Abstract
The "cold" tumor microenvironment (TME) impaired colorectal cancer (CRC) immunotherapy efficacy, while oncolytic viruses (OVs) could "heat" TME and stimulate anti-tumor immunity. Here, it rescued rPR8-CCL19, a recombinant oncolytic influenza virus expressing CCL19, using reverse genetics technology. Gene sequencing and transmission electron microscopy confirmed its genetic stability during serial passaging. Hemagglutination assay, ELISA, transwell, real-time cell analysis (RTCA), and apoptosis detection demonstrated that it selectively infected CRC cells, expressed CCL19 with the function of chemotaxis and activation on immune cells, and exerted killing of significant CRC cells. In syngeneic CRC mouse models, it effectively inhibited tumor growth and metastasis, prolonging survival. By enhancing immune cell infiltration, it remodeled the TME, thereby inducing systemic anti-tumor immunity and even immune memory, without causing severe pathological damage. This study confirmed rPR8-CCL19 as a promising CRC immunotherapy for further exploration.