Breast Tumor Microenvironment Research Articles

Prospective evaluation of the breast microbiome and tumor microenvironment-related biomarkers of response to neoadjuvant systemic therapy in triple negative breast cancer.

TPS615 Background: Predictive biomarkers of response to combination chemotherapy and immune-checkpoint inhibitors are urgently needed to tailor treatment recommendations for patients with early-stage triple negative breast cancer (eTNBC). Our group has demonstrated that tumour-associated microbiota in primary breast tumours represent promising and novel candidate biomarkers for patients with breast cancer. We aim to prospectively interrogate the breast cancer microbiome and tumour microenvironment (TME) of eTNBC treated with neoadjuvant chemo-immunotherapy, and correlate with clinical outcome. Methods: Trial design: This prospective translational biomarker study is enrolling patients with eTNBC suitable for standard neoadjuvant systemic therapy followed by definitive surgery. The primary objective is to evaluate the change in breast cancer microbiome composition pre- and post-therapy. Secondary objectives include correlation of the breast microbiome with pathologic complete response (pCR) and survival outcomes, tumor infiltrating lymphocytes (TILs), PDL-1 and immune subset analysis, extracellular vesicles analysis and analysis of gut microbiome. Tumor tissue samples will be collected at baseline (mandatory research biopsy) and at the time of surgery. Microbiome evaluation will be conducted using metagenomic sequencing to assess changes in the relative abundance of microbial taxa. Blood and stool samples will be collected and analysed at baseline, on-treatment, at the time of surgery and post-operatively for secondary endpoints. Statistical analysis: Hierarchical clustering of samples based on relative abundance of the operational taxonomic units (OTUs) with sample composition at family and genus level will be performed. Alpha Diversity (Shannon Diversity Index) and Beta Diversity (Jaccard Similarity Index) will be analysed. Principal component analysis (PCA) and Random Forest analysis will be performed for classification and clustering analysis. Comparison of taxa or functions between clinical cohorts will be performed (two tailed Z test) and corrected using the false discovery rate to determine Q-values. Thirty patients will be recruited over 18-24 months. Current status: This University College Cork sponsored trial received ethics approval from the CREC in January 2024 and recruitment is ongoing. Those interested can contact uccctg@ucc.ie. This trial will provide a deeper insight into the potential role of the local breast microbiome as a predictive biomarker for response to neoadjuvant therapy in patients with eTNBC. The results will guide further studies exploring optimal immunomodulatory combinations for the treatment of TNBC and may provide evidence regarding future therapeutic targeting of the breast cancer microbiome.

Epigenetics, Microbiota, and Breast Cancer: A Systematic Review.

Breast cancer is the most frequently diagnosed cancer in women worldwide. According to recent studies, alterations in the microbiota and epigenetic modulations are risk factors for this disease. This systematic review aims to determine the possible associations between the intestinal and mammary microbial populations, epigenetic modifications, and breast cancer. To achieve this objective, we conducted a literature search in the PubMed, Web of Science, and Science Direct databases following the PRISMA guidelines. Although no results are yet available in humans, studies in mice suggest a protective effect of maternal dietary interventions with bioactive compounds on the development of breast tumors in offspring. These dietary interventions also modified the gut microbiota, increasing the relative abundance of short-chain fatty acid-producing taxa and preventing mammary carcinogenesis. In addition, short-chain fatty acids produced by the microbiota act as epigenetic modulators. Furthermore, some authors indicate that stress alters the gut microbiota, promoting breast tumor growth through epigenetic and gene expression changes in the breast tumor microenvironment. Taken together, these findings show the ability of epigenetic modifications and alterations of the microbiota associated with environmental factors to modulate the development, aggressiveness, and progression of breast cancer.

Exploring the interplay between triple-negative breast cancer stem cells and tumor microenvironment for effective therapeutic strategies.

Triple-negative breast cancer (TNBC)is a highly aggressive and metastatic malignancy with poor treatment outcomes. The interaction between the tumor microenvironment (TME)and breast cancer stem cells (BCSCs)plays an important role in the development of TNBC. Owing to their ability of self-renewal and multidirectional differentiation, BCSCs maintain tumor growth, drive metastatic colonization, and facilitate the development of drug resistance. TME is the main factor regulating the phenotype and metastasis of BCSCs. Immune cells, cancer-related fibroblasts (CAFs),cytokines, mesenchymal cells, endothelial cells, and extracellular matrix within the TME form a complex communication network, exert highly selective pressure on the tumor, and provide a conducive environment for the formation of BCSC niches. Tumor growth and metastasis can be controlled by targeting the TME to eliminate BCSC niches or targeting BCSCs to modify the TME. These approaches may improve the treatment outcomes and possess great application potential in clinical settings. In this review, we summarized the relationship between BCSCs and the progression and drug resistance of TNBC, especially focusing on the interaction between BCSCs and TME. In addition, we discussed therapeutic strategies that target the TME to inhibit or eliminate BCSCs, providing valuable insights into the clinical treatment of TNBC.

Laminin I mediates resistance to lapatinib in HER2-positive brain metastatic breast cancer cells in vitro

The role of extracellular matrix (ECM) prevalent in the brain metastatic breast cancer (BMBC) niche in mediating cancer cell growth, survival, and response to therapeutic agents is not well understood. Emerging evidence suggests a vital role of ECM of the primary breast tumor microenvironment (TME) in tumor progression and survival. Possibly, the BMBC cells are also similarly influenced by the ECM of the metastatic niche; therefore, understanding the effect of the metastatic ECM on BMBC cells is imperative. Herein, we assessed the impact of various ECM components (i.e., Tenascin C, Laminin I, Collagen I, Collagen IV, and Fibronectin) on brain metastatic human epidermal growth factor receptor 2 (HER2)-positive and triple negative breast cancer (TNBC) cell lines in vitro. The highly aggressive TNBC cell line was minimally affected by ECM components exhibiting no remarkable changes in viability and morphology. On the contrary, amongst various ECM components tested, the HER2-positive cell line was significantly affected by Laminin I with higher viability and demonstrated a distinct spread morphology. In addition, HER2-positive BMBC cells exhibited resistance to Lapatinib in presence of Laminin I. Mechanistically, Laminin I-induced resistance to Lapatinib was mediated in part by phosphorylation of Erk 1/2 and elevated levels of Vimentin. Laminin I also significantly enhanced the migratory potential and replicative viability of HER2-positive BMBC cells. In sum, our findings show that presence of Laminin I in the TME of BMBC cells imparts resistance to targeted therapeutic agent Lapatinib, while increasing the possibility of its dispersal and clonogenic survival.

CD163+ macrophages in the triple-negative breast tumor microenvironment are associated with improved survival in the Women’s Circle of Health Study and the Women’s Circle of Health Follow-Up Study

BackgroundTumor-associated macrophages (TAMs) are a prominent immune subpopulation in the tumor microenvironment that could potentially serve as therapeutic targets for breast cancer. Thus, it is important to characterize this cell population across different tumor subtypes including patterns of association with demographic and prognostic factors, and breast cancer outcomes.MethodsWe investigated CD163+ macrophages in relation to clinicopathologic variables and breast cancer outcomes in the Women’s Circle of Health Study and Women’s Circle of Health Follow-up Study populations of predominantly Black women with breast cancer. We evaluated 611 invasive breast tumor samples (507 from Black women, 104 from White women) with immunohistochemical staining of tissue microarray slides followed by digital image analysis. Multivariable Cox proportional hazards models were used to estimate hazard ratios for overall survival (OS) and breast cancer-specific survival (BCSS) for 546 cases with available survival data (median follow-up time 9.68 years (IQR: 7.43–12.33).ResultsWomen with triple-negative breast cancer showed significantly improved OS in relation to increased levels of tumor-infiltrating CD163+ macrophages in age-adjusted (Q3 vs. Q1: HR = 0.36; 95% CI 0.16–0.83) and fully adjusted models (Q3 vs. Q1: HR = 0.30; 95% CI 0.12–0.73). A similar, but non-statistically significant, association was observed for BCSS. Macrophage infiltration in luminal and HER2+ tumors was not associated with OS or BCSS. In a multivariate regression model that adjusted for age, subtype, grade, and tumor size, there was no significant difference in CD163+ macrophage density between Black and White women (RR = 0.88; 95% CI 0.71–1.10).ConclusionsIn contrast to previous studies, we observed that higher densities of CD163+ macrophages are independently associated with improved OS and BCSS in women with invasive triple-negative breast cancer.Trial registrationNot applicable.

CXCR6 expression correlates with radiotherapy response and immune context in triple-negative breast cancer (Experimental Studies)

Background: The chemokine receptor CXCR6 is critical for sustained tumor control mediated by CD8+ cytotoxic T cells (CTLs) in tumors. Previous studies have shown that ionizing radiation induces an inflamed immune contexture by upregulating CXCR6. However, the clinical significance of CXCR6 expression in triple-negative breast cancer (TNBC) and its correlation with radiotherapy remains unknown. This study aimed to clarify the prognostic value of CXCR6 and its role in the breast tumor microenvironment (TME). Methods: The mRNA and protein expression of CXCR6 in human triple-negative breast cancer and their association with survival were analyzed. The role of CXCR6 in the immune context was investigated using a combination of single-cell RNA sequencing, general transcriptome sequencing data and fluorescence-based multiplex immunohistochemistry (mIHC) techniques. Results: Elevated CXCR6 expression correlated with better clinical outcomes and superior response to adjuvant radiotherapy and immunotherapy in TNBC. CXCR6 fostered an immunostimulatory microenvironment characterized by upregulated cytotoxic markers. We also found that CXCR6 plays a crucial role in regulating the differentiation of CD8+ T cells and the intercellular communication of immune cell subtypes, shaping the anti-tumor microenvironment. Conclusions: This study highlights the emerging role of CXCR6 in shaping the antitumor immune microenvironment, and targeting CXCR6 may be a promising strategy for improving the effectiveness of radiotherapy and immunotherapy in TNBC.

Abstract PO1-14-09: Age-related remodeling of the systemic and breast microenvironment promotes a tumor-permissive locale for ER+ breast cancer in older women

Abstract Introduction: The peak incidence of ER+ breast cancer occurs in women around the age of 70. Compared to younger cohorts, we and others have shown that older patients with ER+ breast cancer have an enrichment of luminal disease and experience fewer recurrences, suggesting overtreatment. Differences in the biological phenotypes of tumors in older women remain poorly understood and treatment remains challenging as older patients are underrepresented in clinical trials. In this study, we hypothesized that aging alters both local and systemic hormones and inflammatory cytokines, creating a permissive environment for tumor formation and growth. To address this hypothesis, we employed a systems biology approach across multiple model systems and tissue samples. Methods: Our study included specimens from two cohorts of patients: (1) n = 90 women without breast cancer obtained from the Komen Tissue Bank, from whom we obtained matched plasma and non-cancerous breast tissue (n = 30 across age groups comprising of young donors (35-45yo), middle-aged donors (55-69yo), and older donors (≥ 70yo); and (2) n = 115 women with ER+ breast cancer (n = 25 young patients, n = 57 middle-aged patients, and n = 33 older patients) obtained from our institutional biobank, including matched plasma, tumor tissue, and tumor-adjacent tissue. From each of these sets of specimens, we measured estrogen disposition using mass spectrometry, a 22-plex panel of inflammatory markers, and transcriptomic changes using RNA-seq. Using scRNA-seq and multiplexed IHC, we further characterized the immune changes that occur with age. Lastly, we used an aged, carcinogen-induced ER+ tumor model in F344 rats (obtained from the NIA) to model tumor development and growth. Results: In the plasma, estradiol (E2) drastically decreased in post-menopausal women, leaving estrone (E1) as the predominant circulating estrogen in older women. However, the breast tumor microenvironment (TME) showed comparable levels of E1 and E2 across age groups. Multi-class concordance analysis of breast cancer tissue from RNA revealed significant increases in gene expression of the HSD17B7 enzyme and decreases in HSD17B2 enzyme across age, likely elevating local E1-to-E2 conversion and yielding high local E2 levels despite low circulating levels specifically in older women. In older patients with ER+ breast cancer, there was a significant increase in a chemokine network characterized by CCL2 and CXCL9 within the TME. scRNA-seq revealed enrichment of inflammatory M2-like macrophages in the aged TME. Immune dysfunction and decreased immunosurveillance in older patients manifested with widespread decreases in the presence of cytotoxic lymphocytes and decreased pathway enrichment of key immune pathways, such as JAK/STAT and IFNy signaling. mIHC analysis revealed decreases in CD4 and CD8 T cells and increases in M2-like macrophages in older patients in both tumor and stromal regions. Lastly, in the carcinogen-induced F344 rat model, aged rats (~ 24mo, human equivalent 60-70yo) had a shorter tumor-free interval than the younger rats (~ 4-6mo, human equivalent 20-30yo), likely due to increases in tumor-promoting inflammation and decreased immune surveillance. Ongoing mechanistic work is focused on macrophage-patient derived organoid co-cultures evaluating the functional relevance of E2, CCL2, and CXCL9 in shaping the aged TME. Conclusions: Our study of systemic and local breast hormones and cytokines in both healthy individuals and breast cancer patients showed that older women with ER+ breast cancer harbor different systemic and local environments compared to younger women. The aged TME is characterized by high E2, cytotoxic lymphocyte depletion, and immune dysfunction, creating a permissive environment for tumor formation. Future translational work should be aimed at chemo-preventative strategies to reduce the age-related chronic inflammation and immune dysfunction. Citation Format: Neil Carleton, Jian Zou, Sanghoon Lee, Dixcy Jaba Sheeba John Mary, Ruxuan Li, Jennifer Atkinson, Ziyu Huang, Hatice Osmanbeyoglu, Peter Lucas, Emilia Diego, Michael Lotze, George Tseng, Jagmohan Hooda, Ioannis Zervantonakis, Priscilla McAuliffe, Steffi Oesterreich, ADRIAN LEE. Age-related remodeling of the systemic and breast microenvironment promotes a tumor-permissive locale for ER+ breast cancer in older women [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 PO1-14-09.

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 PO3-07-01: Multiscale Computational Analysis of Patient-Matched Multi-Modal Imaging of Breast Tissue Microenvironment

Abstract Introduction: Mammographic breast density is a strong quantitative cancer risk factor, but its link to underlying tissue and cellular level changes are weakly understood. Understanding the complex restructuring changes in the breast tumor microenvironment and its impact on breast density is integral to improving risk assessment and treatment strategies. A novel approach was developed to stratify mammographic dense breast tissue into areas of active dense tissue and passive dense tissue. Active dense tissue is structurally reorganizing and links to cancer dynamics, whereas passive dense mammographic tissue remains organized.1,2 A complementary wavelet-based analysis technique to measure the multiscale anisotropy of collagen fibers from Second-Harmonic Generation (SHG) imaging demonstrated clinical cancer diagnostic potential in mouse carcinoma and human pancreatic cancer.3,4 This tool is now leveraged for a multi-modal study in which patient-matched H&E tissue biopsies are imaged using both bright-field microscopy and SHG imaging to compare with the patient’s corresponding mammograms. Stratifying the mammographic density into active and passive dense areas, a correlation between biopsy tissue anisotropy and mammographic density subtypes can be calculated to help understand how tumor microenvironment changes affect a patient’s mammographic breast tissue composition. Methods: SHG and brightfield-imaged biopsy slides from 10 patients (five malignant, five benign) collected from Maine Medical Center (Maine, US) were analyzed using the 2D Wavelet Transform Modulus Maxima (WTMM) Anisotropy Method generating a large-scale and a small-scale anisotropy factor as well as a scale-combined anisotropy calculated from their large- and small-scale anisotropy separation. A multi-modal anisotropy score was calculated by adding the imaged area’s H&E combined anisotropy score with its respective SHG combined anisotropy score. This analysis was done on eight randomly selected ductal areas for each imaging modality on a slide and correlated with the measured areas of density subtypes from the whole mammographic view. Linear regression was used for determining correlation between H&E and SHG anisotropy factors as well as with active and passive mammographic density subtype measurements. Results: The malignant patients’ small-scale H&E anisotropy factor positively correlated with their small scale SHG anisotropy factor (R2=0.571). No correlation was found for the benign patients (R2=0.011). The strongest correlations were found between benign patients’ mammographic passive (positive correlation) and active (negative correlation) dense tissues vs the H&E anisotropy scale-combined factor (R2=0.858 and R2=0.961, respectively). The largest malignant patient correlation was a negative relationship between SHG anisotropy scale-combined factor and overall mammographic density (inclusive of both active and passive dense tissue) (R2=0.891). The multi-modal anisotropy score correlated with overall mammographic density for both benign (positive correlation, R2=0.698) and malignant patients (negative correlation, R2=0.773). Combining the multi-modal anisotropy factor from SHG and H&E with the active dense tissue measurement using K-Means clustering, 5/5 malignant and 4/5 benign patients were correctly classified. Conclusion: Density in mammograms correlates with underlying quantitative changes in a patient’s tissue and collagen anisotropy. Combining both H&E and SHG analysis on tissue biopsy with active and passive density metrics from computational mammography leads to the best predictive potential than any one method on their own.

Abstract PO2-25-04: Anti-TIGIT upregulate CD226 on CD8+T cell as a potential therapeutic manner in HER2 positive breast cancer

Abstract Purpose: HER2 positive breast cancer subtype shows high malignancy, aggressive invasion, frequent recurrence, and low efficacy to PD-1 inhibitor though enriches in immune microenvironment. TIGIT is an immune checkpoint which expresses on T/NK cells and participates in immune evasion of cancer cells. Here, we investigated the effect of anti-HER2 and anti-TIGIT combination regarding to HER2 breast cancer treatment and tumor microenvironment, further exploring mechanisms behind the combination utilization. Methods: We re-analyzed the untreated human and mouse HER2 breast cancer scRNA-seq dataset to explore proportions of unique cell clusters in tumor immune microenvironment and communicated signals between immune cells and tumor cells. Mouse HER2 non-sensitive model and 27 non-pCR patients were utilized to investigate the variation of TIGIT and its ligand CD112/CD155 in HER2 non-sensitive population. Relevance between CD112/CD155 and prognosis was verified by 200 patients tissue microarray IHC. We validated the efficacy of combination of anti-TIGIT and anti-Neu and delineated scRNA-seq landscape via mouse model. Multilayer analysis including CellChat and AuCell were used to investigate difference of immune network between ctrl group, monotherapy and combination. In vitro and in vivo studies were used to verify the efficacy of anti-TIGIT and pivotal clusters. Results: We found that CD8+T cells occupied major proportion in TILs in both human and mouse HER2 breast cancer tumor immune microenvironment. CD8+T cells showed TIGIT signal to malignant epithelial cells instead of PD-1 signal. Increased CD8+TIGIT+T cells were related to poor prognosis in 200-patients cohort. In HER2 non-sensitive mouse model, significantly increased CD8+TIGIT+T cells were found in HER2 non-sensitive group. Multiplex IHF of 27 non-pCR patients also confirmed that CD8+TIGIT+T cells increased after treatment. CD112, ligand of TIGIT on malignant cells, demonstrated higher expression after anti-HER2 treatment in HER2 non-sensitive population and negatively correlated with prognosis. In scRNA-seq about control group, monotherapy and combination group, we defined malignant cells as sensitive subclone, Neu resistant subclone and others according to cell ratio variation in ctrl, monotherapy and combination group. CD112 upregulated in all subclones in monotherapy group. GSVA analysis indicated Neu resistant subclone had highest IFN-γ response among subclones. In Neu resistant subclone, combination group showed highest expression of MHC-I genes and upregulated IFN-γresponse, suggested the restored antigen presenting ability of Neu resistant subclone. Higher CD226, MHC-I and IFN-γ signals between malignant epithelial cells and CD8+T cells was found in combination group via CellChat. AuCell manifested upregulated T cell activation and IFN-γ production pathways in combination group. Public dataset showed favorable results. Increased CD226 expression and promoted cytotoxicity on CD8+T cells can only be induced by anti-TIGIT therapy in vitro and in vivo. 200-patients cohort indicated the predictive values of CD8+CD226+T cells in HER2 breast cancer. Conclusion: In conclusion, engaged immune checkpoint TIGIT shades malignant cells from CD8+T mediated immune surveillance in HER2 breast cancer. Disruption of TIGIT inhibit tumor growth in vivo by immune elimination of malignant cells. Besides, CD8+CD226+T cells induced by combined therapy can predict patient prognosis. Citation Format: Liyi Zhang, Qi Zhang, Zehao Wang, Zhibo Shao, Jingyan Xue, Yayun Chi, Bingqiu Xiu, Jiong Wu. Anti-TIGIT upregulate CD226 on CD8+T cell as a potential therapeutic manner in HER2 positive 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 PO2-25-04.

Abstract PO4-24-03: Adiponectin modulates cell polarity according to ERα expression in breast cancer cells

Abstract INTRODUCTION: Tumor microenvironment (TME) is a complex and heterogeneous network, consisting of stromal and immune cells, embedded with cytokines, growth factors, soluble receptors, and exosomes. Mutual and dynamic crosstalk among cancer, stromal and immune cells predispose cancer cells to metastasis. Particularly, the main component of breast TME is represented by adipocytes, which secretory activity is compromised in obesity. Indeed, hypertrophic, and hyperplastic adipocytes undergo to a modified cytokines secretory pattern, characterized by a reduced secretion of Adiponectin, now recognized as a crucial factor in the pathogenesis of breast cancer. LKB1 is one the most important effector of Adiponectin transduction pathways, and recently it has been demonstrated its role as ERα coactivator in promoting breast cancer cell growth. Moreover, LKB1 is a key regulator in controlling cell polarity along with E-cadherin and Cdc42. Cell polarity has been considered essential for mammary epithelial integrity, and disruption of individual polarity proteins is sufficient to induce tumor progression. The aim of the present study was to investigate the effects of adiponectin on the regulation and alteration of cell polarity, essential for breast cancer cell growth and invasiveness. METHODS: Immunofluorescence analyses were done to detect protein localization in breast cancer cells. Protein expression was performed by immunoblotting in the presence or absence of ERa and LKB1 siRNA. LKB1/Cdc42 and LKB1/E-cadherin interaction was detected by Proximity ligation and immunoprecipitation assay respectively. Wound healing assay was carried out to evaluate GM130 orientation. Growth and metastatic potential of breast cancer cells were assessed in vivo by orthotopic model and metastatic study respectively. RESULTS: In Adiponectin-treated MCF-7 cells LKB1 is mostly present in the nucleus, recruited as ERa-coactivator, while in BT20 cells LKB1 is localized with E-cadherin in the cytosolic compartment, suggesting its role in polarity. Our results showed that in MCF-7 cells Adiponectin increases Cdc42 level depending on LKB1 expression. Moreover, Cdc42 and LKB1 interact and colocalized in the nucleus of Adiponectin-treated MCF-7 cells. On the contrary, LKB1 and Cdc42 are located with E-cadherin in the cytosol of BT20 cells, addressing their cytosolic cooperation in maintaining cell polarity, as confirmed by an increased Cdc42 activity. Immunofluorescence analyses demonstrated a lack of Golgi realignment toward the front of the leading edge in MCF-7 cells treated with Adiponectin, not observed in BT20 cells. All this suggest a loss of cell polarity organization that predispose to cancer growth and progression, as confirmed by an increased tumor volume mass and a major number of lung metastatic events upon tail vein injection of adiponectin-treated ERα-positive cells with respect to untreated cells. CONCLUSION: In ERα-positive breast cancer cells Adiponectin modulates the expression of the main proteins involved in cell polarity, leading to increased growth, progression, and distant metastasis. Citation Format: Giuseppina Daniela Naimo, Martina Forestiero, Alessandro Paolì, Luca Gelsomino, Rocco Malivindi, Adele Elisabetta Leonetti, Francesca Giordano, Maria Luisa Panno, Loredana Mauro, Sebastiano Andò. Adiponectin modulates cell polarity according to ERα expression in breast cancer cells [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 PO4-24-03.

Nanoparticle Retinoic Acid-Inducible Gene I Agonist for Cancer Immunotherapy.

Pharmacological activation of the retinoic acid-inducible gene I (RIG-I) pathway holds promise for increasing tumor immunogenicity and improving the response to immune checkpoint inhibitors (ICIs). However, the potency and clinical efficacy of 5'-triphosphate RNA (3pRNA) agonists of RIG-I are hindered by multiple pharmacological barriers, including poor pharmacokinetics, nuclease degradation, and inefficient delivery to the cytosol where RIG-I is localized. Here, we address these challenges through the design and evaluation of ionizable lipid nanoparticles (LNPs) for the delivery of 3p-modified stem-loop RNAs (SLRs). Packaging of SLRs into LNPs (SLR-LNPs) yielded surface charge-neutral nanoparticles with a size of ∼100 nm that activated RIG-I signaling in vitro and in vivo. SLR-LNPs were safely administered to mice via both intratumoral and intravenous routes, resulting in RIG-I activation in the tumor microenvironment (TME) and the inhibition of tumor growth in mouse models of poorly immunogenic melanoma and breast cancer. Significantly, we found that systemic administration of SLR-LNPs reprogrammed the breast TME to enhance the infiltration of CD8+ and CD4+ T cells with antitumor function, resulting in enhanced response to αPD-1 ICI in an orthotopic EO771 model of triple-negative breast cancer. Therapeutic efficacy was further demonstrated in a metastatic B16.F10 melanoma model, with systemically administered SLR-LNPs significantly reducing lung metastatic burden compared to combined αPD-1 + αCTLA-4 ICI. Collectively, these studies have established SLR-LNPs as a translationally promising immunotherapeutic nanomedicine for potent and selective activation of RIG-I with the potential to enhance response to ICIs and other immunotherapeutic modalities.

A comprehensive single-cell breast tumor atlas defines epithelial and immune heterogeneity and interactions predicting anti-PD-1 therapy response

We present an integrated single-cell RNA sequencing atlas of the primary breast tumor microenvironment (TME) containing 236,363 cells from 119 biopsy samples across eight datasets. In this study, we leverage this resource for multiple analyses of immune and cancer epithelial cell heterogeneity. We define natural killer (NK) cell heterogeneity through six subsets in the breast TME. Because NK cell heterogeneity correlates with epithelial cell heterogeneity, we characterize epithelial cells at the level of single-gene expression, molecular subtype, and 10 categories reflecting intratumoral transcriptional heterogeneity. We develop InteractPrint, which considers how cancer epithelial cell heterogeneity influences cancer-immune interactions. We use Tcell InteractPrint to predict response to immune checkpoint inhibition (ICI) in two breast cancer clinical trials testing neoadjuvant anti-PD-1 therapy. Tcell InteractPrint was predictive of response in both trials versus PD-L1 (AUC= 0.82, 0.83 vs. 0.50, 0.72). This resource enables additional high-resolution investigations of the breast TME.

CAR-based immunotherapy for breast cancer: peculiarities, ongoing investigations, and future strategies.

Surgery, chemotherapy, and endocrine therapy have improved the overall survival and postoperative recurrence rates of Luminal A, Luminal B, and HER2-positive breast cancers but treatment modalities for triple-negative breast cancer (TNBC) with poor prognosis remain limited. The effective application of the rapidly developing chimeric antigen receptor (CAR)-T cell therapy in hematological tumors provides new ideas for the treatment of breast cancer. Choosing suitable and specific targets is crucial for applying CAR-T therapy for breast cancer treatment. In this paper, we summarize CAR-T therapy's effective targets and potential targets in different subtypes based on the existing research progress, especially for TNBC. CAR-based immunotherapy has resulted in advancements in the treatment of breast cancer. CAR-macrophages, CAR-NK cells, and CAR-mesenchymal stem cells (MSCs) may be more effective and safer for treating solid tumors, such as breast cancer. However, the tumor microenvironment (TME) of breast tumors and the side effects of CAR-T therapy pose challenges to CAR-based immunotherapy. CAR-T cells and CAR-NK cells-derived exosomes are advantageous in tumor therapy. Exosomes carrying CAR for breast cancer immunotherapy are of immense research value and may provide a treatment modality with good treatment effects. In this review, we provide an overview of the development and challenges of CAR-based immunotherapy in treating different subtypes of breast cancer and discuss the progress of CAR-expressing exosomes for breast cancer treatment. We elaborate on the development of CAR-T cells in TNBC therapy and the prospects of using CAR-macrophages, CAR-NK cells, and CAR-MSCs for treating breast cancer.

Zinc-Iron Bimetallic Peroxides Modulate the Tumor Stromal Microenvironment and Enhance Cell Immunogenicity for Enhanced Breast Cancer Immunotherapy Therapy.

Immunotherapy has emerged as a potential approach for breast cancer treatment. However, the rigid stromal microenvironment and low immunogenicity of breast tumors strongly reduce sensitivity to immunotherapy. To sensitize patients to breast cancer immunotherapy, hyaluronic acid-modified zinc peroxide-iron nanocomposites (Fe-ZnO2@HA, abbreviated FZOH) were synthesized to remodel the stromal microenvironment and increase tumor immunogenicity. The constructed FZOH spontaneously generated highly oxidative hydroxyl radicals (·OH) that degrade hyaluronic acid (HA) in the tumor extracellular matrix (ECM), thereby reshaping the tumor stromal microenvironment and enhancing blood perfusion, drug penetration, and immune cell infiltration. Furthermore, FZOH not only triggers pyroptosis through the activation of the caspase-1/GSDMD-dependent pathway but also induces ferroptosis through various mechanisms, including increasing the levels of Fe2+ in the intracellular iron pool, downregulating the expression of FPN1 to inhibit iron efflux, and activating the p53 signaling pathway to cause the failure of the SLC7A11-GSH-GPX4 signaling axis. Upon treatment with FZOH, 4T1 cancer cells undergo both ferroptosis and pyroptosis, exhibiting a strong immunogenic response. The remodeling of the tumor stromal microenvironment and the immunogenic response of the cells induced by FZOH collectively compensate for the limitations of cancer immunotherapy and significantly enhance the antitumor immune response to the immune checkpoint inhibitor αPD-1. This study proposes a perspective for enhancing immune therapy for breast cancer.

Abstract 6108: Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach

Abstract Women of African descent are at an increased risk of developing and dying from aggressive subtypes of breast cancer. A connection between aggressive disease and Western Sub-Saharan African ancestry has been postulated, but it remains largely unknown to what extent breast cancer in Africa is reminiscent of breast cancer in U.S. African American (AA) women who experience disproportionately high mortality rates. We performed ATAC- and RNA-sequencing on 9 human triple-negative breast cancer cell lines of U.S. origin and discovered that African ancestry influences the chromatin landscape, leading to disparate transcription factor (TF) activity and downstream gene expression patterns indicative of an aggressive tumor biology. Here, we describe an ambitious study that employs single-nucleus (sn) ATAC- and RNA-sequencing (snMultiome) of frozen breast tumors to characterize chromatin accessibility and gene expression patterns with single-cell resolution in AA (n=33), Kenyan (n=25), and European American (EA, n=24) women in relation to genetic ancestry, risk factor exposures, clinical characteristics, and 5-year survival. To achieve this, we successfully isolated intact, high-quality single nuclei from archival frozen breast tumor tissue through an optimized combination of enzymatic digestion and automated tissue homogenization. We performed snMultiome sequencing of 82 tumors using the 10x Genomics platform. Following filtering, normalization (SCT for snRNA; LSI for snATAC), peak calling (MACS2), and integration (Harmony), our dataset includes a total of 296,557 nuclei. Cancerous (163,419 nuclei) and non-cancerous (133,138 nuclei) cells were distinguished based on DNA copy number (CopyKat). Within the microenvironment, 11 major immune, epithelial, and stromal cell types were successfully annotated, exhibiting distinct patterns by population group (e.g. AA tumors showed markedly increased abundance of myeloid and T-cells, while Kenyan tumors showed increased abundance of pericytes and fibroblasts). A large number of enriched TFs within each cell type varied significantly by population group, suggesting distinct chromatin accessibility patterns related to genetic ancestry. Within cancerous cells, striking intra- and inter-tumoral heterogeneity was observed by genetic ancestry even within molecular subtype groups. Current efforts focus on in-depth molecular characterization of ancestry- and risk factor-related differences in the tumor epithelium and microenvironment and distinct signatures present in lethal disease. This project holds the potential to yield crucial insights into how ancestry or other factors may influence the etiology of different breast cancer subtypes, as well as produce clinically actionable biomarkers and therapeutic targets to enhance precision medicine within patient populations at high risk for aggressive disease. Citation Format: Alexandra R. Harris, Huaitian Liu, Brittany Jenkins-Lord, Tiffany H. Dorsey, Francis Makokha, Shahin Sayed, Gretchen Gierach, Stefan Ambs. Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach [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 6108.

Abstract 284: Crosstalk with postmenopausal obese mammary adipocytes impacts breast cancer cell metabolism and promotes tumorigenesis

Abstract Introduction: Obesity is associated with an increased risk and a poor prognosis of breast cancer (BC) in postmenopausal (PM) women. The mechanism(s) underlying obesity-related PM BC are not clearly understood. We hypothesized that the increased local presence of ‘obese’ mammary adipocytes within the breast tumor microenvironment (TME) promotes the acquisition of an invasive BC cell phenotype and markedly accelerates tumor proliferation and progression. Methods: We asked whether cancer-associated mammary adipocytes (CA-MAd) differ from normal mammary adipocytes (N-MAd) in their ability to promote tumorigenesis of dormant BC cells and if so, what the underlying mechanism(s) might be. Using a 2D transwell model, BC cells (ER- and ER+), were cocultured with obese PM CA-MAd isolated from breast cancer patients or obese mammary N-MAds from PM disease-free women and analyzed for migration, proliferation, phenotypic, signaling and metabolic alterations. Results: Compared to monoculture (MC) conditions, BC cells cocultured (CC) with CA-MAd displayed significantly increased migration and proliferation. Quantitative phase imaging indicated that CA-MAd secretome-treated BC cells displayed increased motility, speed, distance covered and cell size/area compared to N-MAd. Consistent with these results, mesenchymal markers such as fibronectin, vimentin and SNAIL were upregulated whereas the epithelial marker E-cadherin was downregulated in CC vs. MC conditions suggesting that Ads in the TME promote EMT in BC cells. Interestingly, mature Ads CC with BC cells underwent delipidation and dedifferentiation as indicated by reduced Oil Red staining, decreased lipid content and reduced expression of Ad maturation markers such as FABP4, ADIPOQ and PLIN1. Consistently, we determined that CC BC cells exhibited increased lipid uptake and BODIPY staining and upregulated pAMPK, pACC and CPT1A signaling when compared to MC BC cells. Short term treatment of BC cells with both N-MAd and CA-MAd secretomes resulted in increased oxygen consumption rate (OCR), basal respiration, ATP-linked respiration and proton-linked respiration compared to control and as determined by the Seahorse extracellular flux assay. CC with MAds significantly downregulated OCR and all respiration parameters in BC cells suggesting decreased glucose uptake and glycolytic flux. Conclusions: Compared to MC, CC with obese M-Ads promoted functional, phenotypic and metabolic changes in BC cells. Taken together, our results indicate that crosstalk between BC cells and obese MAds promotes delipidation of MAds and a concomitant lipid uptake in BC cells and a switch to lipid metabolism signaling pathways that may, in turn, drive a metabolic shift in BC cells and promote tumor growth. [Supported by NIH RO1CA185530, the Breast Cancer Research Foundation, the Karp Family Foundation and the Nile Albright Research Foundation] Citation Format: Roopali Roy, Rama Aldakhlallah, Stephen Cobbs, Emily Man, Caleb Hallinan, Kwonmoo Lee, Margaret Lotz-Bousvaros, Susan Pories, Marsha A. Moses. Crosstalk with postmenopausal obese mammary adipocytes impacts breast cancer cell metabolism and promotes tumorigenesis [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 284.

Abstract 2705: Hyaluronic acid impairs NK killing towards breast tumor spheroids despite elevated cytotoxicity

Abstract The tumor microenvironment (TME) is often regarded as a critical component in tumor progression and metastasis. Among the main structural components of the TME, such as collagen and glycosaminoglycans (GAGs), hyaluronic acid (HA) is highly elevated in the breast TME, and is associated with poor prognosis. In recent years, immunotherapy has gained tremendous success in the battle against hematological cancers. Yet, solid tumors have remained a significant clinical challenge for immunotherapy due to the physical constraints they impose on immune cells. Mechanically, our previous work has shown that the abundance of high molecular weight (HMW) HA significantly reduces matrix pore sizes and softens the collagen and HA cogel, and impairs breast tumor invasion. However, it is still unclear how modulating tumor invasion through the mechanics of the TME may correlate with the efficacy of immunotherapy. In this study, we aim to investigate the effect of HMW HA on natural killer (NK) cell-based immunotherapy using a tumor spheroid model (GFP-labeled MCF-7). We introduced NK-92 MI cells into tumor spheroid embedded collagen gels with or without HA, and followed spheroid-NK cell interactions through live cell imaging. Preliminary data shows that while tumor spheroids were still able to invade in the collagen only gel, they underwent apoptosis in the presence of NK cells. More importantly, the presence of HA within the ECM reduced NK cells’ ability to kill tumor spheroids. Specifically, when surrounded by NK cells, tumor spheroids remained intact and viable in the collagen-HA cogel, while they underwent apoptosis in collagen only gels. In a parallel experiment, multicolor flow cytometry data showed that HA increased NK activation and certain cytotoxic markers (interferon-gamma, perforin, and granzyme-B), despite the fact that spheroids appeared to be more resistant towards NK cells in the presence of HA. Taken together, we speculate that the presence of HMW HA in the TME may protect solid tumors from immune attacks by modulating the mechanical environment. Citation Format: Brian C. Cheung, Alan T. Li, Jeffrey E. Segall, Mingming Wu. Hyaluronic acid impairs NK killing towards breast tumor spheroids despite elevated cytotoxicity [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 2705.

Abstract 4217: An engineered breast tumor microenvironment model, with single-cell spatial resolution, to assess spatial dynamics of tumor evolution

Abstract Cellular heterogeneity is a prominent feature of the tumor microenvironment, and the organization of these cells has been linked to clinical outcomes such as disease progression and drug resistance. While retrospective analysis of patient tumors has produced a myriad of insights, samples are limited and highly complex making it challenging to validate causality. Here we present an engineered breast tumor microenvironment model, with single cell spatial resolution, to systematically identify which cell phenotypes and their spatial arrangements may be driving ductal carcinoma in situ disease progression. A microfluidic dispenser (Biopixlar, Fluicell) was optimized to enable the spatial patterning of single cells to replicate native histology. To demonstrate the high spatial precision of our method, we first replicated an annotated 2D section of a breast tumor biopsy region of interest (ROI) using MCF10A, MDA-MB-231, primary mammary fibroblasts, THP-1 derived macrophages, and primary mesenchymal stem cells. The XY coordinate of each cell was identified to create a print map matching the original biopsy. Deposited cells adhered to their intended target with an average print fidelity of 2.4 µm. Next, the stromal compartment of the ROI was altered to include phenotypes associated with tumor promoting microenvironments (i.e. cancer associated fibroblasts, M2 macrophages) and the core of the MCF10A ring filled with MDA-MB-231. Each deposited cell contained a fluorescent tag corresponding with its cell type to facilitate real time spatial tracking and annotation. The tumor microenvironments were live cell imaged for 24 hours at 5 minute intervals. MCF10A cells spread to form their junctions and create a cohesive ring, MDA-MB-231 cells proliferated and rapidly moved around within the confined space, and the cells of the stromal compartment spread to create a dense tissue like structure. The microenvironments were cultured for an additional 2-6 days, fixed, and stained for markers of proliferation, phenotype shifts, cell-cell junctions, and morphology. Unlocking the relative contributions of the specific cell types in ductal carcinoma in situ tumor microenvironment to the overall evolution of invasive breast cancer may hold the answer for many questions in early detection, treatment, and cancer avatar models. Citation Format: Haylie R. Helms, Kody A. Oyama, Alexander E. Davies, Ellen M. Langer, Luiz E. Bertassoni. An engineered breast tumor microenvironment model, with single-cell spatial resolution, to assess spatial dynamics of tumor evolution [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 4217.

Abstract 4167: Selective imaging of cancer-associated fibroblasts in the breast cancer microenvironment with near-infrared fluorescent probes

Abstract Tumors consist of a complex mixture of resident and infiltrating cells that can support their survival and evasion of treatment. Selective detection of genetically stable cells in the tumor microenvironment (TME) offers insight into tumor biology beyond the malignant cells, which are the focus of most molecular imaging approaches to cancer detection. Motivated by the goal of non-invasively understanding the primary breast TME, we have developed fluorescent molecular probes that target cancer-associated fibroblasts (CAFs), a major component of the stromal compartment of many tumors, and emit light in the near-infrared (NIR) spectral window, providing access to deeper tissues than visible probes. We synthesized and characterized a series of CAF-targeted antibody- and small molecule-based fluorescent imaging agents utilizing a highly hydrophilic NIR dye and ligands that target Fibroblast Activation Protein (FAP). FAP is a key cell-surface marker of breast cancer CAFs with an S1 phenotype, which are modulators of immunosuppression, metastatic spread, and are associated with worse outcomes, especially in HER2+ and triple-negative breast cancer. Modifications to the chemical structure of the linker and targeting ligands allowed us to alter the specificity and brightness of the CAF-probes in cellulo. Imaging of a co-implanted tumor model of SKBR3 human breast cancer cells and FAP expressing fibroblasts after administering our lead small-molecule CAF imaging agent revealed selective tumor targeting of CAF-rich tumors compared to cancer cell predominant tumors with favorable pharmacokinetics and tumor-to-background ratio (TBR) greater than 2. Similar imaging studies with MMTV-PyMT transgenic mice, an established murine model of breast cancer, showed analogous enhanced uptake in mammary tumors (TBR > 2) and clearance over 24 hours. NIR fluorescence also correlated with CAFs isolated from PyMT tumor tissue via flow cytometry, consistent with our hypothesis that this agent can specifically detect endogenous CAFs. This study identified a novel, bright, and water-soluble FAP-specific NIR agent for in vivo imaging, setting the stage for simultaneous optical imaging of malignant cells and activated fibroblasts in the TME to elucidate cancer-host interactions. Citation Format: Megan S. Michie, Junwei Du, Jiayu (Jennifer) Ye, Kathleen Duncan, Mikhail Y. Berezin, Samuel Achilefu. Selective imaging of cancer-associated fibroblasts in the breast cancer microenvironment with near-infrared fluorescent probes [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 4167.