Myeloid cell receptor tyrosine kinases (RTK) TYRO3, AXL and MERTK and their ligands, Gas 6 and Protein S, physiologically suppress innate immune responses, including in the tumor microenvironment. Here, we showed that myeloid-derived suppressor cells (MDSCs) dramatically upregulated TYRO3, AXL and MERTK and their ligands (M-MDSCs>20-fold, PMN-MDSCs>15-fold) in tumor-bearing mice. MDSCs from tumor bearing Mertk −/− , Axl −/− and Tyro3 −/− mice exhibited diminished suppressive enzymatic capabilities, displayed deficits in T cell suppression and migrated poorly to tumor-draining lymph nodes (TDLNs). In co-implantation experiments, using TYRO3 −/− , AXL −/− and MERTK −/− MDSCs, we showed the absence of these RTKs reversed the pro-tumorigenic properties of MDSCs in vivo. Consistent with these findings, in vivo pharmacologic TYRO3, AXL and MERTK inhibition diminished MDSCs' suppressive capability, slowed tumor growth, increased CD8 + T cell infiltration and augmented anti-PD-1 checkpoint inhibitor immunotherapy. Mechanistically, MERTK regulated MDSC suppression and differentiation in part through regulation of STAT3 serine phosphorylation and nuclear localization. Analysis of metastatic melanoma patients demonstrated an enrichment of circulating MERTK + and TYRO3 + M-MDSCs, PMN-MDSCs and e-MDSCs relative to these MDSC populations in healthy controls. These studies demonstrated that TYRO3, AXL and MERTK

Background: Myeloid-derived suppressor cells (MDSC) have been found to play an important role in limiting immune responses in cancer. Higher circulating MDSC levels have been associated with greater tumor burden, poorer response to immunotherapy, and poorer survival. Optimal measurement of MDSC levels could provide clinicians with a useful prognostic and/or management tool. Methods: A whole blood (WB) nine color, 11 parameter flow cytometric assay was designed, utilizing fluorescentlylabeled antibodies against CD45, CD3, CD19, CD20, CD56, CD16, HLA-DR, CD33, CD11b, CD14 and CD15, and BD Trucount beads for quantitation. Total MDSC were defined as CD45 + CD3 − CD19 − CD20 − CD56 − CD16 − HLA-DR − CD33 + CD11b + cells, while the monocytic (M-MDSC) and polymorphonuclear subsets were defined as CD14+ or CD15+, respectively. Results: A novel gating strategy was devised to eliminate granulocytes and improve consistency in gating. Several preanalytical variables were found to significantly affect MDSC quantitation, including collection tube type and time elapsed between blood collection and testing. Total and M-MDSC levels were a mean of 63% and 73% greater, respectively, with K 2 EDTA compared to Na + heparin collection tubes (N = 5). In addition, time elapsed at room temperature prior to cell labeling affected MDSC quantitation; by 24 h after blood collection, total and M-MDSC levels were a mean of 26% and 57% lower compared to testing as soon as possible after collection (N = 6). Refrigeration of samples at 4°C ameliorated time-dependent effects at both 4 and 8 h, but not 24 h after blood collection. To establish normal ranges for this assay, MDSC levels were quantified in 67 healthy subjects (30 male, 37 female) ages 20-93. No significant differences in total or M-MDSC levels were detected for ages ≤60 compared to > 60 (p = 0.5 and p = 0.8, respectively). Finally, assay results demonstrated significantly higher MDSC levels among patients with hepatocellular carcinoma (N = 55) compared to agematched healthy controls (N = 27) for total and M-MDSC (p = 0.006 and 0.004, respectively). Conclusions: MDSC are a heterogenous group of cells, and their quantitation in WB can be affected by a number of preanalytical variables. Consideration of these factors, and measurement using a material type that has not been manipulated, such as whole blood, is likely to yield the most accurate results.

Background KRAS variant alleles may have differential biological properties which impact prognosis and therapeutic options in pancreatic ductal adenocarcinomas (PDA). Materials and Methods We retrospectively identified patients with advanced PDA who received first-line therapy and underwent blood and/or tumor genomic sequencing at the University of Washington between 2013 and 2020. We examined the incidence of KRAS mutation variants with and without co-occurring PI3K or other genomic alterations and evaluated the association of these mutations with clinicopathological characteristics and survival using a Cox proportional hazards model. Results One hundred twenty-six patients had genomic sequencing data; KRAS mutations were identified in 111 PDA and included the following variants: G12D (43)/G12V (35)/G12R (23)/other (10). PI3K pathway mutations (26% vs. 8%) and homologous recombination DNA repair (HRR) defects (35% vs. 12.5%) were more common among KRAS G12R vs. non-G12R mutated cancers. Patients with KRAS G12R vs. non-G12R cancers had significantly longer overall survival (OS) (HR 0.55) and progression-free survival (PFS) (HR 0.58), adjusted for HRR pathway co-mutations among other covariates. Within the KRAS G12R group, co-occurring PI3K pathway mutations were associated with numerically shorter OS (HR 1.58), while no effect was observed on PFS. Conclusions Patients with PDA harboring KRAS G12R vs. non-G12R mutations have longer survival, but this advantage was offset by co-occurring PI3K alterations. The KRAS/PI3K genomic profile could inform therapeutic vulnerabilities in patients with PDA.