Foxs1-mediated transformation of CD34+ fibroblast to myCAFs promotes tumor growth

Abstract

Cancer-associated fibroblasts (CAFs) represent a major structural component of solid tumors and play crucial roles in cancer progression and drug resistance. However, their developmental origin, differentiation trajectory, and therapeutic potential remain poorly defined. Using advanced approaches—including inducible genetic lineage tracing, single-cell RNA sequencing, and spatial transcriptomic profiling—we identified a population of Cd34+Pi16+ fibroblast progenitors (Cd34+ CAFs) in both melanoma and gastric cancer. We delineated their differentiation trajectory toward Acta2+ CAFs, driven by the upregulation of the transcription factor Foxs1. This work establishes the developmental origin of Acta2+ CAFs and experimentally validates the Cd34+ to Acta2+ transition. By reverse-matching the transcriptional signatures of Acta2+ CAF differentiation with the CMap/LINCS L1000 drug perturbation database, we identified four small-molecule candidates predicted to inhibit tumor-induced Foxs1 upregulation. These compounds effectively suppressed Cd34+ CAF differentiation, maintaining the progenitor-like Cd34+ state. Collectively, this study proposes a novel antitumor strategy that targets CAF lineage development to restrain tumor progression. A comprehensive atlas of CD34⁺ fibroblast progenitors within the tumor microenvironment was generated using single-cell sequencing and lineage tracing, delineating their differentiation trajectory toward myofibroblasts and identifying potential therapeutic targets to block this transition. In situ sequencing reveals that CD34⁺Pi16⁺ CAFs originate adjacent to blood vessels, suggesting a vascular adventitial progenitor origin. Dual-recombinase lineage tracing combined with diphtheria toxin ablation demonstrates that CD34⁺Pi16⁺ cells are an essential source of tumor myofibroblasts required for tumor growth. Foxs1 directly binds the α-SMA promoter to drive CD34⁺ to Acta2⁺ CAF conversion, and its knockdown suppresses tumor growth in vivo. Machine learning-based screening of CMap/LINCS L1000 identifies disulfiram and three other compounds that block CAF differentiation and restrain tumor progression. Pharmacological intervention preserves the CD34⁺Pi16⁺ progenitor state, with single-cell sequencing confirming that WH-4-023 reverses CAF phenotypic transition. In situ sequencing reveals that CD34⁺Pi16⁺ CAFs originate adjacent to blood vessels, suggesting a vascular adventitial progenitor origin. Dual-recombinase lineage tracing combined with diphtheria toxin ablation demonstrates that CD34⁺Pi16⁺ cells are an essential source of tumor myofibroblasts required for tumor growth. Foxs1 directly binds the α-SMA promoter to drive CD34⁺ to Acta2⁺ CAF conversion, and its knockdown suppresses tumor growth in vivo. Machine learning-based screening of CMap/LINCS L1000 identifies disulfiram and three other compounds that block CAF differentiation and restrain tumor progression. Pharmacological intervention preserves the CD34⁺Pi16⁺ progenitor state, with single-cell sequencing confirming that WH-4-023 reverses CAF phenotypic transition. A comprehensive atlas of CD34⁺ fibroblast progenitors within the tumor microenvironment was generated using single-cell sequencing and lineage tracing, delineating their differentiation trajectory toward myofibroblasts and identifying potential therapeutic targets to block this transition.