Purpose <p>Axl is a therapeutic target under active clinical investigation for a variety of cancer indications. While pre-clinical mouse studies have been promising, clinical trials targeting Axl in combination with standard-of-care therapies have yielded mixed results. We hypothesize that this clinical translation gap may in part be due to an incomplete understanding of the effects of Axl inhibition specifically on the myeloid compartment of the human tumor microenvironment, as Axl mediates immunoregulatory signaling in myeloid cells. Utilizing human in vitro melanoma tumor microenvironment model systems, herein we generated and analyzed single-cell RNA-sequencing data to understand the impacts of tumor microenvironment composition and tumor cell prior therapy on myeloid cell response to Axl inhibition.</p> Methods <p>We used our previously established in vitro model system to generate single-cell RNA-sequencing samples of tumor cell-macrophage co-cultures as well as tumor cell-macrophage-dendritic cell tri-cultures from four healthy buffy coat donors. We first applied LIgand-receptor ANalysis frAmework (LIANA) to our transcriptional data to infer potential intercellular crosstalk, complemented by Tensor-cell2cell to identify prevalent patterns of potential communication across the various experimental conditions. We next probed the impact of Axl inhibition on intercellular communication with orthogonalized partial least squares discriminant modeling. Finally, we conducted gene set enrichment analysis to understand how each cell type’s state and signaling activity was impacted by each axis of experimental variation in our complex experimental design.</p> Results <p>Tensor-cell2cell yielded eight patterns of communication in the inferred intercellular interaction data, and these eight patterns were paired based on their communicating cell types as well as their shared top ligand-receptor interactions. These pairings demonstrated the impact of including dendritic cells in the model system and highlighted coordination among the myeloid cells. The specific effects of Axl inhibition on potential intercellular crosstalk were clarified with supervised modeling, demonstrating impaired tumor cell invasion and migration programs, as expected. Intriguingly, gene set enrichment analysis revealed that tumor cell prior injury with standard-of-care therapy impacted myeloid cell interferon signaling in response to Axl inhibition.</p> Conclusions <p>In this work, we demonstrated that tumor microenvironment cellular composition as well as prior treatment with standard-of-care therapy modulate myeloid cell response to Axl inhibition in human in vitro melanoma model systems. While the inferred effects of Axl inhibition on intercellular crosstalk, cell state, and cell activity were largely consistent with prior literature, the impact of tumor cell prior injury on the myeloid interferon signaling response to Axl inhibition underscores the importance of intentionally incorporating treatment history as a design parameter when building model systems. Ultimately, complex human in vitro model systems that consider the nuanced effects of prior therapy on the tumor microenvironment can complement insights gained from pre-clinical mouse studies to better evaluate and iteratively design candidate therapies.</p>