Background Cytokines have a large impact on the tumor microenvironment by affecting cell growth, survival, inflammation, and differentiation. Understanding the spatial biology within the tumor microenvironment, including the cytokine organization within specific cell types, can lead to better treatment plans for patients. We developed a hybrid multiplex panel, using RNA in-situ hybridization (ISH) and immunofluorescence (IF) to investigate the spatial biology of the tumor microenvironment. This panel also enabled us to simultaneously detect the RNA of cytokines and protein of specific immuno-oncology (IO) biomarkers. This allows us to visualize spatially, not only the organization of cell types but also to identify the cellular sources of specific cytokines.

Methods In this study, we analyzed the tumor microenvironment of samples from non-small cell lung cancer (NSCLC) and breast cancer in two panels for cytokine RNAs and IO protein biomarkers. Panel 1 included the cytokine TNFalpha combined with CD68 and CD163 IO protein biomarkers. Panel 2 consisted of the cytokine IL-6 with the CD3 IO protein biomarker. Staining was completed on the Leica Bond RX autostainer, and slides were scanned using the PhenoImager™ Fusion. Visiopharm® software was used to analyze the tumor microenvironment of tissue samples and localize cytokines in specific cell types.

Results CD163 positive macrophages (M2 phenotype) were seen predominantly within the tumors (NSCLC and breast cancer) whereas CD68 positive macrophages were identified towards the periphery in the non-tumor microenvironment. NSCLC tumor cells showed higher expression levels of IL-6 and TNFalpha compared to breast cancer tumor cells. NSCLC harbored a significantly higher number of IL-6 positive T cells compared to breast cancer. Multiplexing enabled assessment of relative expression levels of IL-6 and TNFalpha in the immune cells and tumor cells.

Conclusions Our hybrid multiplex panel can provide insights into the visualization of spatial organization of tumor microenvironment. It also enables semi-quantitative assessments for the expression levels of cytokines and their relative expression within tumor and immune cells, thereby helping to correlate response to treatment, especially with checkpoint inhibitors.