Multiplex imaging of the immune microenvironment after intratumoral immunotherapy.

Understanding the cellular mechanisms initiated by anti-tumor immunotherapies is vital for improving interventions for patients. Here, we characterized the tumor microenvironment following intratumoral injection of agonistic anti-CD40 and Monophosphoryl Lipid A, an agonistic Toll Like Receptor 4 ligand (CM). We used the high-plex protein staining platform IBEX (Iterative Bleaching Extends multipleXity) to image tumors under control and CM-treatment conditions. Based on single cell RNA-Seq data, we focused on key myeloid cells of the innate immune system as possible effectors of antitumor activity following CM treatment.

To determine if CM induces an antitumor effect observable by imaging, we quantified tumor cell apoptosis by combining Hoechst nuclear staining with cleaved caspase 3 (apoptotic marker) staining. This revealed that a significantly larger surface area of CM-treated tumors showed evidence of tumor cell apoptosis as compared to isotype-treated tumors. We observed a distinct overlap of CD177+ neutrophils (PMN) with regions of tumor cell apoptosis, suggesting that PMNs play an important role in enhancing cytotoxic activity against tumor cells. This was consistent with evidence of an increase in the depth to which PMN populations occurred within tumor cell apoptotic regions following CM-treatment. PMN depletion experiments confirmed their presumptive role in the antitumor effect of CM, providing mechanistic insight into a new form of intratumoral immunotherapy.