Effect of sitravatinib combined with PD-1 blockade on cytotoxic T-cell infiltration by M2 to M1 tumor macrophage repolarization in esophageal adenocarcinoma.
Authors: Omstead Ashten , Zheng Ping , Sweeny Ryan , Gorbunova Anastasia , Goel Arul , Wagner Patrick , Kelly Ronan Joseph , Jobe Blair A , Zaidi Ali Hussainy 
Published: January 2024

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Background: Esophageal adenocarcinoma (EAC) is a deadly cancer with a low survival rate and limited treatment options. Receptor tyrosine kinase inhibitor, sitravatinib, selectively targets tumor associated macrophage (TAM) family receptors, AXL, TYRO3 and MERTK, and split family receptors, VEGFR, PDGFR, KIT and MET. In solid tumors, sitravatinib has been shown to potentiate PD-1 blockade by production of pro-inflammatory cytokines and reducing MDSCs, Tregs and TAMs, in the tumor microenvironment (TME). Methods: End-to-side esophagojejunostomy was performed on a cohort of 96 rats to induce gastroesophageal reflux, leading to EAC carcinogenesis. At 32 weeks post-operative, all surviving animals underwent endoscopic examination with biopsy and a pre-treatment MRI. All tumor bearing animals were randomized to a dose of 10mg/kg of sitravatinib (S) or vehicle control (VC) for three cycles, and PD-1 inhibitor, AUNP-12 (IO), was administered on day 12 of each 14 day cycle, at a dose of 3mg/kg. At 38 weeks post-operative, animals received a post-treatment MRI and were euthanized. Safety and efficacy were evaluated by on-treatment mortality, pre- and post-treatment MRI, immunohistochemistry, immunofluorescence and real-time PCR. Results: The S+/-IO groups demonstrated a higher on-treatment mortality when compared to the VC+/-IO groups (p=<0.001). Pre- to post-treatment, mean MRI tumor volume decreased by 32% and 73% in the S-IO and S+IO animals and increased by 90% and 160% in the VC+IO and VC-IO animals, respectively (p=<0.001). Increased apoptosis and decreased proliferation were confirmed through Cas-3 and Ki-67 immunohistochemistry, respectively, in both S treatment groups (p=<0.001). CD8+ T-cell infiltration was upregulated, and M2 to M1 macrophage phenotype repolarization was demonstrated in all treatment groups by immunofluorescence (p<0.01). Downstream gene expression demonstrated upregulation of pro-inflammatory cytokines including TNF-α, IFN-γ, IL-1β, IL-6, IL-12 and downregulation of anti-inflammatory cytokines including TGF-β, IL-4, IL-10, and IL-13 in S treatment groups compared to VC treatment groups (p<0.05). There was no significant difference in pre-treatment PD-L1 levels between any of the treatment groups (p=0.09). Pre- to post-treatment qRT-PCR demonstrated significant inhibition of pathway genes AXL, AKT, MERTK and PI3K in the treatment animals (p=<0.001). Additionally, pre-treatment AXL and PD-L1 levels were significantly higher in major responders (>80% tumor reduction) when compared to the non-responders (<25% tumor reduction or progression), in the combined S+/-IO groups (p<0.05). Conclusions: This study establishes a favorable combinational strategy using sitravatinib to overcome immunosuppression in the TME, providing strong rationale for future clinical strategies in the treatment of EAC.