Background The ability of T cells to mediate anti-tumor immunity has been harnessed to develop successful immunotherapies in recent years. Although direct presentation of tumor antigens by tumor cells plays an important role in the effector function of cytotoxic T lymphocytes (CTLs), cross-presentation by professional antigen presenting cells such as dendritic cells (DCs) is vital for priming naive CD8+ T cells and developing a sustainable cytotoxic response. Natural killer (NK) cells within the tumor microenvironment (TME) recruit a specific population of DCs called conventional type 1 DCs (cDC1s) into the TME by secreting chemokines such as CCL5 and XCL1. However, these cells are very low in abundance and are characterized by the expression of numerous markers, making their detection in the tissue context challenging.

Methods Therefore, to interrogate the presence of cDC1 and NK cells in the TME and reveal their spatial relationship we utilized the highly sensitive and specific RNAscope Multiplex Fluorescence in situ hybridization (ISH) assay. Probes for XCR1, THBD, CLEC9A, and CCR5 were used to identify cDC1 cells within 4 cervical cancer tumors. These tumors were then assessed for the presence of NK cells by using specific marker probes such as CD56 and NCR1 and chemokines XCL1 and CCL5. Finally, CTLs were visualized to determine if there is a correlation between the presence of cDC1 and NK cells and CTL infiltration within the cervical cancer tumors.

Results Our results revealed a strong correlation between the presence of NK cells, cDC1 cells, and CTLs within 3 out of 4 cervical cancer samples. The NK cells showed expression of the chemokines XCL1 and CCL5, which are the ligands for XCR1 and CCR5 respectively, suggesting that the XCR1+/CCR5+ cDC1 cells may have been potentially recruited by these NK cells. Regions high in cDC1 and NK cells also showed significantly higher levels of CTL recruitment, as indicated by the presence of CD8+/IFNG+ T cells. Conversely, 1 of the 4 cervical cancer samples demonstrated relatively lower levels of NK cells which correlated with lower cDC1 cells and in turn lower CTL infiltration.

Conclusions In conclusion, by utilizing the RNAscope Multiplex ISH assay we were able to identify and visualize the spatial relationship between NK cells, CTLs, and cDC1 cells, a rare subset of DC cells that excel at presenting tumor antigens to T cells. Using this technology, it is possible to spatially interrogate the TME and detect specialized immune cells when assessing response to immunotherapies.