Establishment and Characterization of Paired Primary Cultures of Human Pancreatic Cancer Cells and Stellate Cells Derived from the Same Tumor

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extremely poor prognosis, and its treatment remains a challenge. As the existing in vitro experimental models offer only a limited resemblance to human PDAC, there is a strong need for additional research tools to better understand PDAC tumor biology, particularly the impact of the tumor stroma. Here, we report for the first time the establishment and characterization of human PDAC-derived paired primary monolayer cultures of (epithelial) cancer cells (PCCs) and mesenchymal stellate cells (PSCs) derived from the same tumor by the outgrowth method. Characterization of cell morphology, cytostructural, and functional profiles and proteomics-based secretome analysis were performed. All PCCs harbored KRAS and TP53 mutations, and expressed cytokeratin 19, ki-67, and p53, while the expression of EpCAM and vimentin was variable. All PSCs expressed α-smooth muscle actin (α-SMA) and vimentin. PCCs showed a significantly higher growth rate and proliferation than PSCs. Secretome analysis confirmed the distinct nature of PCCs as compared to PSCs and allowed identification of potential molecular regulators of PSC-conditioned medium (PSC-CM)-induced migration of PCCs. Paired primary cultures of PCCs and PSCs derived from the same tumor specimen represent a novel experimental model for basic research in PDAC tumor biology.

Senescent Human Pancreatic Stellate Cells Secrete CXCR2 Agonist CXCLs to Promote Proliferation and Migration of Human Pancreatic Cancer AsPC-1 and MIAPaCa-2 Cell Lines

“…The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Primary hPSCs were isolated from the resected pancreatic tissues of patients who underwent surgery for pancreatic cancer, as previously described [ 55 , 56 ]. hPSCs were maintained in Ham’s F-12/DMEM (1:1) supplemented with 10% FBS and antibiotics in a humidified incubator with 5% CO 2 at 37 °C.…”

Differential Gemcitabine Sensitivity in Primary Human Pancreatic Cancer Cells and Paired Stellate Cells Is Driven by Heterogenous Drug Uptake and Processing

“…Human PDAC-derived paired primary cultures of cancer cells (PCCs) and stellate cells (PSCs) were obtained from the same surgically resected specimens by using the outgrowth method [34,49]. Four paired primary cultures were grown from different PDACs, of which two were treatment-naïve (PC-1, PC-2), and two were neoadjuvantly treated (PC-5, PC-6) with one cycle of Folfirinox followed by five cycles Gemzar-Abraxane (PC-5), and seven cycles Gemzar (PC-6), respectively.…”

“…Four paired primary cultures were grown from different PDACs, of which two were treatment-naïve (PC-1, PC-2), and two were neoadjuvantly treated (PC-5, PC-6) with one cycle of Folfirinox followed by five cycles Gemzar-Abraxane (PC-5), and seven cycles Gemzar (PC-6), respectively. Detailed information on the establishment and characterization of cells, clinicopathological features of the source tumors, patient survival, and treatment is provided in our recent study [34]. The established cultures were designated as PCC-and PSC-1, -2, -5, and -6, respectively.…”

“…In further gemcitabine uptake experiments, the co-culture system of paired PCCs and PSCs was used, as described in our previous study [15]. Briefly, for indirect co-culture experiments, PCCs and PSCs seeded individually at a density of~5000 cells/well in 96-well plates were exposed to conditioned medium from their paired PSCs (PSC-CM) and PCCs (PCC-CM), respectively, for 48 h. Conditioned medium was prepared as described previously [34]. For direct co-cultures, paired PCCs and PSCs were seeded at an equal density of~3000 cells/well in 96-well plates and cultured together for 48 h. The co-cultures were exposed to [ 3 H]-gemcitabine for 4 h and evaluated for intracellular uptake of gemcitabine.…”

“…In a recent report, we demonstrated the establishment of a novel model system comprising human PDAC-derived paired primary cultures of primary cancer cells (PCCs), and PSCs derived from the same resected tumor specimen [34]. In the present study, paired primary cultures of PCCs and PSCs along with three commonly used PDAC cell lines, BxPC-3, Mia PaCa-2, and Panc-1, were used to explore the fate of gemcitabine in human PDAC.…”