Targeting heterogeneous tumor microenvironments in pancreatic cancer mouse models of metastasis by TGFB depletion. Targeting heterogeneous tumor microenvironments in pancreatic cancer mouse models of metastasis by TGFB depletion

The dual tumor-suppressive and promoting function of TGFB signaling has made its targeting challenging. We hereby examined the effects of TGFB depletion by AVID200/BMS-986416(TGFB-TRAP), a TGFB ligand trap, on the tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) murine models with different organ-specific metastasis using single-nuclear RNA-sequencing (snRNA-seq) Overall design: Frozen mouse orthotopic KPC PDAC tumors were homogenized and isolated into single nuclei, modified from a previously described 10x Genomics protocol. Frozen mouse orthotopic KPC PDAC tumors were mechanically homogenized into powder form using mortar and pestle in liquid nitrogen. Lysis buffer containing 0.1% Triton-X (Sigma-Aldrich), 0.2 U/mL NxGen RNase inhibitor (Lucigen), and sucrose density buffer was added to the homogenized tissue and let sit for 3 min on ice. Following lysis, the crude homogenate was pipetted over a 100 mm cell strainer, washed with sucrose density buffer, and centrifuged at 400 rcf for 5 min at 4°C on low brake setting. The supernatant was aspirated, and the pellet was resuspended in resuspension buffer containing PBS + 1% BSA (Sigma-Aldrich) and 0.2 U/mL RNase inhibitor (Lucigen) over a 35 mm filter flow tube. Amplified cDNA was generated from RNA from each single nuclei using the Chromium Next GEM Single Cell V(D)J Reagent Kits v.1.1, per the manufacturer’s instructions.