Isolation of Pancreatic Cancer Cells from a Patient-Derived Xenograft Model Allows for Practical Expansion and Preserved Heterogeneity in Culture

Abstract

Commercially available, highly passaged pancreatic cancer (PC) cell lines are of limited translational value. Attempts to overcome this limitation have primarily consisted of cancer cell isolation and culture directly from human PC specimens. However, these techniques are associated with exceedingly low success rates. Here, we demonstrate a highly reproducible culture of primary PC cell lines (PPCLs) from patient-derived xenografts, which preserve, in part, the intratumoral heterogeneity known to exist in PC. PPCL expansion from patient-derived xenografts was successful in 100% of attempts (5 of 5). Phenotypic analysis was evaluated with flow cytometry, immunofluorescence microscopy, and short tandem repeat profiling. Importantly, tumorigenicity of PPCLs expanded from patient-derived xenografts was assessed by subcutaneous injection into nonobese diabeteic.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ mice. Morphologically, subcutaneous injection of all PPCLs into mice yielded tumors with similar characteristics to the parent xenograft. PPCLs uniformly expressed class I human leukocyte antigen, epithelial cell adhesion molecule, and cytokeratin-19. Heterogeneity within each PPCL persisted in culture for the frequency of cells expressing the cancer stem cell markers CD44, CD133, and c-Met and the immunologic markers human leukocyte antigen class II and programmed death ligand 1. This work therefore presents a reliable method for the rapid expansion of primary human PC cells and, thereby, provides a platform for translational investigation and, importantly, potential personalized therapeutic approaches.

Figure 1

Hematoxylin and eosin-stained micrographs are displayed as follows for (from top to bottom): human pancreatic cancer specimens, PDX tumors, PPCLs, and PPCL-derived subcutaneous xenografts. Scale bars: 200 μm. PDX, patient-derived xenograft; PPCL, primary pancreatic cancer cell line.

Figure 2

PPCL population express markers consistent with pure human PCCs. A: PPCLs were examined for staining of HLA-ABC, EpCam, CK19, and CD45 and α-SMA. Cells were gated according to forward/side scatter profiles, and isotype control histograms are shown in gray. B: PPCLs were analyzed for EpCam, CK18, CK19, and CD45 expression with the use of immunocytochemistry. Each indicated marker is displayed in red. The nuclear stain DAPI is shown in blue. Scale bar, 20 μm. CK19, cytokeratin-19; EpCam, epithelial cell adhesion molecule; HLA, human leukocyte antigen; PPCL, primary pancreatic cancer cell line; α-SMA, α-smooth muscle actin.

Figure 3

Growth dynamics and chemosensitivity of PPCLs. A and B:In vivo (A) and in vitro (B) growth curves of PPCLs through 6 and 40 days, respectively. The growth rate of PPCLs and corresponding tumors were evaluated. Mean tumor volume is presented at each time point. C: Dose-dependent response to gemcitabine treatment of PPCLs, at concentration of 0, 3, 10, 30, 100, 300, and 1000 nmol/L. The IC₅₀ of each line was measured. Viability at each concentration is presented. D: The doubling time, in vitro and in vivo growth rates, and IC₅₀ for each PPCL are summarized. Data are expressed as means ± SEM. n = 3 (A and B); n = 6 (C). All experiments were performed in triplicate. IC₅₀, half maximal inhibitory concentration; PPCL, primary pancreatic cancer cell line.

Figure 4

PPCLs demonstrate heterogeneity in markers associated with cancer stemness and PC-immune cell interactions. A and B: PPCLs were evaluated for the cancer stem cell markers CD44, CD133 (A) and c-Met (B). The percentage of cells positive for both CD144 and CD33 are indicated in the rectangular gate. C: Immunohistochemical staining for PDL1 was performed on patient-derived xenografts from each patient. C–F: In addition, cell populations were analyzed for the expression of the negative costimulatory marker PDL1 (C), antigen presentation machinery (HLA-DR/DP/DQ; D), and the positive costimulatory markers CD80 (E) and CD86 (F). Histograms are shown for isotype controls (gray) and staining for the indicated marker (black). Scale bar = 200 μm (C). HLA, human leukocyte antigen; PDL1, programmed death ligand-1; PPLC, primary pancreatic cancer cell line.