StromaBlast™: A novel CRISPR-based functional genomics platform to identify therapeutic targets within the tumor microenvironment

Background: The solid tumor microenvironment (TME) plays a critical role in regulating tumor progression, metastasis, and therapeutic resistance. It is a complex network of cancer-associated fibroblasts (CAFs), immune cells, endothelial cells, extracellular matrix (ECM), and soluble mediators. Recent advances in oncology have shifted the therapeutic paradigm from a cancer cell-centric to a TME-centric model, underscoring the importance of stromal components in shaping tumor behavior. Among these, CAFs represent the functionally dominant stromal population, driving resistance through multifaceted mechanisms including ECM remodeling, paracrine signaling, and metabolic reprogramming. These processes collectively create a protective niche that fosters drug tolerance and immune evasion. Methods: To overcome the limitations of traditional discovery screens that rely solely on monoculture cancer models, we developed StromaBlast™, a next-generation CRISPR screening platform which incorporates co-cultures of cancer cells, CAFs, and immune cells. Using both Cas9-mediated knockout and dCas9-based activation approaches, we performed a comprehensive CRISPR surfaceome screen in lung cancer cells under conditions that simulate therapeutic antibody exposure and T-cell mediated killing in fibrotic tumors. Our screen was optimized to identify essential interaction networks and novel therapeutic targets involved in CAF-cancer-immune cell crosstalk. Results: The StromaBlast™ screen uncovered several novel and known targets that are essential for cancer cell survival and communication within a CAF-rich microenvironment. Comparative analyses distinguished targets exhibiting CAF dependency from those functioning independently of stromal interactions. Functional validation confirmed the critical role of select targets in sustaining cancer cell viability in the presence of CAFs. These findings nominate actionable targets amenable to development as biologics, including T-cell engagers and antibody-drug conjugates, designed to disrupt CAF-cancer cell interactions and restore anti-tumor immune activity. Conclusion: Fibrotic, CAF-enriched tumors are notoriously resistant to immunotherapy and antibody-based treatments due to CAF-ECM-mediated physical barriers and immune suppression. StromaBlast™ represents a transformative platform for discovering therapeutic targets that can overcome this resistance. Targeting CAF-dependent vulnerabilities identified through this approach holds the potential to convert immune-cold, fibrotic tumors into immune-active, therapy-sensitive states - advancing precision oncology and improving patient outcomes.