Corre, I. | bioRxiv Science

bioRxiv2025

MCL-1 as a molecular switch between myofibroblastic and pro-angiogenic features of breast cancer-associated fibroblasts

Breast cancer-associated fibroblasts (bCAFs) comprise inflammatory CAFs (iCAFs), characterized by the secretion of pro-inflammatory cytokines, and myofibroblastic CAFs (myCAFs), distinguished by their high production of extracellular matrix and their immunosuppressive properties. We previously showed that targeting the anti-apoptotic protein MCL-1 in primary culture of bCAF derived directly from human samples reduces their myofibroblastic characteristics. We herein show by single-cell RNA-sequencing analysis of bCAFs that MCL-1 knock down induces a phenotypic shift from wound-myCAF to IL-iCAF, characterized by the upregulation of genes associated with inflammation as well as angiogenesis-related genes. In vitro, genetic and pharmacologic MCL-1 inhibition increases VEGF secretion by bCAFs, enhancing endothelial cell tubulogenesis. In a chicken chorioallantoic membrane (CAM) model in ovo, co-engraftment of breast cancer cells and bCAFs with reduced MCL-1 expression leads to heightened peritumoral vascular density, driven by VEGF. Mechanistically, the pro-angiogenic phenotype revealed by MCL-1 inhibition is dependent on BAX-BAK activity. It results in NF-{kappa}B activation, inhibition of which by a IKK{beta} inhibitor suppresses the transcription of VEGF and pro-inflammatory factors triggered by MCL-1 inhibition in bCAFs. Chemotherapy induces a downregulation of MCL-1 in bCAFs and it promotes a pro-angiogenic phenotype, counteracted by overexpressed MCL-1. Overall, these findings uncover a novel regulatory function of MCL-1 in determining bCAF subpopulation differentiation and highlight its role in modulating their pro-angiogenic properties, in response to treatment in particular.

cancer biology↗

bioRxiv2024

In vitro models to mimic tumor endothelial cell-mediated immune cell reprogramming in lung adenocarcinoma

Tumor endothelial (TECs) cells play a critical role in regulating immune responses within the tumor microenvironment (TME). However, the mechanisms by which TECs modulate immune cell population remain unclear, particularly in non-small cell lung cancer (NSCLC). Here, we investigated how NSCLC cells tweak normal endothelial cells (NECs) into TECs and the subsequent effects on immune regulation. NECs were cocultured with various NSCLC cell lines, using 2D and 3D coculture models to evaluate TEC-mediated effects on immune cells. We show that direct coculture led to significant transcriptomic, proteomic and kinomic alterations in TECs, especially in pro-inflammatory pathways. We identified a downregulation of the co-stimulatory molecule OX40L in TECs compared to NECs, suggesting impaired T-cell proliferation support. While TECs showed a limited effect on CD8+ T-cell activation, TECs supported CD4 T-cells polarization into Treg and Th22 subsets. Moreover, TECs also promoted M2-like macrophages polarization, thereby potentially contributing to the TME immunosuppression. State-of-the-art single-cell RNA sequencing of 3D multicellular tumor spheroids (MCTS) revealed distinct TEC subpopulations, including an inflammatory subset with UPR signature. The latter was absent in 2D-cultured NECs but present in freshly isolated and 2D-cultured TECs from NSCLC patients. Importantly, we also identified within MCTS a perivascular M2-like macrophage subset, predicted to interact with TECs with MIF and Midkine signaling. In conclusion, TECs in NSCLC tumors play a pivotal role in remodeling the TME immune landscape by promoting immune suppression. This study highlights the complex immunoregulatory functions of TECs within our different in vitro models that mimic aspects of the TME. Our data may provide new insights into potential therapeutic strategies targeting TECs or regulatory signaling to improve the efficacy of immunotherapy in NSCLC.

cancer biology↗