Spatial mapping and significance of IL12 signaling in non-small cell lung cancer

Background: Immunostimulatory therapies targeting T-cell co-inhibitory receptors have transformed the management of patients with non-small cell lung cancer (NSCLC) and revealed the potential of harnessing adaptive immunity to treat aggressive malignancy. However, only ~20-30% of patients with advanced NSCLC show clinical benefit to such therapies, highlighting the need for additional developments. Activation of IL-12 signaling in the tumor microenvironment (TME) induces prominent immunostimulatory responses and anti-tumor effect in pre-clinical models and early clinical trials. Understanding the expression, biological role and clinical significance of the IL-12 pathway in human NSCLC could be used to support optimal therapeutic developments. Strategy: We established multiplexed quantitative immunofluorescence (mQIF) panels to measure and spatially map the IL-12 pathway (DAPI/CK/IL12Rbeta1/pSTAT4/CD8), major tumor associated macrophage subpopulations (TAMs; DAPI/CK/CD14/CD68/CD206), dendritic cells (DCs; DAPI/CK/CD11c/XCR1/HLADR) and tumor-infiltrating lymphocytes (TILs; DAPI/CK/CD8/CD4/CD20) in consecutive sections from >600 primary NSCLCs from 4 independent cohorts represented in tissue microarrays. Western blot analysis and flow cytometry from cultured cell preparations of PC-9 human lung adenocarcinoma cell line stimulated for 48 hours with human recombinant IL-12 or IL-23 were used to assess functional responses and support analytical assay validations. Results: Membranous IL12Rbeta1 expression was seen in >95% of primary NSCLCs (mean 1519 ± 83.22 cell/mm 2 ), with continuous distribution across cases, predominant location in the (intratumoral) stromal compartment and association with pSTAT4 expression and adenocarcinoma histology. No consistent association was seen with other clinicopathologic variables, major oncogenic driver mutations and 5-year overall survival. Elevated abundance of IL12Rbeta1/pSTAT4 expressing cells was consistently associated with higher density of M2/3-like polarized TAMs, DCs and CD4+, CD8+ and CD20+ TILs. Notably, 13.2-29.2% of NSCLCs across the cohorts showed IL12Rbeta1 expression in cancer cells associated with increased pSTAT4 and reduced expression of pancytokeratin. Treatment of IL12Rbeta1+ lung adenocarcinoma cells with human recombinant IL-12 or IL-23 reduced the levels of the epithelial marker EpCAM and increased Ki-67. Supporting enhanced proliferation. Conclusions: IL-12 pathway expression and activation occurs in most primary NSCLCs with variable levels and is associated with adenocarcinoma histology and a favorable immune TME composition. IL12Rbeta1 is expressed in cancer cells from ~23% of tumors and associated with enhanced oncogenic properties, supporting a dual role in immune evasion and malignant progression. These results can support optimal development of IL-12 directed therapies and patient selection strategies.