Primary outgrowth cultures are a reliable source of human pancreatic stellate cells

Abstract

Recent advances demonstrate a critical yet poorly understood role for the pancreatic stellate cell (PSC) in the pathogenesis of chronic pancreatitis (CP) and pancreatic cancer (PC). Progress in this area has been hampered by the availability, fidelity, and/or reliability of in vitro models of PSCs. We examined whether outgrowth cultures from human surgical specimens exhibited reproducible phenotypic and functional characteristics of PSCs. PSCs were cultured from surgical specimens of healthy pancreas, CP and PC. Growth dynamics, phenotypic characteristics, soluble mediator secretion profiles and co-culture with PC cells both in vitro and in vivo were assessed. Forty-seven primary cultures were established from 52 attempts, demonstrating universal α-smooth muscle actin and glial fibrillary acidic protein but negligible epithelial surface antigen expression. Modification of culture conditions consistently led to cytoplasmic lipid accumulation, suggesting induction of a quiescent phenotype. Secretion of growth factors, chemokines and cytokines did not significantly differ between donor pathologies, but did evolve over time in culture. Co-culture of PSCs with established PC cell lines resulted in significant changes in levels of multiple secreted mediators. Primary PSCs co-inoculated with PC cells in a xenograft model led to augmented tumor growth and metastasis. Therefore, regardless of donor pathology, outgrowth cultures produce PSCs that demonstrate consistent growth and protein secretion properties. Primary cultures from pancreatic surgical specimens, including malignancies, may represent a reliable source of human PSCs. Evidence continues to accumulate implicating the intense desmoplastic reaction common to both chronic pancreatitis (CP) and pancreatic cancer (PC) is not simply a bystander process from a dynamic inflammatory state, but rather represents a complex interplay within the microenvironment responsible for the initiation and maintenance of key pathologic feedback systems. 1 Pancreatic stellate cells (PSCs) appear to represent a critical determinant in the development and maintenance of this desmoplastic microenvironment in pancreatic disease states. 2-4 However, recent investigations have questioned the role of PSCs in cancer. Specifically, depletion of activated PSCs in genetically engineered mouse models of PC has paradoxically demonstrated pro-tumor effects. 5,6 While disparate from prior observations showing the tumor promoting abilities of activated PSCs, 3,7-9 these investigations still implicate an interplay between PSCs, other cells within the tumor microenvironment, and the cancer. 5,6 Given these disparate findings, further investigation into the biology of PSCs in various disease states is essential and necessitates a high degree of experimental standardization. To this effect, an expert panel recently published guidelines regarding PSC isolation, identification, and activation. 10 Human PSCs are most typically isolated via the outgrowth method, whereby direct culture of minced pancreatic s...

Interplay between interferon regulatory factor 1 and BRD4 in the regulation of PD-L1 in pancreatic stellate cells

“…Since activated PSCs are the predominant fibroblasts present in the pancreatic stromal reaction 5 , and the immune cells are very likely to encounter these cells in vivo , we evaluated expression of PD-L1 in an immortalized PSC cell line and in primary PSCs isolated from human PDAC tumors using the outgrowth assay 19 . Human PSCs express PD-L1 mRNA, which in most cases exceeded the PD-L1 mRNA levels present in human PDAC cell lines (Fig.…”

BET inhibitors block pancreatic stellate cell collagen I production and attenuate fibrosis in vivo

“…The human PSC line, which was immortalized with telomerase and SV40 large T antigen and characterized previously (26,28,44), was obtained from Rosa F. Hwang (MD Anderson Cancer Center). Primary cancer-associated PSCs were generated from deidentified fresh human PDAC specimens using primary outgrowth cultures, which have been shown to be a reliable source of human PSCs (23). The primary cancer-associated PSCs were used within 5 to 6 passages from isolation.…”

“…The BET inhibitors decreased COL1A1 and COL1A2 mRNA expression and collagen I protein expression ( Figure 1B). We also isolated primary stellate cells from resected human PDAC specimens using the outgrowth culture assay (23). Treatment of primary cancer-associated PSCs with BET inhibitors decreased collagen I mRNA and protein expression (Figure 1, C and D, and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.88032DS1).…”

Explant culture of sarcoma patients' tissue

“…Such stromal cells could be useful to study tumor stroma interaction in two-dimensional (2D) or three-dimensional (3D) co-culture systems in vitro 36 or in vivo. 37 The time to freezing was o1 month for 10% of the samples (not shown) and o2 months for 40% of samples (Figure 1). Therefore, in 57% of bone tumors and 26% of soft tissue tumors a considerable amount of cells could be obtained in o2 months after diagnostic biopsy (eg, for Ewing patient 1, enough BX-derived cells were obtained two months before resection).…”