Abstract A58: Notch signaling is a key regulator for immune-suppressive function of tumor-associated macrophages in pancreatic adenocarcinoma

Objective: Pancreatic ductal adenocarcinoma (PDA) has the most dismal prognosis among all human cancers. The poor survival rate has been attributed to high rate of metastasis and low efficacy of current therapies, which may result from the complex immunosuppressive tumor microenvironment. We explored the role of Notch signaling, a key regulator of immune responses, within the PDA microenvironment. Our goal is to better define these immunosuppressive pathways and to develop effective therapies for patients with PDA. Methods: Two primary mouse PDA cell lines (7940B and mT3-2D) were used for implantation into the pancreata of transgenic CBF:H2B-Venus mice; this enabled single-cell reporting of Notch signaling within the PDA microenvironment. Flow cytometry was used to assess Venus expression in different cell populations. In addition, tumor-associated macrophages (TAMs) were sorted by Notch activity [Venus (+) vs. Venus (-)] to analyze expression of Notch targets and macrophage polarization markers by qPCR. Next, a bone marrow-derived macrophage (BMDM) culture system was developed to investigate Notch activation in vitro. Lastly, we evaluated the therapeutic effect of Notch pathway inhibition as monotherapy and in combination with immune checkpoint programmed death receptor-1 (PD-1) blockade in an orthotopic PDA model. Results: Flow cytometry revealed that TAMs were the most abundant immune cell population in the orthotopic PDA microenvironment. About 50% of TAMs were Venus (+), which had significant enrichment in M2 polarization markers such as Arginase 1 (Arg1) and Notch targets (such as Hes1) when compared to Venus (-) TAMs by qPCR. Immunofluorescence showed co-localization of Venus and Hes1 in F4/80+ TAMs. In vitro experiments identified that Notch activation was only present in BMDMs from Venus mice when co-cultured directly with 7940B PDA cells and that Arg1 expression was only found in Venus (+) macrophages. Notch inhibitor LY3039478 led to decreased expression of both M2 polarization markers such as Arg1, Mrc1, Ym1, and immunosuppressive cytokine TGFβ in co-cultured BMDM. In the orthotopic PDA mouse model, neither LY3039478 nor anti-PD1 was effective in treating PDA in vivo, whereas combined inhibition of the Notch pathway and immune checkpoint resulted in significant reduction in tumor growth. Conclusion: Our data demonstrate that Notch activation within TAMs from murine PDAs is associated with an immunosuppressive (M2-like) phenotype, thereby sugges