Rewiring Cancer Cell Mechano-stemness into “Kill Me” Signal for Immunotherapy

Introduction: : Solid tumors are characterized by complex mechanical microenvironments that profoundly influence cancer cell behavior and therapeutic response. One underexplored dimension is how mechanical cues, such as extracellular matrix (ECM) stiffness, impact cancer cell resistance to immunotherapies. Despite advances in chimeric antigen receptor T (CAR-T) cell therapies, durable responses in solid tumors remain limited, partly due to cancer stem-like cells (CSLCs), which are often resistant to cytotoxic killing. These CSLCs are mechanosensitive and enriched in soft tumor microenvironments. We hypothesized that the mechanical softness of the ECM promotes CSLC phenotypes, enhancing their ability to evade CAR-T-mediated cytotoxicity.

To test this, we developed a genetically encoded Mechano-Recorder—a synthetic biology tool that translates transient calcium signals, elevated under soft ECM conditions, into stable fluorescent barcodes. These recorders enable identification and isolation of mechanosensitive CSLCs. Transcriptomic profiling of Mechano-Recorder-positive cells revealed signatures of epithelial-to-mesenchymal transition (EMT), hypoxia, and KRAS/STAT3 pathway activation, all associated with stemness and therapy resistance.

Building on these findings, we rewired the Mechano-Recorder to drive expression of CD19, a clinically validated CAR-T target. This “Mechano-Reprogrammer” strategy allows CSLCs residing in soft niches to be selectively tagged with CD19, rendering them susceptible to CD19-CAR T cell killing. Our approach transforms mechano-stemness from a resistance trait into a therapeutic vulnerability, offering a potential strategy to overcome intratumoral heterogeneity and resistance mechanisms in solid tumor immunotherapy.

Materials and

Methods: : cancer cells on polyacrylamide hydrogels with defined stiffnesses representing soft and stiff ECM environments. A calcium-sensitive Mechano-Recorder was engineered using an NFAT-rtTA/TRE-mCherry dual-promoter circuit that converts intracellular calcium activity into stable reporter expression under doxycycline gating. Cells were exposed to DOX for 2 hours to initiate recording and harvested after 6 hours for flow cytometry and RNA sequencing. Cancer stem-like cell (CSLC) traits were assessed through mammosphere assays, surface marker expression, and atomic force microscopy (AFM) for cortical stiffness measurements.

To functionally rewire stemness into an immune vulnerability, the mCherry reporter cassette was replaced with a CD19 transgene, creating a Mechano-Reprogrammer system. CD19 expression was induced under soft ECM conditions, and cells were co-cultured with CD19-CAR T cells at various effector-to-target ratios. In vivo validation was performed in NSG mice bearing subcutaneous tumors established from FACS-isolated Mechano-Recorder-positive and -negative cells. Doxycycline was administered intraperitoneally to activate transgene expression in vivo. Tumor growth and transgene activation were monitored via bioluminescence imaging.

Primary patient-derived breast cancer cells of varying aggressiveness were also engineered with the Mechano-Recorder and tested across ECM stiffness conditions. Transcriptomic analyses were performed using DESeq2 and GSEA to assess stemness pathway enrichment. Extracellular ATP and intracellular calcium levels were quantified using luciferase-based assays and GCaMP imaging, respectively. All experiments were performed with at least three biological replicates to ensure reproducibility.

Results, Conclusions, and Discussions:: We discovered that cancer cells cultured on soft extracellular matrix (ECM) are more resistant to chimeric antigen receptor T (CAR-T) cell cytotoxicity, despite no difference in antigen levels. This resistance correlated with elevated intracellular calcium and extracellular ATP levels. To mechanistically trace this phenotype, we engineered a genetic “Mechano-Recorder” that converts transient calcium signaling into a stable fluorescent barcode under doxycycline control. Cancer cells grown on soft ECMs exhibited stronger recorder expression, indicating elevated mechano-sensitive calcium signaling.

RNA sequencing of recorder-positive cells revealed enrichment in cancer stem-like cell (CSLC) pathways, including epithelial-to-mesenchymal transition (EMT), hypoxia, KRAS, and STAT3 signaling. These cells displayed functional stemness features—enhanced mammosphere formation, reduced cortical stiffness, and increased resistance to CAR-T killing—validating the presence of a mechano-induced CSLC state, which we term “mechano-stemness.” This phenotype was also observed in diverse patient-derived breast cancer cells, where more aggressive subtypes exhibited stronger recorder expression under soft ECM conditions.

To therapeutically exploit this state, we rewired the Mechano-Recorder by replacing the reporter gene with a CD19 antigen, creating a “Mechano-Reprogrammer.” Under soft ECM conditions, this system induced CD19 expression in CSLCs, enabling their selective recognition and killing by CD19-CAR T cells. This strategy reversed resistance in vitro and improved tumor clearance in vivo when combined with EGFR-CAR T cells in a dual-targeting cocktail. Mice bearing tumors derived from recorder-positive cells showed significant tumor suppression only when treated with the rewired CAR-T combination.

Overall, this work uncovers mechano-stemness as a functional resistance mechanism and introduces a programmable synthetic biology approach to rewire it into a therapeutic vulnerability. The Mechano-Recorder provides a powerful platform for barcoding and isolating mechanosensitive subpopulations, while its rewiring into customizable antigens like CD19 offers a generalizable strategy for targeting CSLCs. Our findings suggest that leveraging mechanical cues through mechano-genetic circuits can improve CAR-T therapy precision and efficacy in solid tumors, particularly those enriched in stem-like, immune-evasive cells.

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