Population Of Breast Cancer Cells Research Articles

Abstract PO2-24-11: Hedgehog signaling influences tumor immunogenicity by the modulation of T cell populations in triple-negative breast cancer

Abstract Breast cancer is becoming more prevalent as it now makes up 30% of all new cancer cases yearly for U.S. women. Additionally, highly aggressive triple-negative breast cancer (TNBC) presents as immunologically “cold” tumors characterized by limited infiltration of anti-tumorigenic lymphocytes and the promotion of immunosuppressive cell populations. Therefore, it is essential to consider possible combinational therapies in treating primary tumors and in preventing metastases and recurrence. In the breast tumor microenvironment (TME), the presence and activity of regulatory T cells (Tregs) have been associated with poorer prognoses in breast cancer due to their ability to promote the progression of tumor growth and metastasis. Our group has extensively studied how the Hedgehog (Hh) signaling pathway can modulate the aggressiveness and the immune portfolio of the mammary TME, and we have recently reported the role of this pathway in regulating Treg activity and abundance. We have discovered that Hh inhibition can influence Treg-to-Th17 plasticity, promoting Tregs into a pro-inflammatory Th17-like phenotype (Tr17). As Tregs are highly influential to other immune populations of the TME, it is imperative to determine what phenotypic changes are induced in other T cell populations after systemic Hh inhibition in the mammary TME. We hypothesize that systemic Hh inhibition reshapes the tumor-immune microenvironment to be tumor-eradicating by influencing key immune populations of the mammary TME. Therefore, we further investigated how CD4+ and CD8+ T cells are influenced by systemic Hh blockade in a mammary carcinoma mouse model using single-cell RNA-Sequencing (scRNA-Seq) and R programming for data analyses and visualization. We sought to assess transcriptomic changes in tumoral T cells and to interpret how these elicited changes may be influencing the immunogenicity of the primary tumor. In these investigations, we found that systemic Hh inhibition modified the abundance and activation profile of CD4+ and CD8+ T cell populations within the primary tumor. As the infiltration and persistence of certain T cell subsets within the TME can influence tumor growth, dictate tumor recurrence, and contribute to immunotherapy responsiveness, it is essential to determine the mechanisms that modulates tumoral CD4+ and CD8+ T cells in the mammary TME. These studies will expand on the mechanism by which Hh signaling modulates mammary tumorigenesis systemically. This comprehensive work will also contribute to understanding the role of Hh signaling in modulating T cell subsets and inform future studies for innovative combinational therapeutic strategies to treat highly aggressive TNBC. Citation Format: Courtney Swain, Dominique Hinshaw, Heba Alsheikh, Brandon Metge, Lalita Shevde-Samant. Hedgehog signaling influences tumor immunogenicity by the modulation of T cell populations in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-11.

Abstract 6225: Tumor evolution through selection by ECM stiffness

Abstract In this study, we used experimental evolution to investigate the extent to which the mechanical stiffness of the tumor extracellular matrix (ECM) exerts selection pressure on a genetically diverse population of breast cancer cells. Polyacrylamide hydrogels were fabricated and conjugated with collagen type I to create the ECM with different stiffness. MDA-MB-231 cells were continuously cultured on the hydrogels of the same corresponding stiffness. Growth rate was determined at certain time points by harvesting and counting the cells. Abundance of clonal MDA-MB-231 cells labeled with heritable DNA barcodes underwent the same selection was identified with sequencing of DNA barcode tags. Cellular phenotypes were characterized through immunofluorescence staining and confocal microscopy. 3D migration of the cancer cells was examined using Boyden chamber assay. Cellular contractility was measured through traction force microscopy. Whole genome transcriptome sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing, and reduced representation bisulfite sequencing were performed for transcriptomic and epigenetic analysis. Sustained culture of MDA-MB-231 breast cancer cells overall three months on soft ECM resulted in an increased proliferation rate and beta1 integrin-dependent spreading area of the cells. Such changes did not occur in clonal cells with low genetic variation and did not occur in ancestral cells on stiff substrates. Barcode analysis revealed two distinct surviving populations post-genetic selection, with one demonstrating robust proliferation on both soft and stiff ECM, and the other exhibiting preferential adaptation to the soft matrix. The soft-selected populations exhibited nuclear localization of the transcriptional regulator yes-associated protein and less wrinkled nuclei compared with ancestral populations when both populations were grown on soft ECM. Aggressive migration was observed in the soft-selected populations, indicating a more malignant selected phenotype. The soft-selected cells showed increased RhoA activity and contractile tension on a soft substrate compared to the ancestral cells. RNA-seq analysis revealed systematic differences between ancestral, stiff-selected, and soft-selected populations corresponding to enriched biological processes related to cell adhesion and ECM organization. Significant differences in DNA methylation and chromatin accessibility were observed in soft selected populations which were correlated with changes in RNA expression. ECM stiffness exerts selection pressure on genetically variable cancer cell populations, selecting specific genetic variants in a cell population optimally adapted for that stiffness. Selected variants on soft ECM tend to be highly migratory, proliferative and generate high traction even on soft ECM. Our results highlight the importance of ECM stiffness-driven evolution in cancer development. Citation Format: Ting-Ching Wang, Suchitaa Sawhney, Daylin Morgan, Richa Rashmi, Marcos R. Estecio, Amy Brock, Irtisha Singh, Richard L. Bennett, Charles F. Baer, Jonathan D. Licht, Tanmay P. Lele. Tumor evolution through selection by ECM stiffness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6225.

The importance of cancer-associated fibroblasts in targeted therapies and drug resistance in breast cancer.

Cancer-associated fibroblasts (CAFs) play a substantial role in the tumor microenvironment, exhibiting a strong association with the advancement of various types of cancer, including breast, pancreatic, and prostate cancer. CAFs represent the most abundant mesenchymal cell population in breast cancer. Through diverse mechanisms, including the release of cytokines and exosomes, CAFs contribute to the progression of breast cancer by influencing tumor energy metabolism, promoting angiogenesis, impairing immune cell function, and remodeling the extracellular matrix. Moreover, CAFs considerably impact the response to treatment in breast cancer. Consequently, the development of interventions targeting CAFs has emerged as a promising therapeutic approach in the management of breast cancer. This article provides an analysis of the role of CAFs in breast cancer, specifically in relation to diagnosis, treatment, drug resistance, and prognosis. The paper succinctly outlines the diverse mechanisms through which CAFs contribute to the malignant behavior of breast cancer cells, including proliferation, invasion, metastasis, and drug resistance. Furthermore, the article emphasizes the potential of CAFs as valuable tools for early diagnosis, targeted therapy, treatment resistance, and prognosis assessment in breast cancer, thereby offering novel approaches for targeted therapy and overcoming treatment resistance in this disease.

GOWDL: gene ontology-driven wide and deep learning model for cell typing of scRNA-seq data.

Single-cell RNA-sequencing (scRNA-seq) allows for obtaining genomic and transcriptomic profiles of individual cells. That data make it possible to characterize tissues at the cell level. In this context, one of the main analyses exploiting scRNA-seq data is identifying the cell types within tissue to estimate the quantitative composition of cell populations. Due to the massive amount of available scRNA-seq data, automatic classification approaches for cell typing, based on the most recent deep learning technology, are needed. Here, we present the gene ontology-driven wide and deep learning (GOWDL) model for classifying cell types in several tissues. GOWDL implements a hybrid architecture that considers the functional annotations found in Gene Ontology and the marker genes typical of specific cell types. We performed cross-validation and independent external testing, comparing our algorithm with 12 other state-of-the-art predictors. Classification scores demonstrated that GOWDL reached the best results over five different tissues, except for recall, where we got about 92% versus 97% of the best tool. Finally, we presented a case study on classifying immune cell populations in breast cancer using a hierarchical approach based on GOWDL.

Antiproliferative and Antimetastatic Properties of 16-Azidomethyl Substituted 3-O-Benzyl Estrone Analogs.

Four diastereomers of 16-azidomethyl substituted 3-O-benzyl estradiol (1-4) and their two estrone analogs (16AABE and 16BABE) were tested for their antiproliferative properties against human gynecological cancer cell lines. The estrones were selected for additional experiments based on their outstanding cell growth-inhibiting activities. Both compounds increased hypodiploid populations of breast cancer cells, and 16AABE elicited cell cycle disturbance as evidenced by flow cytometry. The two analogs substantially increased the rate of tubulin polymerization in vitro. 16AABE and 16BABE inhibited breast cancer cells' migration and invasive ability, as evidenced by wound healing and Boyden chamber assays. Since both estrone analogs exerted remarkable estrogenic activities, as documented by a luciferase reporter gene assay, they can be considered as promising drug candidates for hormone-independent malignancies.

Single-cell RNA sequencing captures patient-level heterogeneity and associated molecular phenotypes in breast cancer pleural effusions.

Malignant pleural effusions (MPEs) are a common complication of advanced cancers, particularly those adjacent to the pleura, such as lung and breast cancer. The pathophysiology of MPE formation remains poorly understood, and although MPEs are routinely used for the diagnosis of breast cancer patients, their composition and biology are poorly understood. It is difficult to distinguish invading malignant cells from resident mesothelial cells and to identify the directionality of interactions between these populations in the pleura. There is a need to characterize the phenotypic diversity of breast cancer cell populations in the pleural microenvironment, and investigate how this varies across patients. Here, we used single-cell RNA-sequencing to study the heterogeneity of 10 MPEs from seven metastatic breast cancer patients, including three Miltenyi-enriched samples using a negative selection approach. This dataset of almost 65000 cells was analysed using integrative approaches to compare heterogeneous cell populations and phenotypes. We identified substantial inter-patient heterogeneity in the composition of cell types (including malignant, mesothelial and immune cell populations), in expression of subtype-specific gene signatures and in copy number aberration patterns, that captured variability across breast cancer cell populations. Within individual MPEs, we distinguished mesothelial cell populations from malignant cells using key markers, the presence of breast cancer subtype expression patterns and copy number aberration patterns. We also identified pleural mesothelial cells expressing a cancer-associated fibroblast-like transcriptomic program that may support cancer growth. Our dataset presents the first unbiased assessment of breast cancer-associated MPEs at a single cell resolution, providing the community with a valuable resource for the study of MPEs. Our work highlights the molecular and cellular diversity captured in MPEs and motivates the potential use of these clinically relevant biopsies in the development of targeted therapeutics for patients with advanced breast cancer.

Migration speed of captured breast cancer subpopulations correlates with metastatic fitness.

The genetic alterations contributing to migration proficiency, a phenotypic hallmark of metastatic cells required for colonizing distant organs, remain poorly defined. Here, we used single-cell magneto-optical capture (scMOCa) to isolate fast cells from heterogeneous human breast cancer cell populations, based on their migratory ability alone. We show that captured fast cell subpopulations retain higher migration speed and focal adhesion dynamics over many generations as a result of a motility-related transcriptomic profile. Upregulated genes in isolated fast cells encoded integrin subunits, proto-cadherins and numerous other genes associated with cell migration. Dysregulation of several of these genes correlates with poor survival outcomes in people with breast cancer, and primary tumors established from fast cells generated a higher number of circulating tumor cells and soft tissue metastases in pre-clinical mouse models. Subpopulations of cells selected for a highly migratory phenotype demonstrated an increased fitness for metastasis.

APC Loss Prevents Doxorubicin-Induced Cell Death by Increasing Drug Efflux and a Chemoresistant Cell Population in Breast Cancer.

Chemoresistance is a major health concern affecting cancer patients. Resistance is multifactorial, with one mechanism being the increased expression of ABC transporters (such as MDR1 and MRP1), which are drug efflux transporters capable of preventing intracellular accumulation of drugs and cell death. Our lab showed that the loss of Adenomatous Polyposis Coli (APC) caused an intrinsic resistance to doxorubicin (DOX), potentially through an enhanced tumor-initiating cell (TIC) population and the increased activation of STAT3 mediating the expression of MDR1 in the absence of WNT being activated. Here, in primary mouse mammary tumor cells, the loss of APC decreased the accumulation of DOX while increasing the protein levels of MDR1 and MRP1. We demonstrated decreased APC mRNA and protein levels in breast cancer patients compared with normal tissue. Using patient samples and a panel of human breast cancer cell lines, we found no significant trend between APC and either MDR1 or MRP1. Since the protein expression patterns did not show a correlation between the ABC transporters and the expression of APC, we evaluated the drug transporter activity. In mouse mammary tumor cells, the pharmacological inhibition or genetic silencing of MDR1 or MRP1, respectively, decreased the TIC population and increased DOX-induced apoptosis, supporting the use of ABC transporter inhibitors as therapeutic targets in APC-deficient tumors.

Abstract 2367: Hedgehog signaling influences tumor immunogenicity by the modulation of T cell populations in breast cancer

Abstract Breast cancer is becoming more prevalent as it now makes up 30% of all new cancer cases yearly for U.S. women. Additionally, highly aggressive triple-negative breast cancer (TNBC) presents with immunologically “cold” tumors characterized by limited infiltration of anti-tumorigenic lymphocytes and the promotion of immunosuppressive cell populations. Therefore, it is essential to consider possible combinational therapies in treating primary tumors and in preventing metastases and recurrence. In the breast tumor microenvironment (TME), the presence and activity of regulatory T cells (Tregs) have been associated with poorer prognoses in breast cancer due to their ability to promote the progression of tumor growth and metastasis. Our group has extensively studied how the Hedgehog (Hh) signaling pathway can modulate the aggressiveness and the immune portfolio of the mammary TME, and we have recently reported the role of this pathway in regulating Treg activity and abundance. We have discovered that Hh inhibition can influence Treg-to-Th17 plasticity, promoting Tregs into a pro-inflammatory Th17-like phenotype (Tr17), therefore, affecting the immunogenicity of the mammary TME. As Tregs are highly influential to other immune populations of the TME, it is imperative to determine how this phenotypic change in Tregs after Hh inhibition influences other T cell populations of the mammary TME. We hypothesize that systemic Hh inhibition reshapes the tumor-immune microenvironment to be tumor-eradicating by influencing key immune populations of the mammary TME. Therefore, we further investigated how CD4+ and CD8+ T cells are influenced by systemic Hh blockade in a mammary carcinoma mouse model using single-cell RNA-Sequencing (scRNA-Seq), multiparameter flow cytometry, and R programming for data analyses and visualization. We sought to interpret how Hh inhibition influences key T cell populations that can dictate the immunogenicity of the primary tumor and to determine if these changes may be a result of the Treg-to-Th17 transition in response to Hh manipulation. In these investigations, we found that systemic Hh inhibition modified the abundance, metabolic signatures, and effector functions of CD4+ and CD8+ T cell populations within the primary tumor. As Tregs can impair essential tumor-infiltrating lymphocytes and promote the recruitment of other immunosuppressive constituents to the primary mammary tumor and Th17s can promote inflammatory and cytotoxic immune populations, these studies will expand on the mechanism by which Hh signaling modulates mammary tumorigenesis holistically, as well as specifically, through Tregs. This comprehensive work revealing the mechanistic role of Hh signaling in Treg plasticity and other T cell subsets will also inform innovative combinational therapeutic strategies to treat highly aggressive TNBC. Citation Format: Courtney A. Swain, Dominique C. Hinshaw, Brandon J. Metge, Heba M. Alsheikh, Lalita A. Shevde-Samant. Hedgehog signaling influences tumor immunogenicity by the modulation of T cell populations in 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 2367.

Abstract 4479: Targeting of eIF4A1 curtails lung metastases in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer. Patients diagnosed with TNBC benefit less or none from any current targeted therapies and also the standard-of-care neoadjuvant chemotherapies (NACT) which is cytotoxic. TNBC shows an increased risk of tumor relapse, therapy failure, and high proclivity to develop distant metastases. Intrinsic resistance to NACT is mainly due to a small population of breast cancer cells that acquire stem cell-like features (BCSCs). BCSCs are capable of self-renewal, proliferation, plasticity, immune regulation and chemoresistance. These properties mediate tumorigenesis, metastasis, and resistance to NACT and sometimes even to targeted therapy. Overall, FDA-approved drugs that are effective for advanced TNBC are currently unavailable. The eukaryotic translation initiation factor (eIF4A1) is an mRNA helicase that catalyzes the ATP-dependent unwinding of many oncogenic mRNAs which harbor secondary structures in the 5’ leader region. There is preferential reliance on eIF4A1 by tumor cells as many oncogenic mRNAs require the helicase activity of eIF4A1 for their efficient translation. Several eIF4A1-dependent mRNAs (downstream effectors) include: survivin (survival), c-MYC, STAT1 (immunoevasion through transcription and subsequent translation of PD-L1), Rho kinase1 (invasion), and Cyclins D1 and D3. Bioinformatic analyses revealed that higher gene expression of eIF4A1 significantly impacts patient survival. Clinically, an enhanced protein expression of eIF4A1 was evident in histopathologic analyses of drug-resistant human TNBC biospecimens. Importantly, our in vitro data indicated that the viability of BCSCs was significantly reduced when eIF4A1 is targeted by nanomolar levels of Rocaglamide A (RocA). Furthermore, in paclitaxel-resistant TNBC cells, there is an upregulation in the total levels of eIF4A1 and its downstream effectors. Pluripotency transcription factors (e.g., SOX2, OCT4 and NANOG) and drug transporters (ABCB1, ABCG2 and ABCC1) were also increased in an eIF4A1-dependent manner. Genetic ablation and pharmacological targeting of eIF4A1 with RocA reversed the drug-resistant profile. Our in vitro data implied a key role for eIF4A1 in chemoresistance/therapy failure. Targeting of eIF4A1 may be an effective anti-cancer strategy to overcome drug resistance. To this end, we orthotopically implanted CRISPR-control (CC) and eIF4A1-KO (KO) human TNBC cells and evaluated the primary tumor progression and metastases in NOD/SCID-beige mice. Longitudinal tumor progression was followed by IVIS imaging. The tumor volume and the wet weight was significantly higher in CC than KO tumors (n=7 mice). Similarly, the lung metastatic burden is significantly low in KO than CC mice (n=7 mice; p<0.002)). Taken together, we propose that eIF4A1 is an actionable molecular target for drug-resistant TNBC using chemotherapy/immunotherapy. Citation Format: Sangita Sridharan, Shobhit Srivastava, David Terrero, Saloni Malla, Amit K. Tiwari, Dayanidhi Raman. Targeting of eIF4A1 curtails lung metastases in triple-negative 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 4479.

Abstract P2-26-18: Adiponectin regulates stem cell activity in tamoxifen-resistant breast cancer cells

Abstract Introduction: Breast cancer is the most diagnosed neoplasia and the second leading cause of malignancy death in women. Drug resistance is still the major challenge in the clinical management of breast cancer patients. Indeed, despite the improvements in the early diagnosis and in the therapeutic approaches, many breast cancer patients experience disease relapse due to de novo or acquired drug resistance. Growing evidence recognized a small population of breast cancer cells, named Breast Cancer Stem Cells (BCSCs), as the leading cause of tumor progression, metastasis formation and resistance against conventional therapy. BCSCs have some specific properties such as self-renewal, differentiation into different cell types, migration, tumorsphere formation, antioxidative activity, that make tumors more aggressive. Tumor microenvironment plays a crucial role in the regulation of stem cell proliferation and resistance to apoptosis through the secretion of cytokines and growth factors. Adipocytes represent the most abundant cellular component of mammary microenvironment. The excessive fat accumulation in obesity leads to the development of a dysfunctional adipose tissue, producing an unbalance in adipokines secretion. Among the secreted factors, adiponectin plays a crucial role in breast cancer development and progression. The aim of the present study was to investigate the effects of low adiponectin level (5 μl/ml), hallmark of obese status, on BCSCs activity in hormone-resistant cells. Methods: We tested the ability of MCF-7 wild type (WT) and tamoxifen-resistant (TR) to grow as mammospheres (Mammospheres Forming Efficiency, MFE), measuring the ability to maintain cell viability (self-renewal) upon serial non-adherent passages. mRNA levels of stemness, EMT and cell cycle markers were evaluated by qRT-PCR. CD44+/24- cell ratio, ALDH expression, ROS production, cell cycle, were analyzed by flow cytometry. Results: Adiponectin treatment significantly enhanced MFE and self-renewal capacity in TR-MCF-7 cells compared to MCF-7 WT cells. To identify the presence of BCSCs into mammospheres it has been evaluated the CD44+/CD24– biomarker signature. Flow cytometry revealed an enrichment of CD44, a trans-membrane glycoprotein which regulates growth signals in stem cells, in TR-MCF-7 mammospheres, whereas expression levels of the differentiation marker CD24 were decreased. The gene expression of these biomarkers was also analyzed by qRT-PCR. The increased BCSCs subpopulation in adiponectin-treated TR-MCF-7 mammospheres was also confirmed by the enhanced ALDH-expressing cells. qRT-PCR revealed that adiponectin increased the mRNA levels of stemness and EMT markers in TR-MCF-7 cells mammospheres. Interestingly, cell cycle analysis showed in adiponectin-treated TR-MCF-7 mammospheres a reduction of apoptosis. Moreover, flow cytometry analysis displayed a reduction of ROS levels, which generally are assumed as cues determining DNA damage-induced cell death, in adiponectin-treated TR-MCF-7 mammospheres. Conclusions: Our results demonstrated that low adiponectin level, as it occurs in obese breast cancer microenvironment, driving EMT, enhances stem-like features in TR-MCF-7 cells to sustain tumor progression. Citation Format: Giuseppina Daniela Naimo, Martina Forestiero, Alessandro Paolì, Francesca Giordano, Maria Luisa Panno, Loredana Mauro, Sebastiano Andò. Adiponectin regulates stem cell activity in tamoxifen-resistant breast cancer cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-18.

Cell migration capacity alone can be hereditary and predict metastatic burden.

Among all alterations that cells need for colonizing distant organs, migratory proficiency is widely accepted as a key characteristic of highly metastatic cells. However, investigations are almost exclusively based on functional inhibition or genetic knock-out of candidate proteins involved in cell migration to study their impact on in vivo metastasis. Importantly, there is no antibody or gene that allows the labeling of highly migratory cells. Identification and capture of live individual cells in a heterogenous ensemble typically rely on genetic manipulations that encode fluorescent probes. Thus, a precise understanding of how several molecular components interact to yield the phenotype of interest is a prerequisite to distinguish and isolate such target cells. Single cell magneto-optical capture (scMOCa) has the capacity to tag and manipulate cells based on visual phenotypes to subsequently identify and characterize novel molecules that modulate these phenotypes. Here, we capture single cells from a highly heterogeneous triple-negative breast cancer cell population based solely on the analysis of their migration trajectories. We isolate and expand intact cells to generate novel populations defined by their travelled distance alone. We demonstrate that the progeny of these cells stably retains their migratory phenotype and exhibits a dramatically different transcriptomic profile when compared to the parental counterpart. Comparison of RNA sequencing of fast and parental cell populations reveals a motility-related transcriptomic profile that highlights Biological Adhesion Gene Ontology terms. Fast cells possess an increased dynamics and distinct morphology of focal adhesions. Finally, primary tumors established from fast cells generate a higher number of circulating tumor cells and soft tissue metastases in vivo, resulting in shorter time to clinical endpoint following tumor resection. Thus, selection for a highly migratory phenotype alone can confer increased fitness for specific steps in the metastatic cascade.

DNA barcoding reveals ongoing immunoediting of clonal cancer populations during metastatic progression and immunotherapy response

Cancers evade the immune system through the process of cancer immunoediting. While immune checkpoint inhibitors are effective for reactivating tumour immunity in some cancer types, many other solid cancers, including breast cancer, remain largely non-responsive. Understanding how non-responsive cancers evade immunity and whether this occurs at the clonal level will improve immunotherapeutic design. Here we use DNA barcoding to track murine mammary cancer cell clones during immunoediting and determine clonal transcriptional profiles that allow immune evasion following anti-PD1 plus anti-CTLA4 immunotherapy. Clonal diversity is significantly restricted by immunotherapy treatment in both primary tumours and metastases, demonstrating selection for pre-existing breast cancer cell populations and ongoing immunoediting during metastasis and treatment. Immunotherapy resistant clones express a common gene signature associated with poor survival of basal-like breast cancer patient cohorts. At least one of these genes has an existing small molecule that can potentially be used to improve immunotherapy response.

Role of Cancer Stem-like Cells in the Process of Invasion and Mesenchymal Transformation by a Reconstituted Triple-negative Breast Cancer Cell Population Resistant to p53-induced Apoptosis.

We investigated the role of cancer stem cells (CSCs) in a population of triple-negative breast cancer (TNBC) cells that are resistant to apoptosis. A human breast cancer cell population capable of inducing p53 expression with doxycycline (Dox) was created and used as an untreated control (UT). After the addition of Dox to UT for 5 days, the cell population reconstituted with cells showing resistance to apoptosis was named RE. Fluorescence-activated cell sorting (FACS) and immunostaining revealed that after the addition of Dox, the ratio of cells in the S and G2/M phases decreased in UT as apoptosis proceeded, but did not markedly change in apoptosis-resistant RE. CSC-like cells in RE exhibited a cell morphology with a larger ratio of the major/minor axis than UT. FACS showed that RE had a higher proportion of CSC-like cells and contained more CD44+CD24- mesenchymal CSCs than ALDH1A3+ epithelial-like CSCs. In a Matrigel invasion assay, UT was more likely to form a three-dimensional cell population, whereas RE exhibited a planar population, higher migration ability, and the up-regulated expression of epithelial-mesenchymal transition-related genes. These results provide insights into the mechanisms by which TNBC cells acquire treatment resistance at the time of recurrence.

137P Combining protein and mRNA spatial profiles to identify distinct cell population in metaplastic breast cancer

Metaplastic breast cancer (MpBC) is a heterogeneous disease and is characterized by the differentiation of the neoplastic epithelium into squamous cells and/or mesenchymal elements. MpBC is rare but very aggressive and presents high rates of recurrence, poor survival outcomes, and poor response to current chemotherapies optimized for other breast cancer subtypes. To date, there are no established treatments for MpBCs, due to paucity of clinical trials and near absence of preclinical models for investigation.

Mathematical characterization of population dynamics in breast cancer cells treated with doxorubicin.

The development of chemoresistance remains a significant cause of treatment failure in breast cancer. We posit that a mathematical understanding of chemoresistance could assist in developing successful treatment strategies. Towards that end, we have developed a model that describes the cytotoxic effects of the standard chemotherapeutic drug doxorubicin on the MCF-7 breast cancer cell line. We assume that treatment with doxorubicin induces a compartmentalization of the breast cancer cell population into surviving cells, which continue proliferating after treatment, and irreversibly damaged cells, which gradually transition from proliferating to treatment-induced death. The model is fit to experimental data including variations in drug concentration, inter-treatment interval, and number of doses. Our model recapitulates tumor cell dynamics in all these scenarios (as quantified by the concordance correlation coefficient, CCC > 0.95). In particular, superior tumor control is observed with higher doxorubicin concentrations, shorter inter-treatment intervals, and a higher number of doses (p < 0.05). Longer inter-treatment intervals require adapting the model parameterization after each doxorubicin dose, suggesting the promotion of chemoresistance. Additionally, we propose promising empirical formulas to describe the variation of model parameters as functions of doxorubicin concentration (CCC > 0.78). Thus, we conclude that our mathematical model could deepen our understanding of the cytotoxic effects of doxorubicin and could be used to explore practical drug regimens achieving optimal tumor control.

Regulation of p53 and survivin by Curcuma longa extract to caspase-3 dependent apoptosis in triple negative breast cancer cells.

Aim Triple negative breast cancer cells (TNBC) are the population of breast cancer cells that are responsible for cancer recurrence and apoptosis resistance. Unfortunately, current therapies have limited efficacy to TNBC population due to apoptosis resistance and chemoresistance. Tumour suppressor p53 and survivin are primary targets for TNBC therapy. Consequently, a search for a natural compound which targets p53 and survivin is needed to further advance TNBC treatment. Curcuma longa extract (CL), a natural compound induces apoptosis in several cancer cells by targeting various molecules and possess fewer side effects. However, a possible potential of CL as p53- and survivin modulating agent in TNBC cells has not been investigated. Methods MDAMB-231 cells were treated with several concentration of CL, after which, viability, p53 gene expression, surviving protein expression, and caspase-3 protein expression were evaluated. Results After 24-h treatment, CL possessed cytotoxic effect with IC50 value of 13 μg/mL. Treatment with 1.625, 3.25, 6.5, and 13 μg/mL of CL resulted in 2.70-25.80% increase in caspase-3 expression levels followed by 94.60 - 21.60% decrease in survivin protein levels. CL induced remarkably p53 gene expression ratio up to 5-fold at 13 μg/mL. Survivin protein levels were inversely proportional to p53 accumulation levels. Low survivin protein levels combined with high levels of p53 accumulation were correlated to higher apoptotic rates. Conclusion p53 and survivin as molecular targets of CL contribute to caspase-3-dependent apoptosis in TNBC cells and this compound represents an attractive p53- and survivin modulating agent in TNBC.

Catulin reporter marks a heterogeneous population of invasive breast cancer cells with some demonstrating plasticity and participating in vascular mimicry

Breast cancer is the most commonly diagnosed cancer in women worldwide. The activation of partial or more complete epithelial–mesenchymal transition in cancer cells enhances acquisition of invasive behaviors and expands their generation of cancer stem cells. Increased by EMT plasticity of tumor cells could promote vascular mimicry, a newly defined pattern of tumor microvascularization by which aggressive tumor cells can form vessel-like structures themselves. VM is strongly associated with a poor prognosis, but biological features of tumor cells that form VM remains unknown. Here we show that catulin is expressed in human BC samples and its expression correlates with the tumor progression. Ablation of catulin in hBC cell lines decreases their invasive potential in the 3D assays. Using a novel catulin promoter based reporter we tracked and characterized the small population of invasive BC cells in xenograft model. RNAseq analysis revealed enrichment in genes important for cellular movement, invasion and interestingly for tumor-vasculature interactions. Analysis of tumors unveiled that catulin reporter marks not only invasive cancer cells but also rare population of plastic, MCAM positive cancer cells that participate in vascular mimicry. Ablation of catulin in the xenograft model revealed deregulation of genes involved in cellular movement, and adhesive properties with striking decrease in CD44 which may impact stemness potential, and plasticity of breast cancer cells. These findings show directly that some plastic tumor cells can change the fate into endothelial-like, expressing MCAM and emphasize the importance of catulin in this process and breast cancer progression.

Abstract 2936: Multi-omic characterization of transitional cell populations in breast cancer

Abstract Breast cancer is a heterogeneous collection of diseases grouped by hormone receptor status or by expression of key subtype-determining genes. Breast cancer subtypes, in particular basal-like breast cancer and the luminal breast cancer subtypes, differ by hormonal receptor status, proliferation, genomic instability and mutational signatures, treatment response, and prognosis. The highly distinct signatures of basal-like and luminal breast cancer suggest that they may have different cells of origin within the breast duct. As part of the Washington University Human Tumor Atlas Network (WU-HTAN) program, we generated multi-omic data for 53 samples from 37 breast cancer tumors and 4 normal adjacent tissues. Genomic subtyping was applied to both bulk and single-nucleus RNA sequencing. Analysis of single-nucleus gene expression and chromatin accessibility in epithelial cells underscores similarities between basal-like breast cancer and luminal progenitor cells within the breast duct, and between luminal breast cancer and mature luminal ductal cells. This study links distinct breast cancer subtypes to normal breast cell populations and suggests distinct cells of origin for these cancer types. Citation Format: Michael D. Iglesia, Reyka G. Jayasinghe, Daniel Cui Zhou, Nadezhda V. Terekhanova, John Herndon, Alla Karpova, Siqi Chen, Nataly Naser Al Deen, Kazuhito Sato, Feng Chen, Deborah J. Veis, Ryan C. Fields, William E. Gillanders, Li Ding. Multi-omic characterization of transitional cell populations in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2936.

Blocking Gi/o-Coupled Signaling Eradicates Cancer Stem Cells and Sensitizes Breast Tumors to HER2-Targeted Therapies to Inhibit Tumor Relapse.

Simple SummaryCancer stem cells (CSCs) are associated with therapeutic resistance and tumor relapse but effective approaches for eliminating CSCs are still lacking. The aim of this study was to assess the role of G protein-coupled receptors (GPCRs) in regulating CSCs in breast cancer. We showed that a subgroup of GPCRs that coupled to Gi/o proteins (Gi/o-GPCRs) was required for maintaining the tumor-forming capability of CSCs in HER2+ breast cancer. Targeting Gi/o-GPCRs or their downstream PI3K/AKT and Src pathways was able to enhance HER2-targeted elimination of CSCs and therapeutic efficacy. These findings suggest that targeting Gi/o-GPCR signaling is an effective strategy for eradicating CSCs, enhancing HER2+ targeted therapy and blocking tumor recurrence.Cancer stem cells (CSCs) are a small subpopulation of cells within tumors that are resistant to anti-tumor therapies, making them a likely origin of tumor relapse after treatment. In many cancers including breast cancer, CSC function is regulated by G protein-coupled receptors (GPCRs), making GPCR signaling an attractive target for new therapies designed to eradicate CSCs. Yet, CSCs overexpress multiple GPCRs that are redundant in maintaining CSC function, so it is unclear how to target all the various GPCRs to prevent relapse. Here, in a model of HER2+ breast cancer (i.e., transgenic MMTV-Neu mice), we were able to block the tumorsphere- and tumor-forming capability of CSCs by targeting GPCRs coupled to Gi/o proteins (Gi/o-GPCRs). Similarly, in HER2+ breast cancer cells, blocking signaling downstream of Gi/o-GPCRs in the PI3K/AKT and Src pathways also enhanced HER2-targeted elimination of CSCs. In a proof-of-concept study, when CSCs were selectively ablated (via a suicide gene construct), loss of CSCs from HER2+ breast cancer cell populations mimicked the effect of targeting Gi/o-GPCR signaling, suppressing their capacity for tumor initiation and progression and enhancing HER2-targeted therapy. Thus, targeting Gi/o-GPCR signaling in HER2+ breast cancer is a promising approach for eradicating CSCs, enhancing HER2+ targeted therapy and blocking tumor reemergence.