Mammary Fat Pad Research Articles

Understanding the impact of heart failure treatment on cancer growth

Abstract Background Heart failure (HF) is associated with systemic alterations that extend beyond the cardiovascular system. The dysregulation of physiological pathways induced by HF, encompassing inflammatory or neurohormonal pathways, may foster an environment conducive to cancer growth and the spead of metastases. It is unknown whether these alterations are attenuated when HF is treated, and whether certain therapies for HF may yield more significant effects than others. Purpose This study aims to investigate whether myocardial infarction (MI)-induced left ventricular (LV) dysfunction promotes breast cancer growth and triggers the development of lung metastases. Additionally, it compares the potential impact of different therapeutic strategies for LV dysfunction on cancer development. Methods Myocardial infarction (MI) was induced in 11-week-old female BALB/c mice by ligation of the left anterior descending (LAD) coronary artery, leading to LV dysfunction and significant dilation within four weeks. Starting 2 weeks after surgery, mice with ejection fraction (EF) < 40% received daily oral gavage of vehicle or one of three drugs: carvedilol (10 mg/kg), enalapril (20 mg/kg), or empagliflozin (15 mg/kg). Four weeks post-surgery, 2x105 4T1 metastatic breast cancer cells were injected into the 4th mammary fat pad. Tumor volume was measured every two days, starting eight days after injection. Weekly echocardiographic assessments monitored cardiac function until the end of the experiment. Tumors were excised on day 22 after injection. Mice were euthanised on day 28 for post-mortem analysis. MCM2 proliferation staining was performed on lung tissue to quantify metastases. Results Among the HF treatments, only empagliflozin (n=11) induced a significant decrease in both left ventricular end-diastolic and end-systolic volume (Fig. 1.A; p=0.0043; Fig. 1.B; p=0.0186) compared to the vehicle group (n=11). Tumor weight and volume (Fig. 1.C-D) were not different in the HF/vehicle group (n=15) and the sham-operated group (n=13). Carvedilol (n=14) led to a small reduction in tumor weight (Fig. 1.C; p=0.1264), while empagliflozin (n=11) exhibited a significant reduction in both tumor weight (Fig. 1.C; p=0.0306) and volume (Fig. 1.D; p=0.0268). In addition, carvedilol (n=13) significantly reduced lung metastases (Fig. 1.E; p=0.0347) compared to the HF/vehicle group (n=13). Enalapril (n=10) and empagliflozin (n=9) did not significantly impact the development of lung metastases. Conclusion Despite the absence of increased cancer growth post-MI, our findings indicate that specific treatments for HF—namely, carvedilol and empagliflozin—attenuate cancer growth post-MI. Furthermore, carvedilol demonstrated potential in reducing metastatic spread. Additional research is required to uncover the underlying mechanisms both in the presence and absence of HF, and to explore the molecular impact that HF treatments have on various stages of cancer progression.Figure 1

Sorted stem/progenitor epithelial cells of pubertal bovine mammary gland present limited potential to reconstitute an organised mammary epithelium after transplantation.

The development and maintenance of mammary gland tissue depend on the proliferation and differentiation of mammary stem and progenitor cells. Here, we investigated populations of mammary epithelial cells that are potential candidates for bovine mammary gland development using xenotransplantation into mice cleared mammary fat pad. Transplanted mammary explants from 17-month-old Holstein heifers developed outgrowths exhibiting the archetypal morphology and molecular marker distributions of the bovine gland. Xenotransplantation of sorted mammary epithelial cells (CD49fpos) into bovinised fat pads using inactivated bovine fibroblasts resulted in outgrowth developments with 50% take rate, but these lacked the ductal or alveolar epithelial structures of the normal mammary gland. Similar results were obtained with xenografts of candidate bovine mammary epithelial stem cells (CD49fhighCD24pos) or epithelial cells of the basal lineage (CD49fhighCD24neg) which also developed as clumps of cells surrounded by stromal stretches within the mouse adipose tissue. In conclusion, sorted cells showed compromised regenerative potential for epithelial morphogenesis. Further work is therefore needed to identify mammary stem/progenitor cells with full regenerative capabilities for biogenesis of normal mammary gland structure, with milk-secreting function.

Abstract B022: Tumoral GLI1 controls the resident innate and adaptive tumor immune microenvironment in triple negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancer (BC) cases but due to its aggressive nature and the lack of targeted treatment options, represents a disproportionately higher number of BC-related deaths. Compared to other BC subtypes, TNBCs are more inflamed with M2-like tumor-associated macrophages (TAMs) and cytotoxic T-lymphocytes (CTLs) which are associated with poor and good prognosis, respectively. Clinical trials have proven that immunogenic cancers can be effectively treated through immune checkpoint blockade (ICB), a treatment strategy that prevents cancer cells from silencing CTL-mediated attack. However, CTLs can be suppressed by M2-like TAMs which can limit ICB efficacy and support tumor growth. The hedgehog (HH) signaling pathway is highly activated in TNBC and higher expression of GLI1 is associated with worse overall survival. Recent pan-cancer analyses highlight a significant correlation between activated HH signaling and characteristics of immune evasion. We aim to clarify how activated HH signaling regulates resident innate and adaptive immune cells in the tumor microenvironment (TME) in TNBC. Methods: Primary K14-cre; Brca1 fl/fl ; P53 fl/fl (KBP) murine TNBC cells were transfected with CRISPR/Cas9 + sgRNA targeting GLI1. After isolating single cell clones and confirming GLI1 knockout (KO), KBP and KBP-GLI1-KO cells were orthotopically injected into the mammary fat pad of syngeneic immune-competent female mice and allowed to grow for 6-8 weeks. Tumor volume was measured once tumors were palpable, and tumors were harvested once the largest tumor reached ethical endpoint. Tumors were weighed at harvest and fluorescence activated cell sorting (FACS) was performed to determine if there was an effect on the composition of immune populations. Results: At harvest, KBP-GLI1-KO tumors were delayed in growth and significantly smaller than KBP tumors. FACS analysis showed that GLI1-KO tumors had increased CD4+ and CD8+ T cells and a reduced amount of Foxp3+ CD4+ Tregs. Furthermore, GLI1-KO tumors had significantly fewer F4/80+ CD11b+ TAMs within the TME. Amongst TAM populations, there was reduced CD206+ M2-like TAMs and an increase in CD80+ M1-like TAMs upon loss of GLI1. Similar trends were observed when KBP allografts were treated with the GLI1 inhibitor, GANT61. Immunohistochemical and RNAseq analysis are ongoing. Similar experiments with other KBP-GLI1-KO clones are currently being done. Conclusion: Here, we show that tumoral GLI1 not only promotes tumor growth, but also supports an immunosuppressive TME in TNBC. Abolishing GLI1 converted tumors to a pro-inflammatory phenotype marked by an increase in CD4+ and CD8+ T cells and a reduction in CD206+ M2-like TAMs. Future experiments are aimed at revealing the mechanisms by which GLI1 regulates immune cell recruitment to the TME and testing the effect of combination immunotherapy. Overall, these preliminary findings identify GLI1 as a potential therapeutic target to limit tumor growth and promote anti-tumor immunity. Citation Format: Aidan J Gray, Wanda Marini, Kiichi Murakami, Michael Reedijk. Tumoral GLI1 controls the resident innate and adaptive tumor immune microenvironment in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B022.

Establishment of Translational Luciferase-Based Cancer Models to Evaluate Antitumoral Therapies.

Luciferase (luc) bioluminescence (BL) is the most used light-emitting protein that has been engineered to be expressed in multiple cancer cell lines, allowing for the detection of tumor nodules in vivo as it can penetrate most tissues. The goal of this study was to develop an oncolytic adenovirus (OAd)-resistant human triple-negative breast cancer (TNBC) that could express luciferase. Thus, when combining an OAd with chemotherapies or targeted therapies, we would be able to monitor the ability of these compounds to enhance OAd antitumor efficacy using BL in real time. The TNBC cell line HCC1937 was stably transfected with the plasmid pGL4.50[luc2/CMV/Hygro] (HCC1937/luc2). Once established, HCC1937/luc2 was orthotopically implanted in the 4th mammary gland fat pad of NSG (non-obese diabetic severe combined immunodeficiency disease gamma) female mice. Bioluminescence imaging (BLI) revealed that the HCC1937/luc2 cell line developed orthotopic breast tumor and lung metastasis over time. However, the integration of luc plasmid modified the HCC1937 phenotype, making HCC1937/luc2 more sensitive to OAdmCherry compared to the parental cell line and blunting the interferon (IFN) antiviral response. Testing two additional luc cell lines revealed that this was not a universal response; however, proper controls would need to be evaluated, as the integration of luciferase could affect the cells' response to different treatments.

Cutting-Edge Approach to Targeted Therapy: Repositioning of Old Drugs in Combination with Standard Clinical Chemotherapeutics Potentiates a Propitious Novel Targeted Therapy for Human Pancreatic Cancer

Metastatic pancreatic cancer leads to a fatal outcome, with a median progression-free survival of approximately six months when utilizing the most successful combination of chemotherapeutic regimens. When drug resistance develops, it facilitates an increase in primary tumor growth and new and growing metastases. Patients inevitably and quickly succumb to their disease and die. Notably, chemotherapy has an unintended impact on the development of drug resistance through the enhancement of EMT development and the enrichment of cancer stem cells (CSC). Recent report discovered that neuraminidase-1 (Neu-1) regulates EMT induction, angiogenesis, and cellular proliferation by the activation of several receptor tyrosine kinases. Here, the continual therapeutic inhibition of Neu-1 through intravenous administration of oseltamivir phosphate (OP) and aspirin (ASA) alongside GEM treatment significantly inhibits tumor progression, crucial compensatory signaling pathways, EMT program, CSC, and metastasis progression in a preclinical RAG2xCy double mutant BALB/c mouse model of human PANC-1 pancreatic cancer. The tumorigenic and metastatic potential of the xenotumors from the animals treated with the experimental protocols were significantly ablated when transferred into the mammary fat pads of NSG (NOD SCID gamma) branded mice. Keywords: pancreatic cancer; chemoresistance; drug repurposing; EMT.

Targeting cancer stress-associated hyperinsulinemia and abnormal behavior mitigates lung carcinoma in postmenopausal mouse: Intervention function of peimine

Obesity stress and its associated metabolic disruption increase the likelihood and advancement of several forms of malignancies, such as postmenopausal lung cancer in humans. Also, the mechanism of lung cancer stress-related cognitive impairment and rhythm change are still unknown. Peimine (PE), an alkaloid derived from the Fritillaria genus, has the ability to mitigate the metabolic dysfunctions induced by obesity and inhibit tumor growth. The objective of this study was to investigate the regulatory impact of PE on obese mice with postmenopausal lung cancer generated by a high fat diet. Our initial findings indicate that PE can significantly reduce the metabolic dysfunction caused by a high-fat diet in mice. This is demonstrated by better glucose tolerance and insulin resistance, reduced accumulation of lipids in the liver, and improved histological alterations in the mammary fat pad. Subsequently, the treatment of PE suppressed the formation of lung tumors caused by a high-fat diet in mice models placed in their original location. In addition, PE effectively inhibited the formation of pre-existing lung tumors in a mouse model of postmenopausal women who were fed a high-fat diet. This treatment also reduced the severity of metabolic problems. Notably, laboratory studies demonstrated that insulin exposure increased the growth and movement of lung cancer cells, but this effect was significantly reduced when the cells were co-cultured with PE. Meanwhile, PE intervention can significantly alleviate cancer stress-related cognitive impairment and disturbed circadian rhythm changes and associated gene expression abnormalities in model animals. Analysis using bioinformatics and molecular biology techniques revealed that PE has the ability to decrease the expression levels of signal transducer and activator of transcription 3 (STAT3) in the liver, mammary fat pad, and tumor tissues of postmenopausal mice with obesity generated by a high-fat diet. The inhibitory effects of PE on the growth and movement of lung cancer cells induced by insulin were completely eliminated when STAT3 expression was removed. This suggests that the suppression of STAT3 expression is crucial for PE to exert its anti-cancer effects against lung cancer in the presence of high insulin levels. The findings of our study indicate that consuming PE is highly effective in preventing and treating lung cancer that is connected with obesity. This is achieved via regulating the signaling pathway of STAT3.

A nanobody against the V-ATPase c subunit inhibits metastasis of 4T1-12B breast tumor cells to lung in mice.

The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that functions to control the pH of intracellular compartments as well as to transport protons across the plasma membrane of various cell types, including cancer cells. We have previously shown that selective inhibition of plasma membrane V-ATPases in breast tumor cells inhibits the invasion of these cells in vitro. We have now developed a nanobody directed against an extracellular epitope of the mouse V-ATPase c subunit. We show that treatment of 4T1-12B mouse breast cancer cells with this nanobody inhibits V-ATPase-dependent acidification of the media and invasion of these cells in vitro. We further find that injection of this nanobody into mice implanted with 4T1-12B cells orthotopically in the mammary fat pad inhibits metastasis of tumor cells to lung. These results suggest that plasma membrane V-ATPases represent a novel therapeutic target to limit breast cancer metastasis.

The role of selenium intervention in gut microbiota homeostasis and gene function in mice with breast cancer on a high-fat diet.

This study aimed to investigate the effect of selenium on gut microbiota in mice with breast cancer under a high-fat diet. A total of 12 female BALB/c mice were randomly divided into two groups: 4 T1 + selenium+ high-fat diet group and 4 T1 + high-fat diet group. Mice were injected with 4 T1 cells on the right 4th mammary fat pad and kept on a high-fat diet. Fecal samples were collected, and DNA was extracted for metagenomic sequencing and bioinformatics analysis. Relevant target genes and pathways were annotated and metabolically analyzed to explore the intervention effect of selenium on breast cancer in the high-fat diet state. Selenium supplementation in the high-fat diet altered the composition and diversity of gut microbiota in mice with breast cancer. The gut microbial composition was significantly different in the selenium intervention group, with an increased abundance of Proteobacteria, Actinobacteria, and Verrucomicrobia phyla and species such as Helicobacter ganmani, Helicobacter japonicus, and Akkermansia muciniphila, while phyla, such as Bacteroidetes, Firmicutes, Deferribacteres, and Spirochaetes, and species, such as Prevotella sp. MGM2, Muribaculum intestinale, Lactobacillus murinus, and Prevotella sp. MGM1, were decreased. Functional analysis revealed differential expression of genes related to carbohydrate-active enzymes, pathogen-host interactions, cell communication, cell auto-induction, membrane transporters, and virulence factors. Furthermore, 37 COGs and 48 metabolites with rising metabolic potential in the selenium intervention group were predicted. Selenium alters the homeostasis of gut microbiota in mice with breast cancer on a high-fat diet, affecting their composition, abundance, and associated metabolism. These findings suggest that the mechanism involves interfering with gut microbiota homeostasis, leading to altered synthesis of tumor-associated proteins and fatty acids and inducing tumor cell apoptosis and pyroptosis.

A Novel Method for the Early Detection of Single Circulating, Metastatic and Self-Seeding Cancer Cells in Orthotopic Breast Cancer Mouse Models.

Metastasis is the main cause of cancer-related deaths, but efficient targeted therapies against metastasis are still missing. Major gaps exist in our understanding of the metastatic cascade, as existing methods cannot combine sensitivity, robustness, and practicality to dissect cancer progression. Addressing this issue requires improved strategies to distinguish early metastatic colonization from metastatic outgrowth. Luciferase-labelled MDA-MB-231, MCF7, and 4T1 breast cancer cells were spiked into samples from tumour-naïve mice to establish the limit of detection for disseminated tumour cells. Luciferase-labelled breast cancer cells (±unlabelled cancer-associated fibroblasts; CAFs) were orthotopically implanted in immunocompromised mice. An ex vivo luciferase assay was used to quantify tumour cell dissemination. In vitro luciferase assay confirmed a linear and positive correlation between cancer cell numbers and the bioluminescence detected at single cell level in blood, brain, lung, liver, and mammary fat pad samples. Remarkably, single luciferase-labelled cancer cells were detectable in all of these sites, as the bioluminescence quantified in the analysed samples was substantially higher than background levels. Ex vivo, circulating tumour cells, metastasis, and tumour self-seeding were detected in all samples from animals implanted with highly metastatic luciferase-labelled MDA-MB-231 cells. In turn, detection of poorly metastatic luciferase-labelled MCF7 cells was scarce but significantly enhanced upon co-implantation with CAFs as early as 20 days after the experiment was initiated. These results demonstrate the feasibility of using an ultrasensitive luciferase-based method to dissect the mechanisms of early metastatic colonization to improving the development of antimetastatic therapies.

Accumulation of liposomes in metastatic tumor sites is not necessary for anti-cancer drug efficacy

BackgroundThe tumor microenvironment is profoundly heterogeneous particularly when comparing sites of metastases. Establishing the extent of this heterogeneity may provide guidance on how best to design lipid-based drug delivery systems to treat metastatic disease. Building on our previous research, the current study employs a murine model of metastatic cancer to explore the distribution of ~ 100 nm liposomes. MethodsFemale NCr nude mice were inoculated with a fluorescently labeled, Her2/neu-positive, trastuzumab-resistant breast cancer cell line, JIMT-1mkate, either in the mammary fat pad to create an orthotopic tumor (OT), or via intracardiac injection (IC) to establish tumors throughout the body. Animals were dosed with fluorescent and radio-labeled liposomes. In vivo and ex vivo fluorescent imaging was used to track liposome distribution over a period of 48 h. Liposome distribution in orthotopic tumors was compared to sites of tumor growth that arose following IC injection. ResultsA significant amount of inter-vessel heterogeneity for DiR distribution was observed, with most tumor blood vessels showing little to no presence of the DiR-labelled liposomes. Further, there was limited extravascular distribution of DiR liposomes in the perivascular regions around DiR-positive vessels. While all OT tumors contained at least some DiR-positive vessels, many metastases had very little or none. Despite the apparent limited distribution of liposomes within metastases, two liposomal drug formulations, Irinophore C and Doxil, showed similar efficacy for both the OT and IC JIMT-1mkate models. ConclusionThese findings suggest that liposomal formulations achieve therapeutic benefits through mechanisms that extend beyond the enhanced permeability and retention effect.

Obesity Induces Temporally Regulated Alterations in the Extracellular Matrix That Drive Breast Tumor Invasion and Metastasis.

Obesity is associated with increased incidence and metastasis of triple-negative breast cancer, an aggressive breast cancer subtype. The extracellular matrix (ECM) is a major component of the tumor microenvironment that drives metastasis. To characterize the temporal effects of age and high-fat diet (HFD)-driven weight gain on the ECM, we injected allograft tumor cells at 4-week intervals into mammary fat pads of mice fed a control or HFD, assessing tumor growth and metastasis and evaluating the ECM composition of the mammary fat pads, lungs, and livers. Tumor growth was increased in obese mice after 12 weeks on HFD. Liver metastasis increased in obese mice only at 4 weeks, and elevated body weight correlated with increased metastasis to the lungs but not the liver. Whole decellularized ECM coupled with proteomics indicated that early stages of obesity were sufficient to induce changes in the ECM composition. Obesity led to an increased abundance of the proinvasive ECM proteins collagen IV and collagen VI in the mammary glands and enhanced the invasive capacity of cancer cells. Cells of stromal vascular fraction and adipose stem and progenitor cells were primarily responsible for secreting collagen IV and collagen VI, not adipocytes. Longer exposure to HFD increased the invasive potential of ECM isolated from the lungs and liver, with significant changes in ECM composition found in the liver with short-term HFD exposure. Together, these data suggest that changes in the breast, lungs, and liver ECM underlie some of the effects of obesity on triple-negative breast cancer incidence and metastasis. Significance: Organ-specific extracellular matrix changes in the primary tumor and metastatic microenvironment are mechanisms by which obesity contributes to breast cancer progression.

Reduced EO771-induced tumour growth and increased overall-survival of mice ablated for immune cell-specific catalytic subunit Cβ2 of protein kinase A

Ablation of the immune-specific catalytic subunit Cβ2 of protein kinase A is associated with a proinflammatory phenotype and increased sensitivity to autoimmunity in mice. Here we show that tumour growth of the adenocarcinoma cell line EO771 in the breast and in the lung after injection into the mammary fat pad and tail vein, respectively, was significantly reduced in mice ablated for Cβ2 compared to wild-type mice. In both cases, the breast and lung tumours showed increased infiltration of immune cells in the mice lacking Cβ2 compared to wild-type mice. Despite this, it appeared that solid tissue- versus intravenously injected EO771 cells evoked different immune responses. This was reflected by significantly increased levels of splenic proinflammatory immune cells and circulating cytokines in Cβ2 ablated mice carrying breast- but not the lung tumours. Moreover, Cβ2 ablated mice injected with EO771 cells showed increased overall survival compared to wild-type mice. Taken together, our results suggest for a role for immune cell-specific Cβ2 in protecting against tumour growth induced by EO771 cells in mice that is reflected in improved overall survival.

Targeting SIRT3 signaling alleviates lung carcinoma progression by reducing hyperinsulinemia in postmenopausal obese mice: Protective intervention of betaxanthin

Metabolic disturbances associated with obesity increase the risk and advancement of various tumor forms, including postmenopausal lung cancer, in humans. Betaxanthin, a type of yellow-orange pigment found in certain plants, particularly in some flowers, fruits, and vegetables, can be utilized to inhibit the growth of tumors and lessen the metabolic dysfunctions brought on by obesity. However, the functional role of betaxanthin (BET) in suppressing lung cancer progression and its potential mechanisms are still not fully understood. In this study, we examined the regulation of BET in obese mice with postmenopausal lung cancer receiving a high-fat diet (HFD). Initially, we found that BET could significantly mitigate the metabolic dysfunction that a high-fat diet causes in mice. Improved histological changes in the mammary fat pad, lower hepatic lipid deposition, and improved glucose tolerance and insulin resistance demonstrated this. Following that, BET treatment inhibited the formation of lung neoplasms in in-situ cancer animal models that were stimulated by HFD. Furthermore, in a postmenopausal mouse model fed HFD, BET significantly reduced the growth of pre-existing lung tumors, as well as attenuating metabolic abnormalities. Notably, in vitro research revealed that BET co-culture significantly reduced the proliferation and migration of mouse lung cancer cells, whereas insulin exposure increased these processes. Research on animals verified that lung cancer progressed due to hyperinsulinemia; however, BET treatment might inhibit this condition in postmenopausal mice on a high-fat diet. BET could up-regulate sirtuin 3 (SIRT3) expression levels in tumor, liver, and mammary fat pad tissues in postmenopausal mice with HFD-induced obesity, according to a bioinformatic and molecular biology study. Crucially, eradicating SIRT3 expression completely eliminated BET’s inhibitory effects on insulin-stimulated cancer cell proliferation and migration. This suggests that increasing SIRT3 expression may be required for BET to perform its anti-tumor function against lung cancer in conditions of hyperinsulinemia. Our findings showed that by modifying SIRT3 signaling, BET consumption is probably effective in the prevention and treatment of obesity-related lung cancer.

BUB1 regulates non-homologous end joining pathway to mediate radioresistance in triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer subtype often treated with radiotherapy (RT). Due to its intrinsic heterogeneity and lack of effective targets, it is crucial to identify novel molecular targets that would increase RT efficacy. Here we demonstrate the role of BUB1 (cell cycle Ser/Thr kinase) in TNBC radioresistance and offer a novel strategy to improve TNBC treatment.MethodsGene expression analysis was performed to look at genes upregulated in TNBC patient samples compared to other subtypes. Cell proliferation and clonogenic survivals assays determined the IC50 of BUB1 inhibitor (BAY1816032) and radiation enhancement ratio (rER) with pharmacologic and genomic BUB1 inhibition. Mammary fat pad xenografts experiments were performed in CB17/SCID. The mechanism through which BUB1 inhibitor sensitizes TNBC cells to radiotherapy was delineated by γ-H2AX foci assays, BLRR, Immunoblotting, qPCR, CHX chase, and cell fractionation assays.ResultsBUB1 is overexpressed in BC and its expression is considerably elevated in TNBC with poor survival outcomes. Pharmacological or genomic ablation of BUB1 sensitized multiple TNBC cell lines to cell killing by radiation, although breast epithelial cells showed no radiosensitization with BUB1 inhibition. Kinase function of BUB1 is mainly accountable for this radiosensitization phenotype. BUB1 ablation also led to radiosensitization in TNBC tumor xenografts with significantly increased tumor growth delay and overall survival. Mechanistically, BUB1 ablation inhibited the repair of radiation-induced DNA double strand breaks (DSBs). BUB1 ablation stabilized phospho-DNAPKcs (S2056) following RT such that half-lives could not be estimated. In contrast, RT alone caused BUB1 stabilization, but pre-treatment with BUB1 inhibitor prevented stabilization (t1/2, ~8 h). Nuclear and chromatin-enriched fractionations illustrated an increase in recruitment of phospho- and total-DNAPK, and KAP1 to chromatin indicating that BUB1 is indispensable in the activation and recruitment of non-homologous end joining (NHEJ) proteins to DSBs. Additionally, BUB1 staining of TNBC tissue microarrays demonstrated significant correlation of BUB1 protein expression with tumor grade.ConclusionsBUB1 ablation sensitizes TNBC cell lines and xenografts to RT and BUB1 mediated radiosensitization may occur through NHEJ. Together, these results highlight BUB1 as a novel molecular target for radiosensitization in women with TNBC.

A YAP-centered mechanotransduction loop drives collective breast cancer cell invasion

Dense and aligned Collagen I fibers are associated with collective cancer invasion led by protrusive tumor cells, leader cells. In some breast tumors, a population of cancer cells (basal-like cells) maintain several epithelial characteristics and express the myoepithelial/basal cell marker Keratin 14 (K14). Emergence of leader cells and K14 expression are regarded as interconnected events triggered by Collagen I, however the underlying mechanisms remain unknown. Using breast carcinoma organoids, we show that Collagen I drives a force-dependent loop, specifically in basal-like cancer cells. The feed-forward loop is centered around the mechanotransducer Yap and independent of K14 expression. Yap promotes a transcriptional program that enhances Collagen I alignment and tension, which further activates Yap. Active Yap is detected in invading breast cancer cells in patients and required for collective invasion in 3D Collagen I and in the mammary fat pad of mice. Our work uncovers an essential function for Yap in leader cell selection during collective cancer invasion.

Cold-inducible RNA-binding protein as a context-specific functional mediator of breast cancer metastasis.

3136 Background: Cold-inducible RNA-binding protein (CIRBP) is a stress-induced mRNA-binding protein associated with clinical outcomes in a variety of human disease states. CIRBP’s role as a prognostic biomarker in breast cancer (BC) has yet to be established. Methods: We describe a clinically annotated tissue micro-array cohort of 1406 hormone receptor positive (HR+) and 281 triple negative primary breast cancers (TNBC) stained by immunohistochemistry (IHC) for CIRBP. Statistical analyses were performed with the Kaplan-Meier estimator, as well as univariate and multivariate Cox proportional-hazards models. Multivariate models incorporated tumor size, lymph node status, grade and CIRBP expression levels. Co-primary endpoints were overall survival (OS) and progression-free survival (PFS). MDA-MB-231 TNBC cells and MDA-MB-361 HR+ cells were used for CIRBP knockout (KO) by CRISPR/Cas9, and subsequent mammary fat pad injections were performed in immunocompromised mice. Results: In N=281 primary TNBCs, high levels of CIRBP expression by IHC was associated with poor prognosis in multivariate analysis (OS: adjusted hazard ratio (aHR) 2.05, 95% confidence interval (CI) 1.24-3.41, P=0.005. PFS: aHR 2.46, 95% CI 1.33-4.57, P=0.004). However, in N=1406 HR+ primary BC, CIRBP expression was correlated with favorable prognosis (OS: aHR 0.927, 95% CI 0.88-0.98, P=0.05. PFS: aHR 0.904, 95% CI 0.85-0.96, P=0.002). CIRBP KO had minimal impact on mammary fat pad primary tumor growth in both MDA-MB-231 and MDA-MB-361 cell models. However, CIRBP KO resulted in the inhibition of efficient spontaneous metastasis to the liver and lungs in TNBC MDA-MB-231 cells but not HR+ MDA-MB-361 cells. Conclusions: CIRBP expression is associated with poor prognosis in TNBC but not HR+ BC patients, a finding validated with CRISPR/Cas9 KO in representative model systems. This finding highlights the prognostic significance of CIRBP in TNBC and suggests differential underlying mRNA targets bound and modulated by CIRBP in TNBC and HR+ BC, respectively.

Super-Resolution Ultrasound Imaging for Monitoring the Therapeutic Efficacy of a Vascular Disrupting Agent in an Animal Model of Breast Cancer.

Evaluate the use of super-resolution ultrasound (SRUS) imaging for the early detection of tumor response to treatment using a vascular-disrupting agent (VDA). A population of 28 female nude athymic mice (Charles River Laboratories) were implanted with human breast cancer cells (MDA-MB-231, ATCC) in the mammary fat pad and allowed to grow. Ultrasound imaging was performed using a Vevo 3100 scanner (FUJIFILM VisualSonics Inc) equipped with the MX250 linear array transducer immediately before and after receiving bolus injections of a microbubble (MB) contrast agent (Definity, Lantheus Medical Imaging) via the tail vein. Following baseline ultrasound imaging, VDA drug (combretastatin A4 phosphate, CA4P, Sigma Aldrich) or control saline was injected via the placed catheter. After 4 or 24 hours, repeat ultrasound imaging along the same tumor cross-section occurred. Direct intratumoral pressure measurements were obtained using a calibrated sensor. All raw ultrasound data were saved for offline processing and SRUS image reconstruction using custom MATLAB software (MathWorks Inc). From a region encompassing the tumor space and the entire postprocessed ultrasound image sequence, time MB count (TMC) curves were generated in addition to traditional SRUS maps reflecting MB enumeration at each pixel location. Peak enhancement (PE) and wash-in rate (WIR) were extracted from these TMC curves. At termination, intratumoral microvessel density (MVD) was quantified using tomato lectin labeling of patent blood vessels. SRUS images exhibited a clear difference between control and treated tumors. While there was no difference in any group parameters at baseline (0 hour, P > .09), both SRUS-derived PE and WIR measurements in tumors treated with VDA exhibited significant decreases by 4 (P = .03 and P = .05, respectively) and 24 hours (P = .02 and P = .01, respectively), but not in control group tumors (P > .22). Similarly, SRUS derived microvascular maps were not different at baseline (P = .81), but measures of vessel density were lower in treated tumors at both 4 and 24 hours (P < .04). An inverse relationship between intratumoral pressure and both PE and WIR parameters were found in control tumors (R2 > .09, P < .03). SRUS imaging is a new modality for assessing tumor response to treatment using a VDA.

The long noncoding RNA MANCR to promote and stabilize triple negative breast cancer.

e13150 Background: Cancer remains a leading cause of death worldwide, despite many advances in cancer treatment over the past decade. Triple Negative Breast Cancer (TNBC) is among the highest with a poor prognosis. Early diagnosis and modern treatment regimens for breast cancer (BC) have led to a high disease-free survival rate. Still, survival decreases significantly for patients with metastatic disease. Long non-coding RNAs are among a recent class of epigenetic regulators that function in the nucleus to support the stability of the cells and maintain the fidelity of chromatin interactions. Our laboratory discovered the long noncoding RNA MANCR (LINC00704) as being upregulated in human BC tumors and this is indicative of a worse survival rate than in patients with low MANCR-expressing tumors. MANCR is aberrantly expressed in TNBC cells, and these cells are highly dependent on MANCR to retain their tumorigenic characteristics. Methods: Our functional in vitro studies in MDA-MB-231 TNBC cells used GapmerRs to knockdown MANCR. For in vivo studies, TNBC cells were injected into the 4th mammary fat pad of mice, allowed initial tumor formation, and then treated the animals with 2 nmol/g of a negative control or MANCR targeting GapmeR. We also identified genome interaction sites at single nucleotide resolution by chromatin isolation by RNA purification (ChIRP)-seq to determine the mechanism of MANCR activity in TNBC cells. Results: We found that MANCR knockdown promotes DNA damage and decreases cell proliferation, migration, anchorage-independent colony formation, transwell invasion, and overall survival. Additionally, targeting MANCR in animals with existing tumors drastically inhibited tumor growth and the end-point tumor mass over time. The MANCR GapmeR treatment also inhibited the ability of TNBC cells to circulate and disseminate to distant organs. After performing ChIRP-seq in the MDA-MB-231 cells, we identified 988 genome-wide binding sites that exhibit MANCR interactions, of which 79% are intergenic and 21% are genic regions. Furthermore, 127 MANCR ChIRP peaks were found to overlap with fragile sites in the genome, indicating MANCR provides stability to these sites. Conclusions: These data suggest that targeting MANCR has therapeutic potential for patients with “MANCR-high” TNBC tumors by disrupting genome stability. Indeed, our in vivo studies demonstrate that “MANCR-high” TNBC tumors require MANCR to rapidly grow and promote disease progression. Lastly, many of the MANCR-chromatin interactions identified were found in intergenic regions and overlap with fragile sites within the genome. Collectively these data strongly indicate that MANCR stabilizes the TNBC genome, and disrupting genome stability by targeting MANCR has therapeutic potential.

Effect of AURKA targeting on nuclear PD-L1 and the efficacy of immune checkpoint inhibitors in triple-negative breast cancer.

e13153 Background: Aurora-A mitotic kinase (AURKA) plays a role in Triple Negative Breast Cancer (TNBC) progression through activation of epithelial to mesenchymal transition (EMT)-mediated cancer cell plasticity. Cancer cells that undergo EMT acquire a CD44high/CD24low and/or ALDHhigh cancer stem-like phenotype (Cancer Stem Cells) characterized by intrinsic drug resistance. Cancer Stem Cells (CSCs) shows high PD-L1 expression that induces tumor immune escape through CD8+ T-cells exhaustion. Significantly, “undraggable” nuclear PD-L1 may play a key role in induction of immune therapy resistance and cancer cell plasticity. Methods: (1) TNBC Models: MDA-MB 231, unique MDA-MB 231/LM Durvalumab resistant (DUV-R) and TNBC-M40 cells established from metastatic PDXs; (2) In Vitro Studies: TNBC cells were treated with the AURKA inhibitor alisertib (50nM). PD-L1 expression was evaluated by immunoblotting. Apoptosis was measured by immunofluorescence using Cleaved-PARP antibodies. To assess stemness capacity, TNBC cells were cultured under non-adherent conditions to form mammospheres. ALDH activity (functional stemness marker) was measured using the Aldefluor Kit. (3) In Vivo Studies: 1x106 MDA-MB 231/LM cells were injected into the mammary fat pad of humanized NSG-CD34+ female mice. Animals were treated with anti-PD-L1 ICI atezolizumab (20 mg/Kg) and alisertib (25 mg/Kg) as monotherapy or in combination. 1x106 MDA-MB 231/LM cells were also injected into the mammary fat pad of humanized NSG-CD34+ female mice and treated with durvalumab (20 mg/Kg) to develop durvalumab-resistant models. PD-L1, CD44, Vimentin expression and tumor infiltration of CD8+ T-cells were assessed by immunofluorescence. Results: Treatment of MDA-MB 231/LM xenografts with alisertib decreased the levels of CD44 and PD-L1 expression and increased CD8+ T-cells tumor infiltration. Because PD-L1 genetic targeting enhanced in vitro alisertib-induced apoptosis, MDA-MB 231/LM xenografts were treated with alisertib and the anti-PD-L1 ICI atezolizumab. Com- bination of atezolizumab and alisertib resulted in the reduced expression of the EMT marker vimentin that was linked to lack of organ metastasis. Combination of atezolizumab and alisertib also induced a significant reduction of ALDHHigh CSC in ex-vivo 3D-Organoids established from TNBC-M40 cells. MDA-MB 231/LM DUV-R cells showed increased nuclear PD-L1 compared to parental cells. The combination of alisertib with Immune Checkpoint Inhibitors (ICIs) induced apoptosis of MDA-MB 231/LM DUV-R cells through down-regulation of intra-tumoral nuclear PD-L1 expression. Conclusions: This study provides innovative pre-clinical rationale for combining AURKA inhibitors with ICIs to reduce “ undruggable” nuclear PD-L1 expression and impair cancer cell plasticity of metastatic TNBCs that currently have limited effective therapeutic options.

Prostate Specific Membrane Antigen Expression in a Syngeneic Breast Cancer Mouse Model

PurposeProstate specific membrane antigen (PSMA) has been studied in human breast cancer (BCa) biopsies, however, lack of data on PSMA expression in mouse models impedes development of PSMA-targeted therapies, particularly in improving breast conserving surgery (BCS) margins. This study aimed to validate and characterize the expression of PSMA in murine BCa models, demonstrating that PSMA can be utilized to improve therapies and imaging techniques.MethodsMurine triple negative breast cancer 4T1 cells, and human cell lines, MDA-MB-231, MDA-MB-468, implanted into the mammary fat pads of BALB/c mice, were imaged by our PSMA targeted theranostic agent, PSMA-1-Pc413, and tumor to background ratios (TBR) were calculated to validate selective uptake. Immunohistochemistry was used to correlate PSMA expression in relation to CD31, an endothelial cell biomarker highlighting neovasculature. PSMA expression was also quantified by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR).ResultsAccumulation of PSMA-1-Pc413 was observed in 4T1 primary tumors and associated metastases. Average TBR of 4T1 tumors were calculated to be greater than 1.5—ratio at which tumor tissues can be distinguished from normal structures—at peak accumulation with the signal intensity in 4T1 tumors comparable to that in high PSMA expressing PC3-pip tumors. Extraction of 4T1 tumors and lung metastases followed by RT-PCR analysis and PSMA-CD31 co-staining shows that PSMA is consistently localized on tumor neovasculature with no expression in tumor cells and surrounding normal tissues.ConclusionThe selective uptake of PSMA-1-Pc413 in these cancer tissues as well as the characterization and validation of PSMA expression on neovasculature in this syngeneic 4T1 model emphasizes their potential for advancements in targeted therapies and imaging techniques for BCa. PSMA holds great promise as an oncogenic target for BCa and its associated metastases.