Single-treatment in situ photo-vaccination with paclitaxel prodrug-PDT enables durable local and abscopal tumor control in a bilateral Colon 26 model.

In situ photo-vaccination (ISPV) is a promising cancer immunomodulation strategy that combines photodynamic therapy (PDT) with targeted release of chemotherapy to transform tumors into personalized vaccines. However, its efficacy in controlling established, larger distal tumors remains limited, reflecting a significant challenge among therapeutic cancer vaccines. To address this, we used a bilateral Colon 26 murine tumor model to systematically optimize ISPV parameters, including drug-light interval, paclitaxel prodrug dose, light dose, size of the illuminated tumor (related to antigen dose), and checkpoint blockade, and to link these to cellular mechanisms of immunogenic cell death. A low prodrug dose (0.5 μmol/kg compared to ~24 μmol/kg of Paclitaxel in mice) and a fluence rate of 75 mW/cm2 for 30 min with a 30-min drug-light interval produced the strongest systemic responses. Abscopal efficacy was strongly linked to antigen dose: treating larger primary tumors expedited control of distant tumors, allowing the suppression of untreated tumors to a size comparable to the primary tumor. Checkpoint inhibition with anti-CTLA-4 was essential, as its removal eliminated distant tumor control. Importantly, optimized ISPV reproducibly controlled distant tumors of a size equivalent to the treated primary, an outcome rarely achieved with other in situ vaccination approaches. These findings establish a mechanistically informed framework for PDT-driven ISPV and provide a strong rationale for translation of this strategy to the treatment of metastatic cancer.