Breast Cancer Microenvironment Research Articles

Abstract GS1-02: Racial disparity in tumor microenvironment and outcomes in residual breast cancer treated with neoadjuvant chemotherapy

Abstract Background: Black, compared to White women with localized breast cancer have higher mortality and worse distant recurrence free survival (DRFS). This has been attributed to social determinants of health and higher prevalence of triple negative breast cancer (TNBC) in Black compared to White women. Recent studies indicate that racial disparity in outcome is present in patients with estrogen receptor-positive (ER+), but not ER- disease, in particular in patients with residual disease after neoadjuvant chemotherapy (NAC). It has been shown that in some patients NAC may induce pro-metastatic changes in tumor microenvironment, such as increased density of tumor associated macrophages and portals for cancer cell dissemination to distant sites called TMEM doorways (TMEM score). TMEM score correlates with metastasis in patients with ER+/HER2- breast cancer. We hypothesized that racial disparity in DRFS in patients with residual ER+/HER2- disease is due to enhanced pro-metastatic components (macrophage and TMEM doorway density) in the tumor microenvironment post-chemotherapy in Black compared to White women. Methods: We performed a retrospective, multi-institutional study of TMEM score and macrophage density in the residual disease after NAC from 196 patients diagnosed with unilateral invasive ductal cancer of breast between 2004 and 2014. 99 patients self-identified as Black and 97 as White. TMEM doorways were visualized by triple immunohistochemistry for macrophages (CD68), tumor cells (panMena), and endothelial cells (CD31). The evaluation of TMEM score and macrophage density was done using automated image analysis. Tumor characteristics and patient survival were compared between Black and White patients. The relationship between TMEM score, macrophage density and DRFS was examined by log-rank test and multivariate Cox regression model. The covariates in Cox model included TMEM score, age (continuous), race (Black vs White), surgery type (mastectomy vs lumpectomy), tumor stage (T3 vs T1; T2 vs T1), lymph node status (positive vs negative), and tumor subtype (triple negative [TN] vs ER+/HER2-; other vs ER+/HER2-). Results: Black compared to White women were more likely to develop distant recurrence (49.5% vs 34%, p=0.04), receive mastectomy (69.7% vs 51.5%, p=0.014), and have higher grade (p=0.001). Tumors from Black patients had more macrophages and a higher TMEM score in the entire cohort (p=0.004; p=0.001 respectively) and in the ER+/HER2- subset (p=0.008; p=0.008 respectively), but not in the TNBC subset. High TMEM score was associated with worse DRFS in all patients (p=0.004) and in the ER+/HER2- (p=0.03), but not in TNBC. In multivariate Cox model, TMEM score was an independent prognostic factor in the entire cohort (HR, 1.92; 95%CI, 1.15-3.22; p=0.01) and trended towards significance in ER+/HER2- disease (HR, 2.13; 95%CI, 0.96-4.71; p=0.06). TN, compared to ER+/HER2- cancers had higher TMEM score (p=0.01), and macrophage density (p=0.001). Conclusion: Racial disparity in outcome in patients with localized breast cancer may be due to a more pronounced pro-metastatic response to chemotherapy in Black, compared to White patients with ER+/HER2- disease. Thus, higher prevalence of TNBC in Black patients may not be the controlling factor in racial disparity. Citation Format: Burcu Karadal, Gina Kim, Ved Sharma, Jessica Pastoriza, Isabelle Oktay, Yu Lin, Xianjun Ye, Jiyue Qin, Esther Cheng, Nurfiza Ladak, John Condeelis, Esther Adler, Paula Ginter, Timothy D’Alfonso, Xiaonan Xue, David Enterberg, Joseph Sparano, Maja Oktay. Racial disparity in tumor microenvironment and outcomes in residual breast cancer treated with neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr GS1-02.

Abstract P3-06-03: Trilaciclib induces immune changes within the tumor microenvironment in early-stage triple-negative breast cancer

Abstract Background: In early-stage triple-negative breast cancer (TNBC), there is accumulating evidence of a correlation between tumor-infiltrating lymphocytes in tumor tissue and favorable clinical outcomes, with a high CD8+/regulatory T-cell (Treg) ratio after neoadjuvant chemotherapy being predictive of overall survival and associated with pathologic complete response (Ladoire S, et al. Br J Cancer. 2011; Park YH, et al. Nat Commun. 2020). Trilaciclib is a transient inhibitor of cyclin-dependent kinase 4/6 that is administered intravenously prior to chemotherapy. In preclinical studies, trilaciclib has been shown to have immune-enhancing effects by differentially arresting CD8+ T-cell and Treg subsets, which is followed by the faster recovery of CD8+ T cells than Tregs in the tumor microenvironment. Methods: This phase 2, single-arm, open-label study aims to evaluate neoadjuvant, single-dose trilaciclib followed by trilaciclib plus dose-dense anthracycline/cyclophosphamide and taxane in patients with early-stage TNBC (NCT05112536). Patients with previously untreated, non-metastatic, confirmed TNBC and a primary tumor ≥ 1.5 cm of any nodal status receive a single dose of trilaciclib 240 mg/m2 during the lead-in phase, followed by 4 cycles of doxorubicin 60 mg/m2 plus cyclophosphamide 600 mg/m2, and 12 weekly cycles of paclitaxel 80 mg/m2. Trilaciclib 240 mg/m2 is administered prior to the first chemotherapy dose of each cycle. Pembrolizumab 400 mg every 6 weeks starting on day 1, cycle 1, and/or carboplatin AUC 1.5 every week starting on day 1, cycle 5, is allowed per investigator discretion. Tumor biopsies and peripheral blood samples are collected prior to any treatment, 7 days ± 1 day post administration of trilaciclib, and during surgery, with an additional blood sample collection on day 1, cycle 2. The primary objective is to evaluate the immune-based mechanism of action of trilaciclib after a single dose of trilaciclib, as measured by changes in the CD8+/Treg ratio in tumor tissue. Pathologic complete response, safety and tolerability, and additional exploratory immune biomarker endpoints will also be assessed. Results: As of June 3, 2022, 9 patients with early-stage TNBC had been enrolled and 8 patients had received the trilaciclib lead-in dose and initiated doxorubicin/cyclophosphamide. Patients had a median age of 53.0 years, and all had stage II tumors at diagnosis, with 7 having ductal carcinoma. The median number of chemotherapy cycles received was 3 (range 1–6), and all 8 patients received pembrolizumab. Seven patients continue study treatment; 1 patient discontinued due to disease progression. Five patients had an adverse event (AE) related to any study treatment, including 4 patients with ≥ 1 trilaciclib-related AE. There were no grade ≥ 3 treatment-related AEs or serious AEs. On-treatment, post-trilaciclib monotherapy biopsies were available for 4 patients. Following neoadjuvant trilaciclib treatment, the median density of stromal CD8+ T cells increased from 103.1/mm2 at baseline to 229.8/mm2 at day 7. The median CD8+/Treg ratio increased in 2 patients from 1.85 at baseline to 1.90 at day 7. Conclusions: Preliminary analysis of on-treatment tumor biopsies from 4 patients suggests that a single dose of trilaciclib may modulate the immune cell composition in the tumor microenvironment to support antitumor immune responses. The increase in CD8+ T cells following 7-day neoadjuvant treatment with trilaciclib supports previous data suggesting a role in T-cell infiltration. The complete dataset from all patients (estimated enrollment: N ≈ 24) and additional biomarker analyses will be presented. Citation Format: Michael Danso, Joyce O’Shaughnessy, Lisa S. Wang, Kailash Mosalpuria, Sara Hurvitz, Shom Goel, Sarah Ahn, Subing Cao, John S. Yi, Taofik Oyekunle, Amanda Jacobson, Andrew Beelen, Jeremy Force. Trilaciclib induces immune changes within the tumor microenvironment in early-stage triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-06-03.

Abstract P2-21-06: The relationship between the gene expression difference of fibroblasts in the tumor microenvironment and metastasis of triple-negative breast cancer

Abstract Background Triple negative breast cancer (TNBC) is a breast cancer subtype with increased risk of distant metastasis, especially to lung and liver. In addition to the intrinsic characteristics of tumor cells, the molecular features of the tumor microenvironment cells can also regulate the metastasis process of TNBC. In this study, we tried to characterize the transcriptomic features of microenvironment fibroblasts associated with the distant metastasis of TNBC. Method We established PDX models by obtaining tumor tissues from 26 triple-negative breast cancer patients and transplanting them into the mammary fat pads of immunodeficient mice. When the PDX tumors grew sufficiently in the fat pads, we excised them and saved the mice to assess whether each PDX tumor was capable of developing spontaneous distant metastasis. We used bulk RNA sequencing and single cell RNA sequencing to determine the transcriptomic features associated with the distant metastasis. Results We classified the 26 TNBC PDX models based on their in vivo metastasis capacity and the clinical outcomes of the corresponding patients. Non-metastatic PDX models were defined as the lack of developing distant metastasis in either mouse experiment or clinical follow-up data (n=5). PDX models were defined as metastatic models when they developed distant metastasis in both mouse and human (n=4). We performed single cell RNA sequencing using the nine PDX tumors and observed a substantial gene expression differences of fibroblasts in the tumor microenvironment between the metastatic and non-metastatic PDX models. The fibroblasts of the metastatic PDX models showed up-regulation of genes involved in the response to hypoxia, inflammatory response, neutrophil chemotaxis, cellular response to IL-1, positive regulation of cell proliferation, and negative regulation of apoptotic process. Moreover, this difference of gene expression between metastatic and non-metastatic TNBC PDX models were not identified by the bulk RNA sequencing data. Conclusion By using the single cell RNA sequencing using the TNBC PDX models, we identified key features of fibroblasts in the tumor microenvironment that contributes to the metastasis capacity of TNBC. Further studies are on-going to elucidate the underlying mechanisms. Citation Format: Woo Hang Heo, Woochan Lee, SONGBIN LI, Jong-Il Kim, Hyeong-Gon Moon. The relationship between the gene expression difference of fibroblasts in the tumor microenvironment and metastasis of triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-06.

Abstract P3-08-07: Self-renewal signatures of peripheral blood T cells are associated with successful engraftment to establish a humanized mouse model of breast cancer

Abstract Background: Stable humanized immune mice transplanted with peripheral blood mononuclear cells (PBMCs) derived from breast cancer patients are important models for assessing the tumor immune responses and the tumor immune microenvironment in breast cancer, helping to advance both pre-clinical and clinical research. The PBMCs of breast cancer patients exhibit various differences from those of healthy individuals depending on the stage of cancer progression, subtype, and type of treatment. Recent studies indicate an association of the self-renewal signatures of T cells with successful generation of a humanized mouse model; however, the optimal T cell signature for the successful generation of a humanized mouse model derived from cancer patient cells is poorly understood. Therefore, we examined the relationship between the signature of T cell subsets, focusing on the self-renewal signatures, in PBMCs derived from patients with breast cancer and the successful generation of a humanized immune mouse model. Materials and Methods: We collected PBMCs from 12 patients with breast cancer. All samples were stimulated with interleukin-2 and beads coated with CD3 and CD28 agonist antibodies to expand T cells. After washing, 1 × 10^7 cells/mouse were intraperitoneally injected into NOD/Shi-scid IL2rgamma (null) (NOG) mice. Transplants were performed on three mice per case. Successful engraftment of immune cells into NOG mice was defined by the presence of human CD45+ cells in one or more mice. Self-renewal signatures of in vitro expanded T cells before injection into NOG mice, including the markers T cell factor-1 (TCF-1), CD45RA, CCR7, CD95, and CXCR3, were determined using flow cytometry, mass cytometry and quantitative RT-PCR. Comparisons between groups of data were evaluated by t-test. Results: The success rate of engraftment of immune cells derived from breast cancer patients into NOG mice was 66.7% (8 out of 12 patients). After expansion, the magnitude of the CD8+ stem cell memory subset and the TCF-1 expression level on the CD4+ and CD8+ T cells in the engrafted group were significantly higher than those in non-engrafted group. TCF-1 and CCR7 mRNA levels in the engrafted group were upregulated compared with those in non-engrafted group. The success of engraftment was not associated with clinical characteristics such as breast cancer progression, subtype, or prior chemotherapy treatment. Conclusion: The self-renewal signatures of T cells from breast cancer patients were associated with successful engraftment of a humanized mouse model. These results suggest that prior identification of the self-renewal signatures of the T-cell population is of direct relevance to the appropriate design of a pre-clinical model for testing immunotherapies and for elucidating the characteristics of the tumor immune microenvironment. Citation Format: Yukiko Fukui, Kosuke Kawaguchi, Ryuji Murakami, Wei Li, Yuki Nakamura, Yurina Maeshima, Sunao Tanaka, Shinpei Kawaoka, Eiji Suzuki, Masakazu Toi. Self-renewal signatures of peripheral blood T cells are associated with successful engraftment to establish a humanized mouse model of breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-07.

Tspan6 stimulates the chemoattractive potential of breast cancer cells for B cells in an EV- and LXR-dependent manner

The immune microenvironment in breast cancer (BCa) is controlled by a complex network of communication between various cell types. Here, we find that recruitment of B lymphocytes to BCa tissues is controlled via mechanisms associated with cancer cell-derived extracellular vesicles (CCD-EVs). Gene expression profiling identifies the Liver X receptor (LXR)-dependent transcriptional network as a key pathway that controls both CCD-EVs-induced migration of B cells and accumulation of B cells in BCa tissues. The increased accumulation oxysterol ligands for LXR (i.e., 25-hydroxycholesterol and 27-hydroxycholesterol) in CCD-EVs is regulated by the tetraspanin 6 (Tspan6). Tspan6 stimulates the chemoattractive potential of BCa cells for B cells in an EV- and LXR-dependent manner. These results demonstrate that tetraspanins control intercellular trafficking of oxysterols via CCD-EVs. Furthermore, tetraspanin-dependent changes in the oxysterol composition of CCD-EVs and the LXR signaling axis play a key role in specific changes in the tumor immune microenvironment.

Abstract P1-08-05: Educating pathologists in quantitating stromal tumor-infiltrating lymphocytes in breast cancer for artificial intelligence applications

Abstract Background Immune cells in the tumor microenvironment play an important role in cancer development [1]. In triple negative breast cancer (TNBC), stromal tumor-infiltrating lymphocytes (sTILs) have been identified as a biomarker with both predictive and prognostic clinical values [2]. The High Throughput Truthing project collected sTILs density estimates in hematoxylin and eosin stained invasive breast cancer biopsy specimens. The goal of the project is to produce a dataset to validate artificial intelligence and machine learning models [3]. After collecting annotations from pathologists for a pilot study, we observed a high level of interobserver variability in sTILs density estimates. To improve pathologist accuracy in breast cancer sTILs assessment, we created educational materials using an expert panel and pilot study data. Method The pilot study data consisted of 640 unique regions of interest (ROIs) derived from 64 digital whole slide images. We categorized ROIs based on their mean sTILs density as “10% or less”, “11% to 40%”, or “greater than 40%”, and selected 72 unique ROIs from those with the highest and lowest pathologist variability in each density bin. In a series of eight one-hour sessions, each ROI was reviewed in a group setting by at least three members of our expert panel, which consisted of one clinical scientist and seven board-certified pathologists trained in breast cancer sTILs assessment. Experts provided estimates of the percent of tumor-associated stroma and sTILs density, and commentary on features that confound sTILs assessment for each ROI. Results We created a set of educational materials to teach the sTILs assessment methodology in breast cancer. These materials include an introduction to the clinical relevance of tumor infiltrating lymphocytes in the breast cancer microenvironment, a tutorial for assessing sTILs according to published guidelines [4], and a discussion of specific pitfalls that may be encountered. Expert panel annotations, comments, and pitfalls were used to generate a reference document and interactive tests: one with expert feedback on each ROI and one to determine proficiency. Conclusions Educational materials designed by an expert panel will serve as reference materials for learning sTILs assessment in breast cancer. Our work provides valuable education for pathologists, and directly supports their ability to provide up-to-date diagnostic information used in caring for breast cancer patients.

Abstract P2-26-01: Prospective breast biopsy collection at an urban safety-net hospital serving a diverse patient population

Abstract Background: Despite advances in breast cancer imaging, research and treatment, higher mortality among racial/ethnic minority and low-income populations persists. Consent and enrollment of diverse and vulnerable patients into breast cancer research is critical. Given this urgent need for diversity in breast cancer research, successful collection and banking of fresh biospecimens from diverse patients is key to ameliorating some of the cancer disparities. Given our access to a unique and vulnerable patient population, we developed and implemented a clinical information and biospecimen repository for patients evaluated in the breast radiology and breast oncology outpatient clinics at Boston Medical Center (BMC). Our goal was to consent patients for donation of percutaneous breast biopsy samples, surgical tissue, and blood to develop a breast cancer biospecimen bank. Our long-term goal is to provide high-yield human samples for translational research studies in breast cancer from a diverse and vulnerable patient population. Methods: We designed a multi-disciplinary team of clinical providers and basic science researchers to envision a breast cancer biospecimen bank for research. Key stakeholders were identified, workflow was arranged, IRB obtained, and research staff trained. Patients presenting with suspicious mass on breast imaging (BI-RADS 4C and 5 categories) were eligible to enroll. Two percutaneous biopsy cores were obtained for research at the time of ultrasound guided biopsy. We leveraged the Hematology Oncology translational research program at Boston Medical Center and pilot finding to launch the project. Results: Since initiation in April 2021, we approached 68 patients and consented 44, for a successful consent rate of 64.7%. Of the 44 patients consented, 22 (50.0%) identified themselves as Black/African American, 11 (25.0%) as White/Caucasian, 10 (22.73%) as Hispanic and 1 (2.27%) as Asian. Of all patients enrolled, 41 (93.2%) had a breast malignancy, of which 27 (70.7%) were hormone-sensitive and 7 (17.1%) were TNBC. Additionally, of the 44 patients on study, only 9 (20.4%) had commercial insurance or managed care plan on the day of the consent. In total, 84 percutaneous biopsy cores were collected from 42 patients. Corresponding surgical tissue, plasma, serum and PBMCs were also obtained. Conclusion: Our program demonstrates that the enrollment of diverse and vulnerable breast cancer patients onto cancer research is achievable. We successfully created a breast biopsy program to provide access to diverse human samples for breast cancer translational research studies. Currently this repository serves to address many ongoing translational projects to understand: 1) the gap in knowledge of inherent differences in tumor biology and tumor environment, 2) metabolic regulation in the breast cancer microenvironment, and 3) heterogeneity of tumors and its effects on clinical outcomes and microenvironment interactions. Citation Format: Adrian Ilinski, Kiana Mahdaviani, Michael Fishman, Michael Cassidy, Naomi Ko. Prospective breast biopsy collection at an urban safety-net hospital serving a diverse patient population [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-01.

Abstract P2-11-19: Inflammatory indexes as prognostic biomarkers in advanced triple negative breast cancer patients

Abstract Background The identification of treatment selection biomarkers for advanced triple negative breast cancer (aTNBC) patients remains an unmet need. The immune system is known to be involved in the microenvironment of triple negative breast cancer (TNBC). Immune ratios like the neutrophil-to-lymphocyte ratio (NLR), the platelet-to-lymphocyte ratio (PLR), the monocyte-lymphocyte ratio (MLR) and the systemic immune-inflammation index (SII) may reflect the immune system functional status in these patients and the involvement of circulating immune cells in cancer progression. In particular MLR showed to predict overall survival (OS) in aTNBC and to contribute to migration of circulating tumor cells [1]. Methods A retrospective-prospective, observational multicenter study from the GIM-14 experience was performed to investigate the association between inflammatory indexes (NLR, MLR, PLR and SII), as measured at baseline and at progression, and clinical and survival aspects in patients with aTNBC in first line setting. The optimal cutoff values between low and high expression of inflammatory indexes were established on the basis of cut-off values determined in a previously conducted study in order to validate them[1], and was used to predict progression-free survival (PFS) and OS. Time-to-event variables (PFS; OS) were calculated using Kaplan-Meier method, while Cox regression model was used to estimate HRs and their 95% CI. Results 105 consecutive patients with a diagnosis of aTNBC were fully evaluated for the final analysis. The median age at diagnosis was 55 years (33-86). Of them, 80 patients received a neo/adjuvant treatment after diagnosis. At first progression the majority of patients (n= 97) received chemotherapy, while only 8 patients were treated with chemo-immunotherapy due to the programmed death ligand 1 (PD-L1) expression in the immunohistochemical analyses. At a median follow-up of 54 months, median PFS in the whole patients population was 13 months (95% CI 9,4-16,5), while median OS was 17.3 OS (95% CI 13-22,2). All high inflammation based scores evaluated at diagnosis of metastatic disease were significantly associated with lower PFS, in particular high NLR (≥3) and high MLR (≥0.34) (p = 0.0006 and p = 0.011, respectively). Similarly, all high indexes appeared significantly associated with a lower OS, especially NLR (>3), SII (≥836) and MLR (≥0.34) (p < 0.0001, p = 0.0005, p=001 respectively). In particular also NLR and SII evaluated at disease progression after first line treatment were significantly associated with a worse OS (p=0.0006 and p=0.001 respectively). In multivariable analysis for predictors of overall survival, the number of metastatic sites, NLR, SII and MLR remained significant (p< 0.0001, p=0.006, p=0.005 respectively). Conclusions NLR, SII and MLR are predictors of OS in aTNBC. Although our results need validation with larger studies, we suggest that inflammatory ratios could be used as feasible biomarkers of prognosis and treatment efficacy in aTNBC. Further research about HER-2 low categorized patients will be updated and presented at the final meeting. 1. De Giorgi U, Mego M, Scarpi E, Giordano A, Giuliano M, Valero V, Alvarez RH, Ueno NT, Cristofanilli M, Reuben JM. Association between circulating tumor cells and peripheral blood monocytes in metastatic breast cancer. Ther Adv Med Oncol. 2019 Aug 14;11:1758835919866065. doi: 10.1177/1758835919866065. PMID: 31452692; PMCID: PMC6696837. Citation Format: Caterina Gianni, Michela Palleschi, Emanuela Scarpi, Filippo Merloni, Eva Blondeaux, Fabio Puglisi, Elena Collovà, Palma Pugliese, Francesco Cognetti, Irene Giannubilo, Tommaso Ruelle, Claudia Bighin, Lucia Del Mastro, Ugo De Giorgi. Inflammatory indexes as prognostic biomarkers in advanced triple negative breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-19.

Abstract P6-14-01: Invasively distinct subpopulations cooperate via a laminin-332/Rac1 axis in triple-negative breast cancer

Abstract Intratumoral heterogeneity poses a significant hurdle for cancer treatment, yet is under-characterized in the context of tumor invasion. Cancer cells from solid tumors can invade through two predominant modes: collective invasion, whereby cancer cells invade in multi-cellular packs or streams marked by intact cell-cell junctions; and single-cell invasion, whereby cells invade independently without intercellular adhesion. We have observed that collective and single-cell invasion co-occur within the same tumor microenvironment in triple-negative breast cancer, suggesting that invasive heterogeneity supports cooperative behavior between tumor subpopulations. To test this, we used a novel, published technique developed by the lab (SaGA) to isolate pure subpopulations of 4T1 cells that collectively invade (collectives) or single cells that invade alone (singles). 3-D spheroids of SaGA-purified collectives and singles exhibited almost exclusively collective and single-cell invasion, respectively, and these invasive phenotypes were retained over multiple passages. Integration of RNA sequencing and methylation array data obtained from RNA and DNA isolates, respectively, of collectives and singles revealed that collectives exhibit drastic overexpression and promoter hypomethylation of two laminin genes that form the laminin-332 complex, Lama3 and Lamc2. Additionally, an unbiased proteomic analysis of secreted proteins also revealed an overabundance of these laminins in collectives media. We found that singles have increased expression of integrin α6 and β4, which together have been found to specifically bind to laminin-332 to activate the Rac1 GTPase. Interestingly, our RNA sequencing data revealed a binary overexpression of a Rac1 GTPase, Prex1 in singles, suggesting that singles have enhanced Rac1 activation when compared to collectives with the potential for hyperactivation upon laminin-332 binding. Indeed, laminin-332 resulted in higher GTP-bound Rac1 in singles and subsequently increased invasion of singles in 3-D models. Additionally, laminin-332 induced cell elongation at the leading edge of singles spheroids, which was reversable by treatment with a Rac1 inhibitor. Together, our data suggests that distinct subpopulations amidst a heterogeneous tumor cooperate via laminin-332 and Rac1 to facilitate tumor invasion in metastatic triple-negative breast cancer. Citation Format: Sung Bo (Joseph) Yoon, Janna Mouw, Luxiao Chen, Hao Wu, Adam Marcus. Invasively distinct subpopulations cooperate via a laminin-332/Rac1 axis in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-01.

Effect of metabolism on the immune microenvironment of breast cancer.

Breast cancer (BC) is a highly prevalent primary malignancy worldwide with poor prognosis. Despite the development of aggressive interventions, mortality due to BC remains high. BC cells reprogram nutrient metabolism to adapt to the energy acquisition and progression of the tumor. The metabolic changes in cancer cells are closely related to the abnormal function and effect of immune cells and immune factors, including chemokines, cytokines, and other related effector molecules in the tumor microenvironment (TME), leading to tumor immune escape, whereby the complex crosstalk between immune cells and cancer cells has been considered the key mechanism regulating cancer progression. In this review, we summarized the latest findings on metabolism-related processes in the immune microenvironment during BC progression. Our findings showing the impact of metabolism on the immune microenvironment may suggest new strategies for regulating the immune microenvironment and attenuating BC through metabolic interventions.

Abstract P4-09-07: Characterization of Immune Contexture in HR+/HER2- and Triple Negative Breast Cancer in a Real-world Cohort

Abstract Background: Prior studies in breast cancer (BC) have demonstrated variability in the immune microenvironment of BCs across subtypes, with higher levels of immune infiltration observed in triple-negative (TN) BC. Clinical trials of immunotherapy in BC have largely targeted patients with TNBC, however many of these patients do not respond to treatment, suggesting heterogeneity across TN tumors. Further, immune infiltrated hormone receptor–positive, HER2-negative (HR+/HER2-) tumors have been observed. Patients with these tumors may benefit from immune checkpoint blockade (ICB). Identification of patients with immunogenic tumors would be valuable for future patient selection and stratification in clinical trials. In this study, we conducted comprehensive molecular profiling of a set of commercially procured BC samples to characterize the tumor microenvironment (TME) of HR+/HER2- and TN tumors and to identify patient or tumor features associated with immune infiltration. Methods: 163 surgically resected BC samples were used for this study (93 HR+/HER2-, 70 TN). Clinically validated immunohistochemistry (IHC) assays were used to evaluate the expression of two immune checkpoint (IC) molecules, PD-L1 (by VENTANA PD-L1 (SP142) Assay and Agilent PD-L1 IHC 28-8 pharmDx) and LAG3 (antibody clone 17B4). The presence of CD8+ cells was measured by IHC (Dako/Agilent clone C8/144B) and used to derive the spatial location and density (topology) of CD8+ cells within the TME. RNA sequencing (RNA-seq) was used to construct gene expression signatures representative of immune cell types. Furthermore, RNAseq data was used to identify expressed somatic mutations and to calculate the total expressed tumor mutational burden (‘eTMB’). Results: As previously reported, the prevalence of PD-L1 + tumors was higher in TN vs HR+/HER2− BC, however close to half (44.1% SP142 IC> 1%) of HR+/HER2- tumors showed some degree of PD-L1 expression. Similar patterns were observed for LAG3 IHC expression (32.3% and 64.7% of HR+ and TN tumors respectively with >1% expression). Concordant expression of the two markers (SP142 IC> 1% and LAG3 >=1% or < 1% and < 1%) was observed in approximately 80% of tumors in both subtypes. Less than 10% of tumors in both subtypes had high LAG3 (>1%) and low PD-L1(< 1%) expression. Most (82.4%) HR+/HER2- tumors in the dataset were deficient in CD8+ cells in both stroma and tumor parenchyma, however a subset (12.9%) were identified as having an ‘excluded’ CD8 pattern, and four (4.7%) were classified as ‘inflamed’. The frequency of both the excluded and inflamed states was slightly higher among the TN tumors (20.6% and 15.9% respectively). Expression of PD-L1+ occurred more frequently among tumors with higher CD8 expression. Among both TN and HR+ tumors, most ‘inflamed’ tumors were grade 3. We also examined a range of gene expression signatures representative of various immune cell subpopulations, their distribution within each subtype, and relationships with other molecular data. For example, expression of a CXCL13+ T cell signature, a cell type that has been shown to be predictive of response to anti-PD-L1 + chemo combo therapy in TNBC, was significantly higher in TN vs HR+ tumors, however, a large subset of HR+ tumors had levels of expression of the signature above the median observed in TN tumors. In addition, we noted that eTMB, while low overall, was positively correlated with certain immune-related expression signatures across HR+/HER2− samples, including signatures previously associated with improved response to ICB, as well as PD-L1 positivity. Conclusions: We observed heterogeneity in the immune profile of the real-world HR+ and TN tumors in this cohort. A subset of tumors in both subtypes expressed markers or signatures previously reported to be associated with response to ICB. (1) Cancer Cell. 2021 Dec 13;39(12):1578-1593.e8. Citation Format: Aparna Chhibber, Lloye M. Dillon, John B. Wojcik, Vishantie Dostal, George Lee, Fayaz Seifuddin, Scott Ely, Mark D. Stern, Charlie Benson-Garnett, Mustimbo Roberts, Jenny Wu. Characterization of Immune Contexture in HR+/HER2- and Triple Negative Breast Cancer in a Real-world Cohort [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-07.

Abstract P2-21-08: Digital spatial profiling of RNA and protein in the invasive lobular breast cancer microenvironment

Abstract Invasive lobular breast cancer (ILC) is an understudied subtype of breast cancer with late recurrence, metastasis to serosal surfaces, such as the peritoneum, and dismal long-term outcome. We previously reported better patient outcome in ER+ breast cancers with high glucocorticoid receptor (GR) expression (Pan et al 2011). Preliminary data suggests more favorable patient outlook in the ILC subtype when tumors express higher levels of GR. The dynamic interaction between a tumor and its microenvironment leads to phenotypic changes in stromal cells and the extracellular matrix to promote growth or invasion of malignant cells. ILC is histologically distinct from invasive ductal carcinoma and is characterized by discohesive tumor cells that grow as “Indian files” due to lack of the cell adhesion molecule E-cadherin. Because of these differences we expect the tumor microenvironment (TME) to be quite unique in ILC compared to other breast cancer subtypes. We hypothesized that differing levels of expression of nuclear receptors by tumor cells would impact cells residing within the stroma, presumably through paracrine signaling. Immunohistochemistry for 25 ILC biopsies revealed a wide variation in GR expression, ranging from completely negative to strongly positive. Our goal was to gain insight into how GR presence or absence in ILC cells might impact gene and protein expression in malignant cells and their TME. We were inspired to examine how crosstalk between the GR-positive or GR-negative cancer cells and their respective tumor microenvironment (TME) differentially impact stromal cell gene expression as well as the immune cell milieu. Bulk expression profiling is inadequate to examine molecular profiles of the tumor and stroma separately. Therefore, we used nanoString GeoMx® high-plex digital spatial profiling of RNA and protein expression in GR-positive and GR-negative primary ILC. We first performed spatial gene expression using the Whole Transcriptome Atlas (WTA) for six primary ILC, two of which were strongly positive for GR, two GR-negative and 2 tumors harboring both GR-positive and GR-negative regions. To examine the tumor cells and the stroma independently we segmented regions into panCK-positive and panCK-negative segments and analyzed those separately. Intriguing differences were observed between GR-positive and negative ILC, for the tumor cells as well as for the TME. Implementation of the Spatial Deconvolution script embedded within the GeoMx® software revealed striking differences in the abundance of immune cells, endothelial cells and fibroblasts in the stroma of GR-positive vs GR-negative ILC. Macrophages and other myeloid subsets were present in significantly higher numbers in the TME of GR-positive compared to GR-negative ILC. The contribution of B- and T-cell subsets as well as endothelial cells and fibroblasts were also notably different. To more precisely define the spatial distribution and abundance of various cell subtypes and their association with tumor cells, we are including protein DSP as an added layer to complement the WTA. We are also performing RNA and protein DSP for an additional ten GR-positive and ten GR-negative ILC. Thus, we will test our initial observations and expand our investigation to generate comprehensive molecular profiles of ILC tumors and stromal cells and acquire a deeper understanding of how GR expression/activation influences the crosstalk between ILC cells and their TME. We anticipate that differences in gene and protein expression will provide clues as to how GR activation affects proliferative and adhesive properties of ILC through modification of the TME. Citation Format: Lynda B. Bennett, Candace Frerich, Sunati Sahoo, Cheryl Lewis, Indu Raman, Min Xu, Guanchun Chen, Suzanne D. Conzen. Digital spatial profiling of RNA and protein in the invasive lobular breast cancer microenvironment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-08.

Abstract PD9-03: Breast Cancer Microenvironment Change After Neoadjuvant Endocrine Treatment Breast Cancer Microenvironment Change After Neoadjuvant Endocrine Treatment

Abstract Background: Estrogen receptor positive (ER+)/HER-2 negative breast cancer (BC) is considered to be an immunologically cold tumor compared to triple negative breast cancer. Therefore, the tumor microenvironment (TME) of ER+/HER-2 negative BC is understudied. The receptor activator of nuclear factor-kB ligand (RANKL)-RANK pathway was first identified, as mediator of T and dendritic cells interaction, but it is mostly known for its role as key regulator of bone remodeling and pathophysiology of bone metastases. RANK is a member of the tumor necrosis factor receptor (TNFR) superfamily that is activated upon RANKL binding, promoting cell proliferation, survival and differentiation. The RANKL-RANK pathway also emerged as a major mediator of hormone-driven breast carcinogenesis. The aim of this study is to investigate the TME and the immune response during neoadjuvant endocrine therapy (NET) and to correlate this with the treatment response in a real life setting. Methods: Expressions of immune checkpoint receptors and immune cells were examined immunohistochemically in pre- and post- NET on a cohort of 44 ER+/HER-2 negative BC patients. They were treated with tamoxifen (N=8), an aromatase inhibitor (N=36) or a combination of an aromatase inhibitor with a PI3K inhibitor (N= 7) for a median duration of 6 months (range 1-32) months. Monoclonal antibodies for PDL-1, PD-1, TIM-3, LAG-3, CTLA-4, CD4, CD68, FOXP3, RANK and RANKL were used. All staining were done according to validated protocols and scoring was done by a pathologist specialized in breast cancer. Positivity was defined as staining > 1% on TILs. Response to NET was evaluated according to tumor size change on imaging and Ki-67 change. Results: The median age was 62.5 (44–90.3) years. Diameter of tumor size decreased with a mean of 7.818 mm (p < 0.0001) during NET and the value of Ki-67 value decreased significantly after NET (value, p< 0.0004). An increase in PD-L1 expression after NET showed a trend towards significant (p= 0.088) and RANK expression on TILs significantly decreased with a median of 30% (range= -70 to 85) (p= 0.0007) after NET. A good response to NET defined as a decrease in tumor size and/or decrease of Ki-67 was found to be associated with a longer duration of NET, a change of CD4+ T-cells, a change of RANK expression on TILs and a higher number of CD68+ tumor-associated macrophages before the start of NET and also RANK expression on TILs before the start of NET. Conclusion: The immune micro-environment plays an important role in ER+/HER-2 negative BC. NET influences the composition and/or functional state of the infiltrating immune cells. Furthermore, changes in the immune micro-environment are also associated with treatment response. Breast Cancer Microenvironment Change After Neoadjuvant Endocrine Treatment Comparison of continuous and categorical parameters before and after NET. Comparison of the continuous parameters was done using Wilcoxon signed rank test. Comparison of the categorical parameters was done using a Chi-square test. sTIL: stromal tumour infiltrating lymphocytes and NET: neoadjuvant endocrine therapy. Bold values denote statistical significance at the p < 0.05 level. Citation Format: Gizem Oner, Glenn Broeckx, Christophe Van Berckelaer, Sevilay Altintas, Zafer Canturk, Wiebren Tjalma, Karen Zwaenepoel, Zwi Berneman, Marc Peeters, Patrick Pauwels, Peter A van Dam. Breast Cancer Microenvironment Change After Neoadjuvant Endocrine Treatment Breast Cancer Microenvironment Change After Neoadjuvant Endocrine Treatment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-03.

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

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

Obesity-mediated upregulation of the YAP/IL33 signaling axis promotes aggressiveness and induces an immunosuppressive tumor microenvironment in breast cancer.

Obesity is a well-known risk factor for breast cancer formation and is associated with elevated mortality and a poor prognosis. An obesity-mediated inflammatory microenvironment is conducive to the malignant progression of tumors. However, the detailed molecular mechanism is still needed to be clarified. Herein, we identified that breast cancer cells from mice with diet-induced obesity exhibited increased growth, invasiveness, and stemness capacities. A transcriptome analysis revealed that expressions of interleukin 33 (IL33) signaling pathway-related genes were elevated in obesity-associated breast cancer cells. Importantly, IL33 expression was significantly associated with the yes-associated protein (YAP) signature, and IL33 was transcriptionally regulated by YAP. Suppression of IL33 reduced tumor migration and invasion, while the addition of IL33 activated nuclear factor (NF)-κB signaling and revived tumor mobility in YAP-silenced cells. Furthermore, suppression of YAP attenuated IL33 expression which was accompanied by relief of obesity-mediated immunosuppression. Clinical analyses showed that IL33 expression was markedly associated with macrophage and regulatory T cell infiltration. These findings reveal a crucial role of the YAP/IL33 axis in promoting aggressiveness and immunosuppression of obesity-associated breast cancer progression.

The usage of melittin in breast cancer microenvironment targeting

In today’s society, there are more people dying from breast cancer than ever. In fact, it has been projected that 1 in 39 women will die from breast cancer. Over the years, scientists have developed many forms of treatment to help alleviate some of the symptoms and keep the cancer at bay, such as chemotherapy and immunotherapy. Recently, however, a discovery has been made that could have the most efficacy and effectiveness against breast cancer. Melittin, a cationic peptide found in bee venom, is a 26 amino acid peptide that is being studied in many fields of medicine to be used as a new form of treatment in conjunction with the current forms. It is being tested in cancer research because of a special function that it has: the ability to start apoptosis by punching holes in the actual cell itself. In this paper, we will look at an overview of breast cancer’s history and statistics, look through some of the types of treatments that are already being used to fight breast cancer, and analyze five case studies to see how melittin can be used to target the microenvironments of breast cancer cells and figure out which method of treatment melittin will best fit into.

Dynamic NIR Fluorescence Imaging and Machine Learning Framework for Stratifying High vs. Low Notch-Dll4 Expressing Host Microenvironment in Triple-Negative Breast Cancer.

Delta like canonical notch ligand 4 (Dll4) expression levels in tumors are known to affect the efficacy of cancer therapies. This study aimed to develop a model to predict Dll4 expression levels in tumors using dynamic enhanced near-infrared (NIR) imaging with indocyanine green (ICG). Two rat-based consomic xenograft (CXM) strains of breast cancer with different Dll4 expression levels and eight congenic xenograft strains were studied. Principal component analysis (PCA) was used to visualize and segment tumors, and modified PCA techniques identified and analyzed tumor and normal regions of interest (ROIs). The average NIR intensity for each ROI was calculated from pixel brightness at each time interval, yielding easily interpretable features including the slope of initial ICG uptake, time to peak perfusion, and rate of ICG intensity change after reaching half-maximum intensity. Machine learning algorithms were applied to select discriminative features for classification, and model performance was evaluated with a confusion matrix, receiver operating characteristic curve, and area under the curve. The selected machine learning methods accurately identified host Dll4 expression alterations with sensitivity and specificity above 90%. This may enable stratification of patients for Dll4 targeted therapies. NIR imaging with ICG can noninvasively assess Dll4 expression levels in tumors and aid in effective decision making for cancer therapy.

Pathological features and immune microenvironment in HER-2 intratumoral heterogeneous breast cancers

Objective: To observe the clinical pathology features, and immune microenvironment of HER-2 intratumoral heterogeneity breast cancer. Methods: Thirty cases of HER-2 intratumoral heterogeneous breast cancer were retrospectively analyzed in Tianjin Medical University Cancer Institute and Hospital from November 2017 to June 2020. HER-2 expression was detected by immunohistochemistry and verified by dual color silver-enhanced in-situ hybridization (D-SISH). HER-2 intratumoral positive and negative regions were divided. The pathological characteristics, subtype, and the level of tumor infiltrating lymphocytes (TILs) and the expression of programmed cell death-ligand 1 (PD-L1) were evaluated respectively. Results: The proportion of HER-2 positive cells of the breast cancer ranged from 10% to 90%. The pathological type was mainly invasive non-special typecarcinoma. Six cases presented different pathological types between HER-2 positive and negative regions. The HER-2-positive areas included 2 cases of carcinoma with apocrine differentiation, and the negative areas included 2 cases of invasive micropapillary carcinoma, 1 case of invasive papillary carcinoma, and 1 case of carcinoma with apocrine differentiation. In HER-2 positive regions, 17 cases were Luminal B and 13 cases were HER-2 overexpressed types. There were 22 cases of Luminal B and 8 cases of triple negative tumors in the HER-2 negative areas. The levels of TILs in HER-2 positive and negative areas accounted for 53.3% (16/30) and 26.7% (8/30), respectively, with a statistically significant difference (P=0.035). The positive expression of PD-L1 in HER-2 positive area and HER-2 negative area were 6 cases and 9 cases, respectively. Among 8 cases with HER-2 negative regions containing triple negative components, 4 cases were positive for PD-L1 expression. Conclusions: In the case of HER-2 intratumoral heterogeneity, it is necessary to pay attention to both HER-2 positive and negative regions, and evaluate subtype separately as far as possible. For HER-2 intratumoral heterogeneous breast cancer containing triple negative components, the treatment mode can be optimized by refining the intratumoral expression of PD-L1.

Metabolic Reprogramming of Breast Tumor-Educated Macrophages Revealed by NMR Metabolomics

The metabolic crosstalk between tumor cells and tumor-associated macrophages (TAMs) has emerged as a critical contributor to tumor development and progression. In breast cancer (BC), the abundance of immune-suppressive TAMs positively correlates with poor prognosis. However, little is known about how TAMs reprogram their metabolism in the BC microenvironment. In this work, we have assessed the metabolic and phenotypic impact of incubating THP-1-derived macrophages in conditioned media (CM) from two BC cell lines cultured in normoxia/hypoxia: MDA-MB-231 cells (highly metastatic, triple-negative BC), and MCF-7 cells (less aggressive, luminal BC). The resulting tumor-educated macrophages (TEM) displayed prominent differences in their metabolic activity and composition, compared to control cells (M0), as assessed by exo- and endometabolomics. In particular, TEM turned to the utilization of extracellular pyruvate, alanine, and branched chain keto acids (BCKA), while exhibiting alterations in metabolites associated with several intracellular pathways, including polyamines catabolism (MDA-TEM), collagen degradation (mainly MCF-TEM), adenosine accumulation (mainly MDA-TEM) and lipid metabolism. Interestingly, following a second-stage incubation in fresh RPMI medium, TEM still displayed several metabolic differences compared to M0, indicating persistent reprogramming. Overall, this work provided new insights into the metabolic plasticity of TEM, revealing potentially important nutritional exchanges and immunoregulatory metabolites in the BC TME.

CD1C is associated with breast cancer prognosis and immune infiltrates

BackgroundThe tumor microenvironment (TME) in breast cancer plays a vital role in occurrence, development, and therapeutic responses. However, immune and stroma constituents in the TME are major obstacles to understanding and treating breast cancer. We evaluated the significance of TME-related genes in breast cancer.MethodsInvasive breast cancer (BRCA) samples were retrieved from the TCGA and GEO databases. Stroma and immune scores of samples as well as the proportion of tumor infiltrating immune cells (TICs) were calculated using the ESTIMATE and CIBERSORT algorithms. TME-related differentially expressed genes (DEGs) were analyzed by a protein interaction (PPI) network and univariate Cox regression to determine CD1C as a hub gene. Subsequently, the prognostic value of CD1C, its response to immunotherapy, and its mechanism in the TME were further studied.ResultsIn BRCA, DEGs were determined to identify CD1C as a hub gene. The expression level of CD1C in BRCA patients was verified based on the TCGA database, polymerase chain reaction (PCR) results, and western blot analysis. Immunohistochemical staining (IHC) results revealed a correlation between prognosis, clinical features, and CD1C expression in BRCA. Enrichment analysis of GSEA and GSVA showed that CD1C participates in immune-associated signaling pathways. CIBERSORT showed that CD1C levels were associated with tumor immune infiltrating cells (TILs), such as different kinds of T cells. Gene co-expression analysis showed that CD1C and the majority of immune-associated genes were co-expressed in BRCA. In renal cell carcinoma, patients with a high expression of CD1C had a better immunotherapy effect.ConclusionCD1C is an important part of the TME and participates in immune activity regulation in breast tumors. CD1C is expected to become a prognostic marker and a new treatment target for breast cancer.