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Background While immune checkpoint blockade has catapulted immunotherapy to the frontier of cancer therapeutics, its success is limited to patients with prior T cell infiltration. 1 2 To expand the applicability of this therapy, immunologically cold tumors need to be converted to hot ones. Axl is a receptor tyrosine kinase that has gained attention as a therapeutic target, as its inhibition is hypothesized to promote tumor cell death and myeloid cell activation – both prerequisites to T cell infiltration.3–5 However, Axl inhibition has had mixed clinical results,6–8 necessitating a deeper understanding of the mechanistic effects of Axl inhibition on myeloid cell activation in the tumor microenvironment.Methods We generated injured and untreated A375 human malignant melanoma populations by treating cells with trametinib + dabrafenib or DMSO, respectively, for 24 hours followed by 24 hours of rest. We then co-cultured each A375 population with macrophages for 24 hours with or without the Axl-specific small molecule inhibitor bemcentinib. For tri-culture experiments, we added dendritic cells (DCs) to these co-cultures for an additional 24 hours. Cells were then harvested for flow cytometry and single-cell RNA-sequencing.Results We found that prior treatment of A375 cells with targeted therapy was the primary source of variability in macrophage surface marker expression in both the co- and tri-culture systems ( figures 1A and 1B).9 Axl inhibition via bemcentinib appeared to have a secondary effect in the co-culture system, shifting the macrophages toward an immunostimulatory state (figure 1A).9 This trend was diminished upon the addition of DCs to the system (figure 1B), suggesting that DC inclusion dampens the macrophage response to bemcentinib.9 Separation of macrophages by A375 state was further reflected at the transcriptional level, as evaluated via single-cell RNA-sequencing (figure 2A), although this distinction was again clearer in co-cultured macrophages than in tri-cultured macrophages. Gene set enrichment analysis revealed overall diminished inflammatory activity in macrophages from the tri-culture system as compared to the co-culture system (figure 2B), further suggestive of a dampened macrophage response.Conclusions Overall, our results demonstrate that the cellular composition of the microenvironment and prior targeted therapy treatment impact the myeloid response to Axl inhibition. Analysis of changes in cell-cell communication that may underlie these observations is actively underway.Acknowledgements Thank you to the Koch Institute Flow Cytometry Core staff as well as the MIT BioMicro Center staff for their technical expertise and guidance in data acquisition.References Robert C. A decade of immune-checkpoint inhibitors in cancer therapy. Nat Commun. 2020;11(1):1–3.Ouyang P, Wang L, Wu J, Tian Y, Chen C, Li D, Yao Z, Chen R, Xiang G, Gong J, Bao Z. Overcoming cold tumors: a combination strategy of immune checkpoint inhibitors. Front Immunol. 2024;15:1344272.Akalu YT, Rothlin CV, Ghosh S. TAM receptor tyrosine kinases as emerging targets of innate immune checkpoint blockade for cancer therapy. Immunol Rev. 2017;276(1):165–77.Rothlin CV, Ghosh S. Lifting the innate immune barriers to antitumor immunity. J Immunother Cancer. 2020;8(1):1–15.Engelsen AST, Lotsberg ML, Abou Khouzam R, Thiery JP, Lorens JB, Chouaib S, Terry S. Dissecting the role of AXL in cancer immune escape and resistance to immune checkpoint inhibition. Front Immunol. 2022;13:869676.Byers LA, Gold KA, Peguero JA, Johnson ML, Nieva JJ, Harb WA, Chiappori A, Rybkin II, Strauss JF, Gerber DE. Ph I/II study of oral selective AXL inhibitor bemcentinib (BGB324) in combination with erlotinib in patients with advanced EGFRm NSCLC: end of trial update. J Clin Oncol. 2021;39(15_suppl):9110–9110.Straume O, Schuster C, Aziz S, Davidsen K, Karlsen J, Rayford AJ, Siraji MI, Svendsen HL, Reisæter L, Gjertsen BT, Akslen L, Lorens J. LBA52 A randomized phase Ib/II study of the selective small molecule AXL inhibitor bemcentinib in combination with either dabrafenib/trametinib or pembrolizumab in patients with metastatic melanoma. Ann Oncol. 2023;34:S1291.Borghaei H, de Marinis F, Dumoulin D, Reynolds C, Theelen WSME, Percent I, Calderon VG, Johnson ML, Madroszyk-Flandin A, Garon EB, He K, Planchard D, Reck M, Popat S, Herbst RS, Leal TA, Shazer RL, Yan X, Harrigan R, Peters S. SAPPHIRE: phase III study of sitravatinib plus nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer. Ann Oncol. 2024;35(1):66–76.Datta A, Bahlmann LC, Gong DN, Tevonian EN, Lorens JB, Lauffenburger DA. Axl inhibitor-mediated reprogramming of the myeloid compartment of the in vitro tumor microenvironment is influenced by prior targeted therapy treatment. Front Immunol. 2025;16:1601420.Ethics Approval This study was exempt from review by the Massachusetts Institute of Technology Committee on the Use of Humans as Experimental Subjects; exemption number E-5296.Abstract 874 Figure 1Prior targeted therapy treatment of A375 tumor cells drives separation of macrophages. Prior targeted therapy treatment of A375 tumor cells drives separation of macrophages as assessed by principal component analysis of surface marker expression data. n = 5-6 independent donors. Data as presented in Datta et al.9Abstract 874 Figure 2Macrophage transcriptional signature is impacted by A375 state and the addition of dendritic cells. Macrophage transcriptional signature is impacted by A375 prior targeted therapy treatment, the presence of dendritic cells in the system, and Axl inhibition. Red ellipses indicate macrophage clusters of interest. n = 4 independent donors