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Background The extracellular matrix (ECM) is a highly organized non-cellular network consisting of structural proteins, growth factors, cytokines and other secreted molecules. The ECM is highly dynamic, a critical player in regulation of local invasion and metastasis, and an important immunosuppressive component of the tumor microenvironment (TME). 1–4 However, the reciprocal effects of the immune system on the tumor-associated ECM are largely unexplored. Regulatory T (Treg) cells function to enforce peripheral tolerance and are potent suppressors of tumor immunity. We have previously shown that Treg cells promote tumor growth in murine breast cancer models by favoring alternative activation of tumor-associated macrophages (TAMs) via suppression of IFN-γ.5–8 Here, we investigate whether immune suppression mediated by Treg cells is linked to alterations in the ECM, and whether these changes have a functional role in promoting metastatic dissemination.Methods We performed histological comparisons of the ECM in models of breast cancer on control and Treg cell ablated tumors, 9 and functional ECM analysis through tumor decellularization and bioengineered 3D-chip models to evaluate EMT and collective tumor cell migration.10 We compared post-resection circulating tumor cells (CTCs) and spontaneous metastasis analysis, and compared the matrisome gene expression changes by bulk and single cell RNA-sequencing (scRNA-seq).11 Finally, we use bioinformatics to derive a Treg cell dependent matrisome signature and evaluate overall survival in clinical sample datasets.Results Treg cell ablation leads to changes in amounts of collagen, fibronectin, and laminin. Tumor cells seeded on tumor-derived decellularized ECM matrices results in reduced epithelial-mesenchymal transition transcription factor expression, and profound collective migration impairment. In vivo, Treg cell ablation in a neo-adjuvant setting followed by primary tumor resection resulted in significant reduction of CTCs and impairment of lung metastatic disease. Additionally, transcriptomic analysis showed a Treg cell-driven matrisome signature that correlates with long-term improved survival in a cohort of breast cancer patient samples. Through genetic knock-out models and in-vivo depletions, we showed Treg cell-dependent changes to ECM-related phenotypes are mediated through IFN-γ-signaling. scRNA-seq of murine breast tumors showed that the matrisome signature is highly upregulated in TAMs, compared to other TME cell types. Lastly, using conditional genetic knock-out models (IFNγ-R2flox LyzMCRE) we show the ECM-driven tumor cell migration changes are driven in large part through IFN-γ-dependent signaling in TAMs.Conclusions Altogether, we identify a novel metastasis-promoting effect of Treg cells in the breast cancer microenvironment through regulation of ECM dynamics that contributes to tumor cell dissemination, beyond their described effects on primary tumor growth.References Winkler J, Abisoye-Ogunniyan A, Metcalf KJ, Werb Z. Concepts of extracellular matrix remodelling in tumour progression and metastasis. Nat Commun. 2020;11:5120.Sutherland TE, Dyer DP, Allen JE. The extracellular matrix and the immune system: a mutually dependent relationship. Science. 2023;379. Shao X, Gomez CD, Kapoor N, Considine JM, Grams C, Gao YT, Naba A. MatrisomeDB 2.0: 2023 updates to the ECM-protein knowledge database. Nucleic Acids Res. 2023;51, D1519-D1530. Hynes RO, Naba A. Overview of the matrisome--an inventory of extracellular matrix constituents and functions. Cold Spring Harb Perspect Biol. 2012;4; a004903.Naba A, Clauser KR, Hoersch S, Liu H, Carr SA, Hynes RO. The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices. Mol Cell Proteomics. 2012;11:M111 014647.Bos PD, Plitas G, Rudra D, Lee SY, Rudensky AY. Transient regulatory T cell ablation deters oncogene-driven breast cancer and enhances radiotherapy. J Exp Med. 2013;210, 2435–2466.Clark NM, Martinez LM, Murdock S, deLigio JT, Olex AL, Effi C, Dozmorov MG.,Bos, P.D. Regulatory T Cells Support Breast Cancer Progression by Opposing IFN-gamma-Dependent Functional Reprogramming of Myeloid Cells. Cell Rep. 2020;33:108482.Munoz-Rojas AR, Mathis D. Tissue regulatory T cells: regulatory chameleons. Nat Rev Immunol. 2021;21:597–611.Bos PD. T(REG) Cells in Cancer: Beyond Classical Immunological Control. Immunol Invest. 2016; 45:721–728.Sanon S, Bos PD. In vivo imaging to measure spontaneous lung metastasis of orthotopically-injected breast tumor cells. J Vis Exp. 2022;184.Link PA, Pouliot RA, Mikhaiel NS, Young BM, Heise RL. Tunable hydrogels from pulmonary extracellular matrix for 3D cell culture. J Vis Exp. 2017;119.Hwang PY, Brenot A, King AC, Longmore GD, George SC. Randomly distributed K14(+) breast tumor cells polarize to the leading edge and guide collective migration in response to chemical and mechanical environmental cues. Cancer Res. 2019;79:899–1912.Ethics Approval Animal studies were conducted in concordance with Virginia Commonwealth University’s Division of Animal Research (DAR) and Institutional Animal Care and Use Committee (IACUC) approved protocol (AD10001219).