Abstract 6582: HD-PTP modulates anti-tumor immunity via extracellular vesicles in triple-negative breast cancer

Abstract The tumor immune microenvironment (TIME) is a critical driver for the progression of triple-negative breast cancer (TNBC), directly impacting patient prognosis and treatment response. One emerging mechanism underlying immune evasion in TNBC involves extracellular vesicles (EVs), which act as carriers of immunosuppressive factors (ISFs) that shape the immune landscape. Here, we identify HD-PTP, an ESCRT-associated protein encoded by the PTPN23 gene, as a key regulator of EV-mediated immune modulation. Clinically, we reveal that HD-PTP loss is frequently observed in TNBC and correlates with immune cold microenvironment, as shown by reduced tumor infiltration levels of lymphocytes, poor response to immune checkpoint inhibitors, and worse overall survival. Additionally, we observe that knockdown of HD-PTP promotes systemic tumor growth only in a syngeneic, immunocompetent TNBC mouse model, but not in the matched, immunodeficient nude mice. Through proteomic profiling of the EVs purified from HD-PTP proficient and deficient cells, we find that HD-PTP downregulation dramatically changes EV protein composition and identify PD-L1 as a major ISF cargo selectively incorporated into EVs upon loss of HD-PTP. Using a co-culture assay with antigen-presenting B cells and PD-1 overexpressing T cells, we show that EVs derived from HD-PTP knockdown TNBC cells significantly reduce IL-2 secretion and impair antigen-dependent T cell activation via EV-associated PD-L1. Mechanistically, HD-PTP inhibition redirects PD-L1 from the plasma membrane to the endosomes, which increases PD-L1 co-localization with the EV marker CD63 and upregulates its subsequent release into EVs. However, blocking EV biogenesis in HD-PTP deficient cells – either by genetic knockdown of Alix or syntenin, or by pharmacological inhibition with GW4869 – reduces EV release of PD-L1, confirming that HD-PTP plays an essential role in regulating EV-associated PD-L1. Perturbation of either PD-L1 expression or EV release by Rab27A in the HD-PTP knockdown tumors abolishes the accelerated growth observed in the immunocompetent mice. Furthermore, we demonstrate that HD-PTP knockdown tumors had significantly fewer infiltrating T cells and altered macrophage polarization, while such immune exclusion was partially reversed by PD-L1 or Rab27A disruption. Collectively, our results unveil a novel tumor suppressor pathway controlling specific ISF cargos in EVs, while HD-PTP loss leads to altered EV-mediated signaling that facilitates immune suppression, remodels TIME, and fosters tumor growth, which eventually leads to inferior patient outcomes. Citation Format: Chenxu Guo, Sheng Sun, Nayana Thimmiah, Ilze Smidt, Shufeng Zhou, Thomas J. Kania, Taisha Joseph, Mark N. Melkonyan, Siang B. Koh, Leif W. Ellisen. HD-PTP modulates anti-tumor immunity via extracellular vesicles in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6582.