the basis of fibroblast markers, epithelial markers, microarray analysis, soft agar assay and in vivo tumorigenecity studies. Our results show that these CAF populations from primary breast tumors are pure populations of CAF cell cultures. These CAFs provide a model that allows us to examine the role of CAF interaction with ER+ breast cancer cells regarding activation of MAPK and subsequent repression of ER expression. We demonstrate that treatment of the ER+ MCF-7 cell line with conditioned media (CM) from the CAFs results in transient activation of MAPK signaling and subsequent repression of ER. Continuous exposure of ER+ breast cancer cells to soluble factors from CAFs also results in activation of MAPK and down-regulation of ER expression. Importantly, CM from human mammary fibroblasts (HMFs) and CAFs generated from an ER+ tumor do not down-regulate ER expression. Gene expression analysis and cytokine arrays indicate key differences in gene and cytokine expression between HMFs and the ER+ tumor CAFs compared to the two CAFs generated from ER- tumors. We have previously identified a miRNA signature associated with hyperactivation of MAPK signaling (hMAPK). This signature identifies the majority of ER- breast cancers as well as a population of ER+ breast cancers that express lower levels of ER. Like the ER- breast cancers identified by this signature, ER+ cancers identified by this signature exhibit gene expression patterns indicative of activated MAPK signaling, and exhibit significantly increased disease recurrence and significantly reduced disease survival. Here we show that breast tumors cancers bearing this hMAPK-miRNA signature have elevated expression of numerous stromal markers associated with poor clinical outcome, as well as a number of microRNAs that are differentially expressed between normal human mammary fibroblasts (HMFs) and mammary tumor carcinoma CAFs. Some of these miRNAs have been established as targeting ER. Using reporter constructs to investigate microRNA regulation of ER, we have observed that treatment of ER+ breast cancer cells with CM from ER- CAFs results in enhanced microRNA-dependent repression of ER. These data are suggestive of a role for CAF secreted factors in activating MAPK in breast cancer cells leading to repression of ER. In addition, CAFs are able to increase the expression of hMAPK-miRNAs that can down-regulate ER expression either via the activation of MAPK in the cancer cell or secreted directly from the CAFs. We hypothesize that CAFs may contribute to an ER-negative phenotype in breast cancer cells not only by activating MAPK signaling in the tumor cells via secretion of growth factors, but also potentially by transfer of MAPK-regulated miRNAs to the breast cancer cells as well. Citation Format: Sanket H. Shah, Philip Miller, Katherine Drews-Elger, Joeli Brinkman, Stefania Lairet, Alana Steinberg, Dorraya El-Ashry. Cancer-associated fibroblasts contribute to establishment of ER-negative breast cancer phenotype through secreted factors and miRNAs. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B108.

High-Density Gene Expression Analysis of Tumor-Associated Macrophages from Mouse Mammary Tumors

Stromal Collagenase in Melanoma: A Vascular Connection

An ideal mouse model should closely mimic a clinical situation. However, for most models available, this is not the case since clinical trials frequently fail to reproduce the highly encouraging therapeutic results obtained from pre-clinical studies performed using mouse models. In this study, in the process of extending the application of our previously established breast tissue-derived orthotopic and metastatic (BOM) model, the human breast cancer cell line MDA-MB-231 failed to exhibit any osteotropic features that differed from previous studies. Our observations suggest that a human tissue-specific microenvironment could be an essential requirement for a successful mouse model of breast cancer. Here, multiple in vivo breast cancer models were used to confirm this hypothesis. Human breast tissue and cancellated bone were transplanted subcutaneously to female severe combined immunodeficiency disease (SCID) mice by different assemblies, to build several mouse models termed 'breast-breast', 'breast-bone', 'bone-bone', 'MFP (mouse mammary fat pad)-bone', and 'MFP-breast' models. Two human breast cancer cell lines, MDA-MB-231 and MDA-MB-231BO, and the mouse breast cancer cell line TM40D were used. All cancer cells were labeled with GFP for gross observation. In addition, transplanted human tissues and various mouse tissues including bone, lung, liver, mesentery were examined microscopically. Based on morphological, immunohistochemical, and enzymohistochemical evidence obtained from several comparative experiments in 'breast-breast', 'breast-bone' and 'bone-bone' models, the BOM model was proved to be feasible and reliable. The organ tropism of the breast cancer cell line, which was derived from a mouse model by intracardiac inoculation in a pure mouse microenvironment, was reconsidered. The behavior of breast cancer cells in the mouse model was altered in response to the varying microenvironment. The results in this study suggest the human tissue-specific micro-environment is most likely an essential requirement in mouse models of breast cancer.

Glioma is the most common malignant central nervous system tumor with significant mortality and morbidity. Despite considerable advances, the exact molecular pathways involved in tumor progression are not fully elucidated, and patients commonly face a poor prognosis. Long non-coding RNAs (lncRNAs) have recently drawn extra attention for their potential roles in different types of cancer as well as non-malignant diseases. More than 200 lncRNAs have been reported to be associated with glioma. We aimed to assess the roles of the most investigated lncRNAs in different stages of tumor progression and the mediating molecular pathways in addition to their clinical applications. lncRNAs are involved in different stages of tumor formation, invasion, and progression, including regulating the cell cycle, apoptosis, autophagy, epithelial-to-mesenchymal transition, tumor stemness, angiogenesis, the integrity of the blood-tumor-brain barrier, tumor metabolism, and immunological responses. The well-known oncogenic lncRNAs, which are upregulated in glioma, are H19, HOTAIR, PVT1, UCA1, XIST, CRNDE, FOXD2-AS1, ANRIL, HOXA11-AS, TP73-AS1, and DANCR. On the other hand, MEG3, GAS5, CCASC2, and TUSC7 are tumor suppressor lncRNAs, which are downregulated. While most studies reported oncogenic effects for MALAT1, TUG1, and NEAT1, there are some controversies regarding these lncRNAs. Expression levels of lncRNAs can be associated with tumor grade, survival, treatment response (chemotherapy drugs or radiotherapy), and overall prognosis. Moreover, circulatory levels of lncRNAs, such as MALAT1, H19, HOTAIR, NEAT1, TUG1, GAS5, LINK-A, and TUSC7, can provide non-invasive diagnostic and prognostic tools. Modulation of expression of lncRNAs using antisense oligonucleotides can lead to novel therapeutics. Notably, a profound understanding of the underlying molecular pathways involved in the function of lncRNAs is required to develop novel therapeutic targets. More investigations with large sample sizes and increased focus on in-vivo models are required to expand our understanding of the potential roles and application of lncRNAs in glioma.

Over the past decade an accumulating body of evidence has demonstrated the pivotal role of stromal cells in promoting breast cancer progression. In breast cancer, the stroma is mainly comprised of carcinoma-associated fibroblasts (CAFs). CAFs secrete various growth factors and cytokines that are known to activate MAPK pathway. We have previously established that hyperactivation of MAPK in breast cancer cells leads to repression of estrogen receptor (ER) expression. Aberrant mitogenic signaling also affects expression of microRNAs (miRNAs). We have recently identified a miRNA signature associated with hyperactivation of MAPK signaling (hMAPK-miRNAs). Most ER- breast cancers and a population of ER+ breast cancers are identified by this signature, and these hMAPK-miRNA tumors exhibit significantly decreased disease-free survival and overall survival compared to tumors that are not hMAPK-miRNA. Some of these hMAPK-miRNAs have been established as targeting ER. To further investigate the contribution of CAFs in activation of MAPK signaling in cancer cells, we isolated CAF cell cultures from three different primary breast tumors: CAF19 (from a luminal A tumor), CAF21 (from a Her-2 like tumor) and CAF23 (from a basal like tumor) and tumor cell cultures (DTs) from 5 different primary breast tumors. There is differential expression of hMAPK miRNAs between these CAFs and DTs, including those that target ER. Thus, we propose that interactions between cancer and stromal cells lead to activation of MAPK signaling in the cancer cell and aberrant miRNA expression within the TME that contribute to the biology of ER-negative tumors. To further investigate the connection between hMAPK-miRNAs and stroma, we analysed TCGA breast cancer datasets and found that hMAPK-miRNAs are significantly associated with tumors that possess high stromal scores. Co-culture of CAFs with ER+ breast cancer cells, as well as treatment of ER+ breast cancer cells with conditioned media (CM) from CAFs, induces both MAPK activation and altered expression of miRNAs, specifically MAPK miRNAs within the breast cancer cells. Treatment of ER+ breast cancer cells with CM from CAFs represses both ER protein and mRNA expression, and importantly, this ER repression is specific to CAFs isolated from ER-negative breast tumors (CAF21 and CAF23) compared to the CAFs obtained from ER-positive tumors (CAF19). Additionally, CM from CAF21 and CAF23 caused repression of an ER 3'UTR reporter construct transfected into ER+ breast cancer cells, indicating a role for miRNAs either secreted into the CM or induced in the cancer cell in repressing ER. To investigate a role for hMAPK-miRNAs secreted by CAFs in mediating ER repression, nanostring analysis of miRNAs isolated from ER+ breast cancer cells treated with CAF CM was performed and differentially expressed miRNAs between CM from CAFs obtained from ER-positive (DT19) vs ER-negative tumors (DT21 and DT23) were identified. qPCR analysis of conditioned media also confirmed the presence of hMAPK-miRNAs in CAF CM. Collectively, these data indicate a role for CAFs in regulating hMAPK-miRNAs, and thus in establishing an ER-negative breast cancer phenotype, and importantly, differences between CAFs from ER-negative as compared to ER-positive tumors in this role. They further suggest that hMAPK-miRNAs secreted from CAFs participate in the induction of the ER-negative phenotype induced by MAPK. Citation Format: Sanket H. Shah, Phil Miller, Emilio Issa, Katherine Drews-Elger, Dorraya El-Ashry. Stromal miRNAs as mediators of mitogen activated protein kinase-induced estrogen repression in breast cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B39. doi:10.1158/1538-7445.CHTME14-B39

While RAS mutations rarely occur in breast cancers, RAS signaling is well-known to be involved in breast cancer development resulting from constitutive activation of receptor tyrosine kinases. The Wnt/β-catenin pathway is also activated and associated with more aggressive forms of breast cancer. We found that the cyclic nucleotide degrading enzyme, phosphodiesterase 10A (PDE10), is overexpressed in breast cancer cell lines and tumors compared with normal human mammary epithelial cells (HMEC) or tissues, respectively. PDE10 selective inhibitors and gene silencing inhibited the growth of cancer cell lines expressing PDE10, while normal cells lacking PDE10 were relatively insensitive. We identified a novel PDE10 inhibitor, ADT-030, that potently and selectively inhibited breast cancer cell growth by synthesizing and screening a focused library of indenes, followed by chemical optimization for oral bioavailability and metabolic stability. ADT-030 binding to recombinant PDE10 was confirmed by enzymatic assays measuring cyclic nucleotide hydrolysis and in intact cells by cellular thermal stability assays. The growth inhibitory activity of ADT-030 was associated with cGMP/PKG activation, blockage of RAS-MAPK/AKT signaling, and suppression of Wnt/β-catenin transcriptional activity, all of which occurred within the same concentration range. ADT-030 strongly inhibited primary tumor growth, blocked lung metastasis, and increased survival in the orthotopic 4T1 mouse model of breast cancer. The antitumor activity was observed at dosages causing no discernable toxicity. ADT-030 also inhibited tumor growth in mouse lung, colon, and pancreatic cancer models. Notably, ADT-030 caused tumor regression in patient-derived xenograft mouse models of pancreatic cancer, increased survival in orthotopic mouse models of lung cancer with “cures,” and synergized with anti-PD1 in the CT26 mouse model of colon cancer to enhance survival. Other studies revealed a significant impact of ADT-030 on the tumor immune microenvironment by inducing apoptosis of myeloid-derived suppressor cells (MDSC), accompanied by increased tumor infiltration of effector CD8+ T cells. Furthermore, ADT-030 treatment of cancer cells led to immunogenic cell death and enhanced dendritic cell activation. These results support further development of ADT-030, a novel PDE10 inhibitor capable of suppressing oncogenic RAS and β-catenin signaling, as a monotherapy or combined with immunotherapy or standard-of-care drugs for treating breast or other cancers with activated RAS and β-catenin signaling. Citation Format: Gary A. Piazza, Khalda Fadlalla, Adam B. Keeton, Yulia Y Maxuitenko, Xi Chen, Kristy L. Berry, Md Yeashin Gazi, Gang Zhou. A novel 1st-in-class RAS/β-catenin inhibitor concurrently targets cancer cells and MDSC to reverse the immunosuppressive tumor microenvironment: antitumor activity in mouse models of breast and other cancers [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-02-27.

Does a young woman living with a smoker or taking a job working in a smoky bar have a greater chance of developing breast cancer? Some scientists believe that such situations can indeed raise a woman’s risk of developing breast cancer before the age of 50. Because epidemiological and toxicological studies show that women’s breast tissue may be especially sensitive to exposure to carcinogens prior to first pregnancy, these researchers contend that public education should be directed at alerting adolescents and young women to the potential risk. However, not everyone in the international public health community agrees that the evidence to date supports a link between passive smoking and breast cancer, and some say that women are being alarmed unnecessarily. This disagreement has sparked debate that is sometimes heated. The stakes are high because breast cancer is the most common cancer in women in industrialized countries, according to the WHO. It is the leading cancer killer of nonsmoking women, and second only to lung cancer deaths among women who smoke. Among the researchers interviewed for this article who disagree that there is enough evidence to link secondhand smoke (SHS) with breast cancer, the majority call the evidence to date “suggestive but not sufficient,” as the Surgeon General’s 2006 report, The Health Consequences of Involuntary Exposure to Tobacco Smoke, put it. That characterization is based largely on the fact that the research considered when the Surgeon General’s report was being amassed did not clearly link even active smoking to breast cancer. Researchers in this camp do, however, stress that ongoing campaigns to prohibit smoking in public will protect the whole of society against the wide variety of ills proven to be caused by SHS. These include lung cancer, cardiovascular disease, and sudden infant death syndrome, among others. A smaller group contends that the question of whether or not SHS causes breast cancer is a political issue with the potential to compromise the scientific process. “A premature decision about causality could jeopardize the credibility of the entire review process and all of the other, established effects of secondhand smoke,” says Michael Thun, national vice president of epidemiology and surveillance research for the American Cancer Society. Adds Valerie Beral, director of the University of Oxford Cancer Research UK Epidemiology Unit, “To prematurely come to conclusions about the causation when there is a big division in the scientific community . . . is bad science.” Thun debated the subject in a series of public forums held in conjunction with scientific meetings. Taking the opposing view was Kenneth C. Johnson, a research scientist with the Public Health Agency of Canada, who was one of the first scientists to discern a potential link. During the debates, Johnson pointed out there are about the same number of studies linking breast cancer to passive smoking as there were linking lung cancer to SHS in 1986, when the Surgeon General concluded that passive smoking caused lung cancer. Johnson also says that more of the breast cancer studies are statistically significant, and that the estimated risk for breast cancer is higher.

Introduction: Combining CDK4/6 inhibitors (CDK4/6i) with endocrine therapy (ET) is a standard-of-care treatment modality for hormone receptor-positive/HER2-negative breast cancer in the metastatic and adjuvant settings. Identifying biomarkers of response to CDK4/6i in breast cancer to improve their application is a subject of great clinical interest. We present a therapeutic and transcriptomic analysis of six Genetically Engineered Mouse Models (GEMMs) of breast cancer treated with abemaciclib to shed further light on tumor cell and microenvironment features correlating with response that may mirror similar patterns in human breast cancer. Methods: Mice from six GEMMs –TP53null-2153 (luminal), TP53null-2208L (luminal), MMTV-PyMT (luminal), MMTV-Neu (luminal), C3-Tag (basal), and RBnull/TP53null-626 (claudin-low) - were used to evaluate tumor response to abemaciclib vs. no treatment (control). Acute changes in tumor volume with treatment were measured, and survival monitored. Tumor specimens from each GEMM were obtained before treatment and at day 7, whereupon they were evaluated with bulk mRNA-sequencing (mRNAseq) using 873 published gene expression signatures, and with single-cell RNASeq (scRNAseq). Results: Tumors from 3 GEMMs demonstrated statistically significantly less growth in tumor volume (p<0.01) on abemaciclib vs. control – 2153 (p<0.0001), MMTV-PyMT (p=0.003), and MMTV-Neu (p=0.001). Only these same 3 models also demonstrated significantly improved survival with abemaciclib vs. control as assessed by the log-rank test (p <0.0001, p=0.009, and p<0.0001, respectively). Notably, two non-responder GEMMs (C3-Tag and 626) are known RB1-deficient models. Differential expression analysis of gene expression signatures using supervised learning (FDR 0%) and hierarchical clustering revealed that for the 3 responder GEMMs, abemaciclib-treated tumors had lower expression of proliferation signatures and higher expression of innate immunity-related signatures at day 7 compared to untreated tumors. The 3 non-responder GEMMs showed no significantly differentially expressed signatures between treated and untreated tumors at day 7. We next compared the pretreatment tumor profiles of the responder vs. non-responder GEMMs and identified higher expression of multiple luminal-related signatures and lower expression of innate and adaptive immunity-related signatures in responder GEMMs. All six models were also assayed using scRNAseq, which is currently being analyzed and will be presented. Conclusions: Tumors from GEMMs with known RB1 loss were insensitive to abemaciclib, and tumors from responder GEMMs had greater luminal gene expression features, despite being ER-negative. Responder GEMMs had lower tumor immune-related gene expression than those that did not respond, and adaptive immunity-related signatures did not change significantly with abemaciclib vs. control in responders, whereas innate immunity-related signatures showed increased expression with treatment. These findings add to a growing body of evidence, including correlative analyses from human clinical trials, which support RB1 loss as a biomarker of resistance and luminal intrinsic subtype and low tumor immune features as biomarkers of response to CDK4/6i in breast cancer. Funding: Supported by the Breast Cancer Research Foundation, Grant DRC-20-004. Citation Format: Yash Agrawal, Kevin Mott, Tulay Yilmaz-Swenson, Charles M. Perou. Leveraging bulk and single-cell RNA sequencing to identify features predicting response and survival to abemaciclib in six mouse models of breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-02-24.

Background/Hypothesis: The amino acid transporter SLC6A14 is upregulated in estrogen receptor (ER)-positive breast cancer, a subtype that represents 75-80% of all breast cancers. Obesity/overweight increases the risk of breast cancer due to estrogen generated from fat cells that fuel the growth of the ER-positive breast cancer. Previous studies from our lab have demonstrated that blockade of the amino acid transporter SLC6A14 by the small molecule α-methyltryptophan reduces the growth of ER-positive breast cancer in mouse xenografts of human breast cancer cells and in a spontaneous mouse model of ER-positive breast cancer. We have also discovered that α-methyltryptophan functions as a potent weight-loss agent. We believe that this compound would be effective for the treatment of ER-positive breast cancer, especially in obese/overweight women. α-methyltryptophan is metabolized by aromatic amino acid decarboxylase. Therefore, combination of this compound with carbidopa, a well-known inhibitor of this enzyme, might potentiate the anticancer efficacy of α-methyltryptophan. We tested this idea in the present study using a syngeneic mouse model of ER-positive breast cancer using the mouse breast cancer cell line AT3. Results: A syngeneic tumor-cell transplant mouse model was used for ER-positive breast cancer. AT3 cells were injected into the mammary fat pad of C57BL/6 mice. This allows to monitor the growth of AT3 tumor growth in an immunologically competent mammary tissue environment. Drug treatment in drinking water was started after tumor growth was noticeable (100 mm3). There were four groups: (i) control; (ii) α-methyltryptophan at 0.5 mg/ml; (iii) carbidopa at 0.25 mg/ml; (iv) α-methyltryptophan at 0.5 mg/ml + carbidopa at 0.25 mg/ml. Tumor growth was monitored every three days. The mice were killed after 4 weeks of treatment. Tumors were excised and weighed. These studies showed that α-methyltryptophan and carbidopa were independently effective in reducing the tumor (48% reduction, p<0.05 for α-methyltryptophan; 50% reduction, p<0.05 for carbidopa), but the combination was the most effective (92% reduction, p<0.01). These effects were evident irrespective of whether the monitored parameter was tumor weight or tumor volume. Conclusion: The combination treatment significantly decreased the tumor size and volume compared to carbidopa and α-methyltryptophan alone. We do not know yet if the improved efficacy of the combination of the two drugs is due to a simple additive effect or due to a synergistic effect. Studies have shown that carbidopa promotes degradation of ER, indicating that carbidopa has its own independent anticancer effect that is relevant to ER-positive breast cancer. Further studies are needed to differentiate between the effect of carbidopa as an independent anticancer agent and its ability to potentiate the anticancer efficacy of α-methyltryptophan by preventing its metabolic degradation. Citation Format: Sirin Falconi, Sabarish Ramachandran, Vadivel Ganapathy. Carbidopa/α-methyltryptophan as an ideal combination therapy for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2785.

The clinical successes of immunotherapy in immunologically “hot” cancers such as kidney, bladder and lung cancers has lead researchers to pursue strategies to improve the initiation of immune responses in immunologically more “cold” cancers such as breast and ovarian cancer. We have characterized two syngeneic mouse models of breast cancer, 4T1 and E0771, and an ovarian cancer model, ID8, for their baseline immune profile and responsiveness to various immunotherapy approaches in an effort to enable rational combination therapy. The 4T1 model has useful traits for immuno-oncology research including a highly metastatic phenotype. However, our data illustrate that the tumors have a paucity of CD8 T cells and a highly immunosuppressed microenvironment with Tregs and ~80% G-MDSCs of CD11b+ cells leading to primary resistance to checkpoint blockade. Radiation can induce changes in an immunosuppressive microenvironment and radiotherapy remains an important clinical modality for the treatment of breast cancer. Treatment of 4T1 tumors with radiation resulted in a ~15% reduction of total MDSCs in the mice receiving 8Gy daily for three days. For the purpose of guiding future immunotherapy combinations, we established a focal beam radiation dose response and found single dose 5Gy had no activity while 10 or 20Gy resulted in increasing anti-tumor effects. The baseline immune cell profiling for E0771, a triple negative breast cancer, revealed notable differences to 4T1. While both models have a similar proportion of CD4 T cells and Tregs, 4T1 has ~55% G-MDSCs of CD11b+ cells while E0771 has ~0.4%. The content of M-MDSCs is nearly reciprocal with E0771 having ~49% M-MDSCs of CD11b+ cells and 4T1 having ~7%. The E0771 model also has a higher proportion of CD8 T cells than 4T1. Based on its immune profile, E0771could be a better candidate for responding to checkpoint inhibition. An efficacy study in the E0771 model demonstrated high sensitivity to inhibitors of PD-1, PD-L1, and CTLA-4, although the activity of anti-PD-1 on established tumors was more modest. Costimulatory agonistic antibodies to OX40, CD137 and GITR were also highly active. These data suggest that E0771 may represent a more immunologically “warm” breast cancer. We have developed the ID8-Luc-mCh-Puro syngeneic mouse cell line as an orthotopic (intraperitoneal) model of ovarian cancer wherein tumor burden is monitored by bioluminescence imaging. Immune profiling on ascites from ID8-Luc-mCh-Puro bearing mice shows heterogeneity in the profiles between mice; although, all mice have robust CD8+ T cell infiltration with favorable CD8+ T cell/Treg ratios. The activity of checkpoint inhibitors against orthotopic ID8-Luc-mCh-Puro is currently being evaluated. Together, these models of breast and ovarian cancer can be used to evaluate anti-tumor immune responses in immunologically more quiescent indications. Citation Format: Dylan Daniel, Sumithra Urs, Kevin Guley, Sarah Krueger, David Draper, Alden Wong, Hillary Evens, Claire Higginbottom, Dan Saims, Scott Wise, Maryland Rosenfeld Franklin. Evaluation of immunomodulatory agents in classically immunologically 'cold' cancers using syngeneic mouse models of breast and ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5691.

Anti-tumor effect of a recombinant Bifidobacterium strain secreting a claudin-targeting molecule in a mouse breast cancer model

Over the past decades, reasonable progress has been made in the understanding of breast cancer biology and the treatment of the primary tumour. However, the molecular contribution of multiple cancer cell clones on the various steps of tumour progression is still poorly understood. Aside from that, standard of care treatments, like the chemotherapeutic reagents cyclophosphamide and docetaxel, are rarely able to cure breast cancer patients and the overall survival rates for metastatic disease remain poor. Only in 2018, more than 620,000 women lost their live to breast cancer, mostly due to the presence of tumour heterogeneity, an emerging drug resistance and the formation of secondary lesions. This exemplifies the unmet medical need to an in-depth understanding of tumour heterogeneity during the progression of metastatic breast cancer to finally develop new targeted therapies for this presently incurable disease. The first project has aimed to assess clonal heterogeneity during tumour progression using the MMTV-PyMT mouse model of metastatic breast cancer expressing the Confetti lineage reporter. For this purpose, mammary epithelial cells have been induced to express one of the four Confetti reporter fluorescent proteins. The outgrowth of clonal cell populations has been analysed when the maximum tumour volume comprising all stages (normal, hyperplasia, adenoma, carcinoma, pulmonary metastases) had been reached. The Confetti lineage tracing system initially visualized the emergence of clonal heterogeneity, which culminated in clonal restriction during carcinogenesis and pointed towards a polychromatic metastatic spread. Laser capture microdissection, RNA sequencing and comparative gene expression analysis of various clonal lesions indicated a substantial level of heterogeneity across and also within the various stages of tumour progression. This intra-stage tumour heterogeneity manifested by differences in proliferation, oxidative phosphorylation and cell death and could also be observed in human breast cancer biopsies. This novel understanding of clonal variation and intra-stage heterogeneity needs to be implemented in diagnosis and therapeutic options. In the past years, rising efforts have been made to develop agents targeting molecules and signalling pathways that are specifically present in breast cancer cells. Previous studies have linked an overexpression of focal adhesion kinase (FAK) – a cytoplasmic tyrosine kinase – with the initiation and progression of a wide variety of malignancies, including breast cancer. This correlation of FAK and cancer, together with its role in cell migration, invasion, and proliferation, propose FAK as an attractive target for cancer therapy. In collaboration with a pharmaceutical company, we have assessed and characterized the therapeutic potential and the biological effects of BI 853520, a novel, potent and selective small chemical inhibitor of FAK, in vitro and in several preclinical mouse models of breast cancer. We observed a significant reduction in primary tumour growth driven by an anti-proliferative effect of BI 853520. In contrast, dissecting its influence on metastasis revealed heterogenous effects at different levels of the metastatic cascade. Hence, manipulation of FAK activity with the novel FAK-inhibitor BI 853520 offers a promising anti-tumour approach for breast cancer therapy. In summary, my Ph.D. work delivered new insights into: - The existence of an intra-stage tumour heterogeneity, which is conferred by clonal variations in proliferation, oxidative phosphorylation and cell death. This novel understanding of an intra-stage heterogeneity could have a significant impact on a patient’s diagnosis and therapeutic response and should be implemented in clinical decision-making. - The therapeutic potential and biological effects of the novel FAK-inhibitor BI 853520 in vitro and in preclinical mouse models of breast cancer. This highlighted BI 853520 as a promising anti-proliferative approach for cancer therapy.