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) 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.