Professor Massachusetts Institute of Technology, United States
Introduction:: Immune checkpoint inhibitors have gained significant traction over the last few years as cancer therapeutics but have yielded remission in only a subset of patients. It is now appreciated that the efficacy of these inhibitors relies on T cell infiltration of the tumor prior to treatment. There is thus a need to perturb the tumor microenvironment (TME) to transform immunologically cold tumors to immunologically hot ones. The Tyro3, Axl, and MerTK (TAM) receptor tyrosine kinase family is a promising target for inducing such a transformation, as these receptors are checkpoints of the innate immune system. They help mediate efferocytosis, which is the non-immunogenic clearance of apoptotic cells, to maintain innate immune homeostasis. TAM receptor inhibition can facilitate activation of macrophages and dendritic cells, which in turn activate and recruit T cells. Furthermore, the TAM receptors have been implicated in sustaining cancer progression as well as promoting therapeutic resistance. Clearly, the TAM receptors are dual targets, but the changes to the tumor immune landscape resulting from their inhibition remain incompletely understood. In this study, we develop a robust method to interrogate changes in macrophage activity that result from the inhibition of TAM receptor-mediated efferocytosis in an in vitro system. When efferocytosis is inhibited, apoptotic cells undergo secondary necrosis, yielding an increase in inflammatory signals in the microenvironment. These inflammatory cues can then tune macrophage activity to a pro-inflammatory, anti-tumor state. Hence, TAM receptor inhibition may alter macrophage state and promote an anti-tumor immune landscape.
Materials and Methods:: A549 tumor cells were grown to confluency prior to collection of cell lysate and media supernatant. The cell lysates were assessed for quantification of TAM receptors via ELISA. The media supernatant was assessed via Luminex for secreted cytokines. THP-1 monocytes were differentiated into macrophages via treatment with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) for 24 hours. Following an additional 24 hours of rest, THP-1 macrophages were treated with A549 media supernatant for 24 hours and lysed. The cell lysates were then assessed for TAM receptors via ELISA. Prior to coculture, A549 tumor cells were labeled with Vibrant DiO while THP-1 macrophages were labeled with CellTrace Violet. Phagocytosis was assessed via flow cytometry, and cells that stained double positive were considered to be THP-1 macrophages that phagocytosed or efferocytosed A549 tumor cells. Primary human-monocyte derived macrophages (HMDMs) were generated by culturing CD14+ monocytes with 25 ng/mL M-CSF for 6 days. As with THP-1 macrophages, HMDMs were cultured with A549 media supernatant prior to lysis. The cell lysates were then assessed for TAM receptors via ELISA.
Results, Conclusions, and Discussions:: We selected the A549 non-small cell lung cancer cell line for our model because A549 tumor cells overexpress Axl (Figure 1A) and secrete cytokines that promote tumor progression and metastasis, including MCP-1, IL-6, and IL-8 (Figure 1B). We utilized THP-1 macrophages in our initial model of tumor-macrophage interactions because these cells express both Axl and MerTK (Figure 1c). Tyro3 was consistently below the limit of detection, so we did not pursue studying Tyro3 in this system any further. Interestingly, upon treatment with A549 media supernatant, THP-1 macrophage expression of both Axl and MerTK increased. This trend was also observed in primary HMDMs (Figure 1D), reinforcing the claim that the effects of TAM receptor inhibition need to be studied in both the tumor and immune compartments of the TME. The predominant explanation for such an increase in Axl and MerTK expression is type I interferon, i.e., IFN-a and IFN-b, signaling. However, A549 tumor cells do not secrete these cytokines (Figure 1B), meaning that the observed increase in Axl and MerTK expression is not mediated through cytokine signaling. It may instead be mediated through toll-like receptor signaling, which is also known to increase TAM receptor expression in phagocytes, but this remains to be explored. Given our ultimate interest in studying the effects of TAM receptor inhibition in tumor-immune crosstalk, we cocultured the two cell types to assess our ability to detect THP-1 macrophage phagocytosis of A549 tumor cells. Our preliminary data demonstrate that we are indeed able to detect THP-1 macrophage phagocytosis of A549 tumor cells, as indicated by cells that are double positive for the stains of each cell type (Figure 1E). This robust phagocytosis detection method enables future studies to be more high-throughput in nature, as the cells can simultaneously be stained for surface markers to ascertain immune cell state. Furthermore, the media from the cocultures can be harvested and assessed via Luminex to detect changes to the secretome and immune cell activity. Having established our pipeline with THP-1 macrophages, we are currently working with HMDMs to elucidate the effects of TAM receptor inhibition on tumor-macrophage crosstalk and anti-tumor immunity.
