The immunogenic clonal-fraction threshold in heterogeneous solid-tumor required to induce effective bystander-killing of non-immunogenic subclones is unknown. Pancreatic cancer poses crucial challenges for immune therapeutic interventions due to low mutational-burden and consequent lack of neoantigens. Here, we designed a model to incorporate artificial-neoantigens into genes-of -interest in cancer-cells and to test their potential to actuate bystander-killing. By precisely controlling a neoantigen's abundance in the tumor, we studied the impact of neoantigen frequency on immune-response and immune-escape. Our results showed single, strong, widely-expressed neoantigen could lead to robust antitumor response when over 80% of cancer cells express the neoantigen. Further, immunological assays demonstrated T-cell responses against non-target self-antigen on KRAS-oncoprotein, when we inoculated animals with a high frequency of tumor-cells expressing test-neoantigen. Using nanoparticle-based gene-therapy, we successfully altered tumor-microenvironment by perturbing interleukin-12 and interleukin-10 gene-expression. The subsequent microenvironment-remodeling reduced the neoantigen frequency threshold at which bioluminescent signal intensity for tumor-burden decreased 1.5-log-fold, marking robust tumor-growth inhibition, from 83% to 29%. Our results thus suggest bystander killing is inefficient in immunologically-cold tumors like pancreatic-cancer and requires high neoantigen abundance. However, bystander killing mediated antitumor response can be rescued by adjuvant-immune therapy.
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