Collagen-anchored interleukin-2 and interleukin-12 safely reprogram the tumor microenvironment in mouse melanomas

Mouse B16F10 melanoma tumors were analyzed for gene expression changes following intratumoral treatment with collagen-anchored IL-2 and IL-12 therapy for comparative analysis to expression changes observed in canine soft tissue sarcomas. Tumors were collected at different intervals following treatment to assess persistence of any therapy-driven gene expression changes. Twelve C57BL/6 mice (Taconic) bearing day 6 B16F10 flank tumors were assigned to cohorts to receive intratumoral doses of interleukin-2 and interleukin-12 collagen-binding fusion proteins or saline (n=3/group). Cohorts were designed to mimic the treatment groups from the canine portion of this work. The first cohort (n=3) had tumors collected 2 days after treatment; the second cohort (n=3) had tumors collected 8 days after treatment; and the third cohort (n=4) received a second cytokine dose 6 days after the first dose, with tumors collected two days later (day 8 post-first dose). Tumor tissue was formalin-fixed and paraffin-embedded prior to RNA extraction (Qiagen FFPE RNEasy) and QC on Bioanalyzer (Agilent). Gene expression was analyzed through hybridization to the Nanostring Mouse PanCancer Immune Profiler nCounter panel set probes, with quantification by Nanostring Digital Analyzer. Expression data was analyzed using Nanostring nSolver software.