Mesothelin Enhances Tumor Vascularity in Newly Forming Pancreatic Peritoneal Metastases

Abstract

Over 90% of pancreatic ductal adenocarcinomas (PDAC) express mesothelin (MSLN). Overexpression or knockdown of MSLN has been implicated in PDAC aggressiveness. This activity has been ascribed to MSLN-induced activation of MAPK or NF-κB signaling pathways and to interaction of MSLN with its only known binding partner, MUC16. Here, we used CRISPR/Cas9 gene editing to delete MSLN from PDAC, then restored expression of wild-type (WT) or Y318A mutant MSLN by viral transduction. We found that MSLN KO cells grew in culture and as subcutaneous tumors in mouse xenografts at the same rate as WT cells but formed intraperitoneal metastases poorly. Complementation with WT MSLN restored intraperitoneal growth, whereas complementation with Y318A mutant MSLN, which does not bind MUC16, was ineffective at enhancing growth in both MUC16(+) and MUC16(-) models. Restoration of WT MSLN did enhance growth but did not affect cell-to-cell binding, cell viability in suspension or signaling pathways previously identified as contributing to the protumorigenic effect of MSLN. RNA deep sequencing of tumor cells identified no changes in transcriptional profile that could explain the observed phenotype. Furthermore, no histologic changes in tumor cell proliferation or morphology were observed in mature tumors. Examination of nascent MSLN KO tumors revealed decreased microvascular density as intraperitoneal tumors were forming, followed by decreased proliferation, which resolved by 2 weeks postimplantation. These data support a model whereby MSLN expression by tumor cells contributes to metastatic colonization. IMPLICATIONS: MSLN confers a growth advantage to tumor cells during colonization of peritoneal metastasis. Therapeutic blockade of MSLN might limit peritoneal spread.

Figure 1.

Synthesis and validation of MSLN KO cells and effect on tumor growth. A, Immunoblot of cell culture lysates showing increased expression of MSLN in KLM1 compared with PK1. GAPDH was used as loading control. B, Surface expression of MSLN as measured by flow cytometry on parental KLM1, Mock, KO#1 and KO#2 cell lines. Solid line shows cells stained with anti-MSLN primary antibody and dotted line shows stained with isotype control. C, Immunoblot of cell culture lysates for MSLN (40 kDa). Actin was used as loading control (D). Average MPF concentration in the media of cells cultured for 72 hours was measured by ELISA. Data are the results of triplicate readings from a representative experiment. E, Parental KLM1, Mock, KO#1 and KO#2 were treated with a concentration gradient of the MSLN-targeting immunotoxin, LMB-100 (left), or the transferrin receptor-targeting immunotoxin LMB-74 (right). Representative experiment shows n = 6 replicates per data point. F, In vitro growth rates of cell lines. Biological triplicates from a representative experiment are shown. Cell numbers at 96 hours were not statistically different between groups as determined by ANOVA (N.S.). G, Nude mice were inoculated subcutaneously into the flank with equal numbers of Mock, KO#1 or KO#2 cells and then tumor volume was serially measured by digital calipers over 25 days (average shown for n = 10 mice per group). No statistically significant difference seen by ANOVA (N.S.). H, Nude mice were inoculated orthotopically into the pancreas with equal numbers of Mock or KO#2 cells (Mock n = 19, KO#2 n = 17 over three separate experiments). Mice were euthanized after 5 to 6 weeks. Pancreata were collected and weighed. Dotted line indicates average normal pancreas weight of nude mice (0.12 g; *, P < 0.05; Mann–Whitney nonparametric test).

Figure 2.

Effect of MSLN KO on intraperitoneal metastasis model. A, Nude mice were inoculated intraperitoneally with 3 × 10⁶ parental KLM1, Mock, KO#1, or KO#2 cells. Mice were euthanized after approximately 6 weeks and all visible tumor in the abdominal cavity was harvested and weighed. Shown are composite results from two experiments (n = 18 for Mock, KO#1 and KO#2; n = 17 for Parent). **, P < 0.01, ****, P < 0.0001 (Mann–Whitney non-parametric test; B), Representative histology stainings of tumors (n = 3–6 mice per group) from (A); scale bars, 300 μm. Samples were analyzed by a consultant veterinary pathologist who found no differences (tumors from n = 3–5 mice obtained from two independent experiments). Quantitation is shown below for staining of Ki-67, SMA (a marker of cancer-associated fibroblasts), collagen on Masson Trichrome and CD31 (a vascular endothelial marker). N.S. = not significant. C, Schema of inoculations for D. D, Experiment in A was repeated but some KO tumors were grown for 3 additional weeks beyond original endpoint (n = 10–12/ group over 3 independent experiments). *, P < 0.05, N.S. = not significant (Mann–Whitney nonparametric test. E, Location of tumor deposits found in animals from D. F, Lysates from harvested KLM1 KO#2 intraperitoneal tumors grown for 9 weeks (9wo) were immunoblotted with anti-MSLN antibody to demonstrate loss of expression of MSLN in vivo. KLM1 Mock intraperitoneal tumors grown for 6 weeks (9wo) were used as positive control for MSLN expression. GAPDH was used as loading control.

Figure 3.

Role of MSLN in establishment of intraperitoneal tumors. A, Schema of time-course experiments. A total of 3 × 10⁶ Luc/GFP cells were inoculated into the mouse intraperitoneal cavity. (n = 4–8/ group over three independent experiments; B–D) Histologic analyses of intraperitoneal tissues containing tumors of (B) microvessel density at 3 (D3), 7 (D7), and 14 (D14) days, green lines show tumor regions selected for pathological analysis (C) proliferation;*, P < 0.05; **, P < 0.01, N.S. = not significant (Mann–Whitney nonparametric test) and (D) invasion of tumor tissue at 7 days after intraperitoneal tumor cell inoculation. 4/5 (80%) Mock and 1/6 (17%) KO#2 tumor tissues showed invasion. Insets (B–D) show magnification of region in red box; scale bars, 600 μm.

Figure 4.

Expression of WT but not Y318A MSLN enhances tumor cell growth. A, Schema of the engineered constructs. B, Growth rate of KLM1 cell lines (Mock, KO#2, and the transduced cell lines KO#2+WT or KO#2+Y318A) in culture was measured by counting triplicate wells on the indicated days. There was no statistically significant difference in cell count at 96 hours as determined by ANOVA (N.S.). C, Nude mice were inoculated into the abdominal cavity with 3 × 10⁶ cells of the indicated cell types. Mice were euthanized after approximately 6 weeks. All visible tumor in the abdominal cavity was harvested and weighed. Figure shows average for 10 mice per group; **, P < 0.01; ***, P < 0.001, N.S. not significant (Mann–Whitney nonparametric test). D, Growth rate of parent MIA PaCa-2 cells and those transduced with WT or Y318A mutant MSLN was measured by counting triplicate wells on the indicated days. A statistically significant increase in growth was found in +WT cells compared with parent and +Y318A (*, P < 0.05 on post hoc comparison). E, Intraperitoneal growth of MIA PaCa-2–derived cells was assessed as in C. Figure shows average for 9–10 mice per group; *, P < 0.05 and **, P < 0.01, N.S. = not significant (Mann–Whitney nonparametric test).

Figure 5.

Identifying a mechanism for MSLN tumorigenicity. A, Lysates from harvested KLM1 intraperitoneal tumors were immunoblotted with anti-MSLN antibody to demonstrate continued expression of the transgene in vivo. B and C, Tumor cells were plated at equal numbers onto ultra-low adherence plates. B, After 24 hours, the cells were stained with crystal violet and photographed. C, Viability was measured by colorimetric assay after 72 hours. N.S. = no significant difference. D, Cell membrane expression of MUC16 was measured by flow cytometry (black outline). Shaded peak shows control where primary antibody was omitted. Immunoblots were performed to examine: E, Total MUC16 expression and associated quantitation (F) total and phosphorylated ERK and p38, and (G) total OCT-2 expression and associated quantitation in KLM1 and/or MIA PaCa-2 cultured cell lysates. GAPDH was used as loading control in A and E–G.

Figure 6.

Identifying a novel mediator for MSLN activity using RNAseq and qPCR. A, Analysis schema for RNAseq transcriptional profiling. Thirteen genes were identified to be differentially expressed with at least a 1.5-fold change in expression and P < 0.05 in both KO#1 and KO#2 cell lines and tumor tissues as compared with Mock. B, qPCR showing PPP1R1B relative gene expression in KLM1 cell lines ***, P < 0.001, N.S. = not significant (Student t test with Welch correction). C, Immunoblot showing PPP1R1B expression in KLM1-cultured cell lysates. GAPDH was used as loading control.