Extended scRNA-seq analysis of mimicked cell states in the Pal and colleagues human breast tumor scRNA-seq dataset

Dedifferentiation programs are commonly enacted during breast cancer progression to enhance tumor cell fitness. Increased cellular plasticity within the neoplastic compartment of tumors correlates with disease aggressiveness, often culminating in greater resistance to cytotoxic therapies or augmented metastatic potential. In this study, we found that subpopulations of dedifferentiated neoplastic breast epithelial cells express canonical leukocyte cell surface receptor proteins and have thus named this cellular program "immune mimicry." Analysis of public human breast tumor single-cell RNA sequencing datasets and histopathologic breast tumor specimens, as well as functional experiments in vitro in breast cancer cell lines and in vivo in murine transgenic and cell line-derived mammary cancer models, showed that neoplastic cells engaged in immune mimicry. Immune-mimicked neoplastic cells harbored hallmarks of dedifferentiation and were enriched in treatment-resistant and high-grade breast tumors. In aggressive breast cancer cell lines, antiproliferative cytotoxic chemotherapies drove epithelial cells toward immune mimicry. The expression of the CD69 leukocyte activation protein by neoplastic cells conferred a proliferative advantage that facilitated early tumor growth. Together, these findings suggest that neoplastic breast epithelial cells upregulating leukocyte surface receptors potentiate malignancy and that neoplastic immune mimicry has potential clinical utility for patient prognosis and stratification.Significance: A subset of neoplastic breast epithelial cells express surface receptors canonically attributed to leukocytes and are associated with therapy resistance and aggressive tumor behavior.

Metastasis is a major cause of cancer-related mortality, yet targeting metastatic cells directly has been largely unsuccessful due to their plasticity and heterogeneity. Myeloid cells play diverse pro- and anti-metastatic functions and are an attractive alternative target for treating metastasis, but how tumor heterogeneity influences myeloid cell phenotypes during metastasis remains poorly understood. Here, we profiled myeloid cells from primary tumors and matched metastatic lungs of 12 heterogeneous and differentially metastatic patient-derived xenograft models of breast cancer. Comparative analysis of cell type abundances revealed distinct myeloid remodeling specific to primary tumors or metastatic lungs. Beyond compositional differences, we identified gene expression programs that were associated with metastatic burden, such as number or size of metastatic nodules, indicating distinct microenvironmental requirements for metastatic seeding and outgrowth. Examining these metastasis-associated programs using time-course datasets, we discovered an evolution from anti- to pro-metastatic monocyte phenotypes during metastatic progression. We further showed that this phenotypic shift was driven by an increase in two distinct myeloid-derived suppressor cell signatures, and a transcriptionally regulated impairment of monocyte differentiation leading to the depletion of non-classical monocytes. Our results comprehensively dissect the heterogeneity of myeloid cell phenotypes across primary tumor and metastatic sites, opening novel avenues for myeloid-targeting therapies specific to metastasis.

Extended data for MMTV-PyMT-CD69KO/KO experiments

Extended scRNA-seq analysis of mimicked cell states in the Wu and colleagues human breast tumor scRNA-seq dataset

A pro-tumorigenic role for adipocytes has been identified in breast cancer, and reliance on fatty acid catabolism found in aggressive tumors. The molecular mechanisms by which tumor cells coopt neighboring adipocytes, however, remain incompletely understood. Here, we describe a direct interaction linking tumorigenesis to adjacent adipocytes. We examine breast tumors and their normal adjacent tissue from several patient cohorts, patient-derived xenografts, and mouse models, and find that lipolysis and lipolytic signaling are activated in neighboring adipose tissue. We find that functional gap junctions form between breast cancer cells and adipocytes. As a result, cAMP is transferred from breast cancer cells to adipocytes and activates lipolysis in a gap junction-dependent manner. We find that connexin 31 (GJB3) promotes receptor triple negative breast cancer growth and activation of lipolysis in vivo. Thus, direct tumor cell-adipocyte interaction contributes to tumorigenesis and may serve as a new therapeutic target in breast cancer.

Dedifferentiation programs are commonly enacted during breast cancer progression to enable novel cellular phenotypes. Increased cellular plasticity within the neoplastic compartment of tumors correlates with disease aggressiveness, often culminating in greater resistance to cytotoxic therapies or the augmented ability to metastasize to distant organs. Here we report that subpopulations of dedifferentiated neoplastic breast epithelial cells express canonical leukocyte cell surface receptor proteins and have named this unique cellular program ‘immune mimicry.” We have documented neoplastic cells engaging in immune mimicry within public human breast tumor single-cell RNA-seq datasets, histopathological breast tumor specimens, breast cancer cell lines, as well as in murine transgenic and cell line-derived mammary cancer models. Immune-mimicked neoplastic cells harbor hallmarks of dedifferentiation and appear enriched in treatment-resistant and high-grade breast tumors. We corroborated these observations in aggressive breast cancer cell lines where growth-arresting cytotoxic chemotherapies drove epithelial cells toward immune mimicry. Moreover, in subsequent proof-of-concept studies, we demonstrate that expression of the CD69 leukocyte activation marker by neoplastic cells confers a proliferative advantage that enhances early tumor growth. We conclude that neoplastic breast epithelial cells upregulating leukocyte surface receptors potentiate malignancy. Moving forward, neoplastic immune mimicry should be evaluated for prognostic utility in breast cancer to determine whether it stratifies patients with increased risks for tumor recurrence and metastasis. Eric B. Berens, Sokchea Khou, Elaine Huang, Amber Hoffman, Briana Johnson, Nell Kirchberger, Sam Sivagnanam, Nicholas Calistri, Daniel Derrick, Tiera A. Liby, Ian C. McLean, Aryn A. Alanizi, E. Shelley Hwang, Pepper Schedin, Hugo Gonzalez, Zena Werb, Laura M. Heiser, Lisa M. Coussens. Neoplastic immune mimicry is a generalizable phenomenon in breast cancer and epithelial CD69 enables early tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB178.

Metastasis is the leading cause of cancer-related deaths. It is unclear how intratumor heterogeneity (ITH) contributes to metastasis and how metastatic cells adapt to distant tissue environments. The study of these adaptations is challenged by the limited access to patient material and a lack of experimental models that appropriately recapitulate ITH. To investigate metastatic cell adaptations and the contribution of ITH to metastasis, we analyzed single-cell transcriptomes of matched primary tumors and metastases from patient-derived xenograft models of breast cancer. We found profound transcriptional differences between the primary tumor and metastatic cells. Primary tumors upregulated several metabolic genes, whereas motility pathway genes were upregulated in micrometastases, and stress response signaling was upregulated during progression. Additionally, we identified primary tumor gene signatures that were associated with increased metastatic potential and correlated with patient outcomes. Immune-regulatory control pathways were enriched in poorly metastatic primary tumors, whereas genes involved in epithelial-mesenchymal transition were upregulated in highly metastatic tumors. We found that ITH was dominated by epithelial-mesenchymal plasticity (EMP), which presented as a dynamic continuum with intermediate EMP cell states characterized by specific genes such as CRYAB and S100A2. . Elevated expression of an intermediate EMP signature correlated with worse patient outcomes. Our findings identified inhibition of the intermediate EMP cell state as a potential therapeutic target to block metastasis.

Abstract Dedifferentiation programs are commonly enacted during breast cancer progression where they enable novel cellular phenotypes. Increased cellular plasticity within the neoplastic compartment of tumors correlates with disease aggressiveness, often manifesting as greater resistance to cytotoxic therapies or increased ability to metastasize to distant organs. Here we report that subpopulations of ostensibly stem-like neoplastic breast epithelial cells express canonical leukocyte cell surface receptor proteins, and have named this unique cellular program ‘immune mimicry’ (IM). We have documented neoplastic cells engaging in IM by examination of histopathological breast tumor specimens and their derived cell lines, public human breast tumor single-cell RNA-seq datasets, and in murine transgenic and cell line-derived mammary cancer models. Immune-mimicked neoplastic cells harbor hallmarks of dedifferentiation and appear enriched for developing aggressive and high-grade tumors. Experimental studies with breast cancer cell lines revealed that neoplastic cells expressing leukocyte surface features are elicited by growth-arresting conditions, such as conventional cytotoxic chemotherapeutic treatments and during metastatic dissemination in mice. In addition, using proof-of-concept studies, we leveraged the canonical leukocyte activation marker CD69, to demonstrate how its expression by neoplastic epithelial cells confers a proliferative advantage under low-density conditions. We conclude that neoplastic breast epithelial cells expressing leukocyte surface receptors may signify a particularly malignant and tumor-initiating cell state. Moving forward, neoplastic IM will be evaluated for prognostic utility in breast cancer to determine whether this unique cell state stratifies patients with increased risks for tumor recurrence and metastasis. The authors acknowledge NIH/NCI (K00 CA212132, T32 CA254888, P30 CA069533), the Collins Medical Trust, the Susan G Komen Foundation, and the National Foundation for Cancer Research for funding. Citation Format: Eric B. Berens, Sokchea Khou, Elaine Huang, Amber Hoffman, Briana Johnson, Zena Werb, Laura M. Heiser, Lisa M. Coussens. Neoplastic immune mimicry potentiates breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB224.

Supplementary Figure 4 from Matrix Metalloproteinases Contribute Distinct Roles in Neuroendocrine Prostate Carcinogenesis, Metastasis, and Angiogenesis Progression

Supplementary Figures 1-6 from CSN5 Isopeptidase Activity Links COP9 Signalosome Activation to Breast Cancer Progression

Supplementary Figure 1 from Matrix Metalloproteinases Contribute Distinct Roles in Neuroendocrine Prostate Carcinogenesis, Metastasis, and Angiogenesis Progression

Brain metastasis (BrM) is the most common form of brain cancer, characterized by neurologic disability and an abysmal prognosis. Unfortunately, our understanding of the biology underlying human BrMs remains rudimentary. Here, we present an integrative analysis of >100,000 malignant and non-malignant cells from 15 human parenchymal BrMs, generated by single-cell transcriptomics, mass cytometry, and complemented with mouse model- and in silico approaches. Specific single-cell interrogation of metastatic tumor cells provides a framework of 8 functional cell programs that coexist or anticorrelate. Collectively, these programs delineate two functional BrM archetypes, that co-exist within each metastatic tumor, one proliferative and the other inflammatory, that are evidently shaped through tumor-immune interactions. Our study provides a foundation to understand the molecular basis of BrM in patients with tumor cell-intrinsic and host environmental traits. For more details please refer to the original study: H. Gonzalez, et al. Cellular architecture of human brain metastases Cell, 185 (2022), pp. 729-745. Citation Format: Hugo Gonzalez, Wenbin Mei, Isabella Robles, Catharina Hagerling, Breanna Allen, Trine Hauge Okholm, Ankitha Nanjaraj, Tamara Verbeek, Sandhya Kalavacherla, Merel van Gogh, Stephen Georgiou, Mariza Daras, Joanna J. Phillips, Matthew H. Spitzer, Jeroen P. Roose, Zena Werb. Cell archetypes in human brain metastases: Connecting programs in tumor cells with the microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr IA022.

Supplementary Tables 1-6 from Matrix Metalloproteinases Contribute Distinct Roles in Neuroendocrine Prostate Carcinogenesis, Metastasis, and Angiogenesis Progression

PDF file - 117K, Microarray target genes from MCF7 siRNA. Target genes identified by microarray expression analysis from MCF7 cells Znf217 siRNA

PDF file - 12MB, Movie of SCp2 cells infected with vector (left movie) or Znf217 (right movie) following a scratch with a pipette tip. This movie ran for 20.25 hours