Such findings further support an immunosuppressive role of PMN-MDSCs. 2.6. Abstract (KPC) mouse [16]. Seven days post-transplantation, mice were treated with chemotherapeutics (gemcitabine plus paclitaxel-like Epothilone A), < 0.05 compared to untreated; = 10C20 mice per group. Digital spatial profiling (DSP) of immune-related protein markers across experimental mouse groups and PDAC patient tissue samples was performed. Regions of interest (ROIs) were selected based on tumor (PanCK), immune (CD8, CD45), and stromal (SMA high) regions (Figure S1DCG). DSP demonstrated that in areas of decreased stromal cell infiltration in response to cabo+PD-1Inh treatment, there was an infiltration of CD8+GZMB+Ki67+ cytotoxic T lymphocytes and decreased immunosuppressive immune cell populations (Figure 1E,F). In particular, compared to controls, mice treated with either cabo+PD-1Inh, or chemo+cabo+PD-1Inh triple treatment, significantly decreased tumor weights correlated with a significant decrease in stromal markers alpha smooth muscle actin (Figure 1I), fibronectin and vimentin, and PMN-MDSCs (Figure 1F,H) infiltration, with an increase in CD8+ infiltrating cells (Figure 1ECG). In support of these observations, quantitative RT-PCR confirmed the observations made by the DSP analyses in that a significant increase in CD8 (Figure 1J) and granzyme (Figure 1K) expression in tumors collected from cabo+PD-1Inh, or chemo+cabo+PD-1Inh triple-treated mice, correlated with a decrease in fibronectin expression (Figure 1L). To validate the findings that decreased tumor weights have a strong negative correlation with CTL HSP70-IN-1 proliferation (CD8+/BrdU+ Cells, Figure 1A,D) in the combination-treated mice, a regression analysis was performed between the two variables (tumor/body weight vs. cell proliferation, Figure S2ACH) of all experimental groups. The data strongly support the finding that combination-treated mice possessed an increased number of CD8+/BrdU+ cells (90, )Figure S2H) with a decreased tumor mass (900 mg) compared to their untreated control (Figure S2A). The summarized column-line graph (Figure S2I) clearly showed the inverse relationship between tumor weight and CTL proliferation. 2.2. Organoids Derived from Cabozantinib-Treated Mouse Tumors Exhibit a Decreased Stromal Cell Compartment That Correlates with Increased CD8+ Cells Organoids were derived from tumor tissues collected from the eight experimental groups shown in Figure 1. Light micrographs of organoids in culture (Figure 2A) and H&E stains of embedded HSP70-IN-1 organoids (Figure 2B) demonstrated morphological changes and decreased efficiency of growth in cultures derived from cabo+PD-1Inh, and chemo+cabo+PD-1Inh-treated mice. Cultures were then directly analyzed by flow cytometry for PMN-MDSCs, CD8+ and SMA+ cells carried forward from tumor tissues into the organoid cultures (Figure 2). Organoids derived from mouse groups treated with cabozantinib showed with a significant decrease in PMN-MDSCs reflective of decreased cell viability (Figure 2C,E). The decrease in PMN-MDSCs HSP70-IN-1 correlated with a significant increase in CD8+ cells in cultures derived from cabo+PD-1Inh and chemo+cabo+PD-1Inh-treated mice (Figure 2D,E). An increase in CD8+ cells that were carried forward from tumor tissues to organoid cultures, correlated with a significant decrease in SMA-positive cells (Figure 2D,E). Overall, cabozantinib treatment resulted in a decrease in the number of SMA-positive cells observed in organoid cultures (Figure 2D,E). Open in a separate window Figure 2 Changes in PMN-MDSC, CD8 and SMA cell compartments in organoids directly derived from mouse tumors in response to experimental treatments. (A) Light micrographs of cultured organoids and (B) H&E staining of embedded organoids that were derived from mouse tumors in response to experimental treatments. Flow cytometric contour plots demonstrating the changes in (C) PMN-MDSC, (D) CD8 and SMA cell populations in organoids derived from mouse tumors in response to experimental treatments. Quantification (% cell populations) is shown in (E). * < 0.05 compared to untreated; = 10 mice per group. Collectively, our in vivo and in vitro studies in the PDAC orthotopic mouse and organoid models demonstrate that PMN-MDSCs are likely to contribute to tumor growth, suppression of CD8+ T cell proliferation and effector function that may lead to disruption of the efficacy of checkpoint inhibition. We also documented a significant reduction in the stroma, both in vivo and in vitro, in response to cabozantinib HSP70-IN-1 treatment. 2.3. PMN-MDCSs Disrupt the Efficacy of Checkpoint Inhibition in Mouse-Derived Organoid/Immune Cell Co-Cultures To investigate whether PMN-MDSCs disrupt the efficacy of checkpoint inhibition in Rabbit Polyclonal to KAPCB PDAC tumor survival, we developed a pancreatic cancer organoid/CTL/MDSC co-culture. Figure 3A is an overview of the experimental approach developed by the research team to co-culture pancreatic cancer organoids with autologous immune cells. The protocol is executed, and data analyzed within 10 days of the start of organoid and immune cell cultures (Figure 3A). Importantly,.