Construction And Mechanism Exploration Of Tumor Budding Organoids (TBO)

To establish a CAF-driven OSCC tumor budding organoid model (TBO) replicating physiological tumor invasion patterns and decode underlying mechanisms for clinically actionable therapeutic discovery. 1) Fabricate TBO models using photolithography-designed microfluidic chips; 2) Identify CAF subtypes inducing TB and corresponding TB cell subtypes through integrated single-cell RNA sequencing and in situ sequencing; 3) Isolate target CAF subtypes via Flow cytometry sorting; 4) Map protein spatial distribution using multiplex immunofluorescence staining. 1. CAFs guided OSCC budding invasion, with leading-edge contact between CAFs and OSCC cells (termed "tumor budding cells," TB). 2. scRNA-seq revealed a CAF subpopulation with enhanced ECM remodeling and TB cells subpopulation with elevated motility and adhesion. ISS confirmed their colocalization. 3. PDGFRα+ CAFs were identified as the key subtype inducing TB; 4. CellPhoneDB analysis identified integrin α2β1 (CAF) / E-cadherin (OSCC) as a key ligand-receptor pair, validated by colocalization on CAF – TB interfaces. 5. Mechanotransduction-mediated YAP activation by CAFs induced p-EMT in TB cells, as demonstrated by SNAIL nuclear translocation. PDGFRα+ CAFs promote OSCC budding invasion through integrin α2β1/E-cadherin mechanosignaling, activating YAP - driven EMT. Targeting PDGFRα+ CAFs and disrupting the integrin α2β1/E-cadherin-YAP axis effectively blocks invasion. The TBO model establishes a robust platform for mechanobiology-targeted drug screening.