Three-Dimensional Primary Cell Culture: A Novel Preclinical Model for Pancreatic Neuroendocrine Tumors

Abstract

Molecular mechanisms underlying the development and progression of pancreatic neuroendocrine tumors (PanNETs) are still insufficiently understood. Efficacy of currently approved PanNET therapies is limited. While novel treatment options are being developed, patient stratification permitting more personalized treatment selection in PanNET is yet not feasible since no predictive markers are established. The lack of representative in vitro and in vivo models as well as the rarity and heterogeneity of PanNET are prevailing reasons for this. In this study, we describe an in vitro 3-dimensional (3-D) human primary PanNET culture system as a novel preclinical model for more personalized therapy selection. We present a screening platform allowing multicenter sample collection and drug screening in 3-D cultures of human primary PanNET cells. We demonstrate that primary cells isolated from PanNET patients and cultured in vitro form isletlike tumoroids. Islet-like tumoroids retain a neuroendocrine phenotype and are viable for at least 2 weeks in culture with a high success rate (86%). Viability can be monitored continuously allowing for a perwell normalization. In a proof-of-concept study, islet-like tumoroids were screened with three clinically approved therapies for PanNET: sunitinib, everolimus and temozolomide. Islet-like tumoroids display varying in vitro response profiles to distinct therapeutic regimes. Treatment response of islet-like tumoroids differs also between patient samples. We believe that the presented human PanNET screening platform is suitable for personalized drug testing in a larger patient cohort, and a broader application will help in identifying novel markers predicting treatment response and in refining PanNET therapy Molecular mechanisms underlying the development and progression of PanNET are still insufficiently understood. Efficacy of currently approved PanNET therapies is limited. While novel treatment options are being developed, patient stratification permitting more personalized treatment selection in PanNET is yet not feasible since no predictive markers are established. The lack of representative in vitro and in vivo models as well as the rarity and heterogeneity of PanNET are prevailing reasons for this.

EZH2 Inhibition as New Epigenetic Treatment Option for Pancreatic Neuroendocrine Neoplasms (PanNENs)

“…For primary cell isolation, viability measurement, micro-cell block manufacture, and quantification, we followed the described protocol [ 24 ]. Fresh human PanNET tissue was obtained from patients diagnosed with PanNETs undergoing surgery at the Inselspital Bern, Switzerland, or at the Pancreatic Surgery Unit, Pancreas Translational and Clinical Research Center, San Raffaele Scientific Institute, Milan, Italy, as previously described [ 24 ]. Cryopreserved tumor tissues of six PanNET patients were used for in vitro drug screening.…”

“…Cells were isolated and cultured as previously reported [ 24 ]. In brief, washed pieces of 1 mm 3 were dissociated in digestion medium (10 mg/mL collagenase IV (Worthington, USA), 0.25% Trypsin-EDTA (Sigma-Aldrich, Switzerland), and 0.2 mg/mL DNase (Roche, Switzerland) in advanced DMEM-F12, Hepes 10 mM, 1% L-glutamine, 1% penicillin-streptomycin-amphotericin B) using a gentle MACSTM dissociator (Miltenyi Biotec, Switzerland).…”

“…A 5-point, 625-fold concentration range was used to calculate reliable absolute IC50 values [ 25 ]. For IC50 calculation, RLU values from a 7 day treatment were weighted and normalized using 6 h RTG-baseline measurements for each well, as described earlier [ 24 ]. Data points were fitted in a four-parameter linear (4PL) regression model with two constraints, Top = 100% and Bottom = 0%, to estimate the corresponding IC50 [ 26 , 27 ].…”

Models of Gastroenteropancreatic Neuroendocrine Neoplasms: Current Status and Future Directions

“…Despite the difficulties encountered when growing NETs as pdx or permanent cell lines, NET cells can be maintained as primary cultures for several days and be subjected to pharmacologic testing [32, 71-74]. In general, proliferation is low or absent, making the amount of primary tissue a major limitation for all subsequent analyses.…”

“…A most promising approach to in vitro drug screening of patient-derived pNET primary cultures was developed by combining a short- to mid-term culture approach with 3D culture conditions and optimized growth factor supply. Organoids formed, which faithfully reproduce proliferation rates and differentiation characteristics of the original tumor tissue, and differentially responded to established clinical drugs, for example, sunitinib, everolimus, and temozolomide [74]. So far, we still lack studies that connect such in vitro drug screening of primary cultures with clinical drug responses, but in principle, they offer the prospect of personalized in vitro drug trials.…”