Digital Spatial Profiling of immuno-oncology proteins in mouse hepatocellular carcinoma xenograft treated with THIO-based combination immune therapie

THIO (6-thio-dG) is a nucleoside analog that induces telomeric DNA damage and triggers activation of anti-tumor immunity in several cancer types such as lung and colorectal cancers. To evaluate its effect in hepatocellular carcinoma (HCC), THIO in combination with the current standard care first-line therapy for advanced HCC, anti-PD-L1 and anti-analyze, was tested in immunocompetent mice subcutaneously implanted with syngeneic HCC cell line (Hep55.1c). Formalin-fixed paraffin-embedded (FFPE) tumor tissues were collected after the treatment, and therapeutic modulations of the tumor immune landscape were assessed with Digital Spatial Profiling of 38 immuno-oncology-related proteins in region of interest (ROI) and intra-ROI segments enriched for T cells, other leukocytes, and HCC/stromal cells. Immunocompetent black 4 mice were subcutaneously implanted with a syngeneic mouse HCC cell line (Hep55.1c). After tumor nodules were formed, mice were treated with vehicle control, THIO, anti-PD-L1 + anti-VEGF, or anti-PD-L1 + anti-VEGF + THIO (n=1 per group). Mice were sacrificed at the end of the treatment, and FFPE tumor tissue blocks were generated. One representative animal was selected for each treatment group, and subjected to Digital Spatial Profiling of 38 immuno-oncology-related proteins. Immunofluorescent (IF) staining of morphology markers (CD3, CD45, pan-cytokeratin [Pan-CK]) was first performed, and based on the IF and H&E staining, region of interest (ROI) was defined in each tissue section. Within each ROI, segments were subsequently selected based on the IF staining enriched for T cells (CD3+), other leukocytes (CD3-/CD45+), and HCC/stromal cells (CD3-/CD45-/Pan-CK+/-), and abundance of the 38 immuno-oncology-related proteins was determined by using the GeoMx Digital Spatial Profiler (NanoString).