Cancer-associated fibroblasts (CAFs) phenotype is associated with poor prognosis in the vast majority of solid tumors, as they contribute to hallmarks of cancer progression, such as inflammation, metastasis, angiogenesis, and critically, immune evasion. Yet, the fundamental cues that control fibroblast into CAF malignant state remain unclear. In the lab, we showed that macrophages from embryonic origin (Alveolar Macrophages or AMs) closely interact with fibroblasts present in the tissue before the onset of cancer. Interestingly, longitudinal confocal imaging, flow cytometry of lung tumor stroma and macrophage-depleted models, established that AMs play an important role in the malignant conversion from naïve fibroblast into CAF in NSCLC.

Single-cell RNA sequencing (scRNA-seq) was performed on both CD45+ and CD45- compartments of lung tissue to investigate stromal populations targeted by AMs. A new mouse model, Dpt-CreERT2-LSL-TdTomato crossed with CD169DTR/+ mouse (AMs-depleted upon Diftheria toxin administration), was generated to track universal fibroblast to CAF conversion in tumors with/without AMs (CD169+). Flow cytometry and confocal imaging were used to analyze this conversion. CellChat Ligand-receptor analysis was conducted to identify potential AM-secreted factors regulating CAF fate.

We found that AM-depleted lungs showed an expansion of universal fibroblasts (Pdgfra+Pdpn+Dpt+) but completely lacked CAF-activated phenotypes. Importantly, AM-depleted tumors confirmed significant downregulation of canonical CAF activation genes, as Acta2, Tgfb1, or Fn1 compared with AM-sufficient tumors. Noteworthy, CAF activation profile remained unchanged in mice missing Monocyte-derived macrophages Mo-Macs (CCR2KO/KO), suggesting AM-specificity in the universal to CAF fate conversion. Using flow cytometry and confocal imaging on the fibroblasts tracking model Dpt-CreERT2-LSL-TdTomato crossed with CD169DTR/+ mouse, we showed that universal fibroblast contributes to CAFs and more importantly, that universal fibroblasts into-CAF conversion is unequivocally controlled by AMs. CellChat Ligand-receptor analysis based on our scRNA-seq dataset identified AM-derived fibronectin (Fn1) and efferocytic Pros1 as macrophage factors interacting with CD44, integrins and AXL in fibroblasts. Finally, as AM-depleted tumors are significantly infiltrated with CD4 and CD8 T cells resulting in a longer survival, we plan to interrogate the impact of early AXL inhibition (Bemcentinib) in KP-tumors to boost T cell surveillance in otherwise T cell dessert tumors.

Altogether, our work highlights a novel regulatory axis between AM and CAF that can be therapeutically exploited to promote immune T cell surveillance in NSCLC. We identified AMs as drivers of pathological responses in CAFs via AXL activation.