Low serum mesothelin in pancreatic cancer patients results from retention of shed mesothelin in the tumor microenvironment

Abstract

Mesothelin (MSLN) is overexpressed by many cancers, including pancreatic ductal adenocarcinoma (PDAC) and has consequently become a target for anti-cancer therapeutics. Mature, membrane bound MSLN is cleaved by proteases, releasing a shed form that transits to the circulation. Many patients with mesothelioma and ovarian cancer have abnormally high serum MSLN concentration. However, serum MSLN concentration in PDAC patients rarely exceeds levels of healthy controls. Here, serum MSLN concentration in advanced PDAC patients was examined pre- and post-treatment. Serum MSLN did not correlate with tumor MSLN expression, nor with changes in tumor burden as assessed by PDAC serum tumor marker CA19-9. Subsequently, tumor-bearing mouse models were used to investigate the fate of shed MSLN in PDAC versus a control cervical cancer model. Efficiency of MSLN secretion into the serum was cell-line dependent. Tumors from some PDAC lines had poor MSLN secretion efficiency although these lines had similar or higher MSLN shedding rate, total and surface MSLN expression. Measurements of compartment-specific MSLN concentration taken at equilibrium suggested that tumors with poor MSLN secretion efficiency trapped shed MSLN in the tumor microenvironment (TME), a finding confirmed by dynamic experiments using a doxycycline-inducible MSLN expression system. Tumors with the poorest MSLN secretion efficiency had higher collagen density and increased abundance of MSLN binding partner MUC16. The tumor with the worst secretion efficiency could rebind shed MSLN to the cancer cell surface. Altogether, these data suggest that PDAC can trap shed MSLN within the TME. This finding has potential significance for design of MSLN-targeted therapeutics.

Keywords: MUC16; Mesothelin; Microenvironment; Pancreatic adenocarcinoma.

Fig. 1

A) Baseline serum MSLN and MPF in PDAC patients treated on Phase I/II study of mesothelin-targeted immunotoxin LMB-100 with or without nab-paclitaxel. B) Correlation between serum MSLN and serum MPF at baseline. C) Relationship between cancer cell expression of MSLN in archival tissue samples versus serum MSLN at baseline. D) Relationship between change in serum MSLN with treatment to change in bona fide PDAC tumor marker CA 19–9. Only patients with detectable CA 19–9 that trended with disease burden were considered evaluable for this analysis.

Fig. 2

Efficiency of MSLN secretion into serum is characteristic of cell type. A & B) Concentration of hMSLN in the serum of athymic nude mice bearing subcutaneous tumors was analyzed by ELISA. A) For each mouse in four independent experiments, serum MSLN concentration was plotted versus tumor weight. Slope [95% CI] expressed in ng/mL serum MSLN per g tumor: KB 181 [128–233], KLM1 6.75 [3.80–9.71], T3M4 65.3 [45.5–85.1], AsPC1 97.0 [76.1–118] B) Secretion efficiency of MSLN, defined as serum MSLN per tumor weight, is a constant value the four cell types tested. C) Flow cytometry tracings showing surface MSLN on the indicated cell types. Representative image from at least 3 runs is shown. Graph below compares geometric means of surface MSLN expression normalized to value for KB for each experiment. Meso29 is a mesothelioma cell line used as a positive control. D) MSLN shedding rate was calculated by serial ELISA measurements of MSLN concentration over 6 h in a known volume of medium for a set number of cells.

Fig. 3

Compartment-specific measurements of soluble MSLN and MPF at steady state. Nude mice were inoculated subcutaneously with the indicated cell types and tumors were permitted to grow to size >100 mm³. Harvested tumors were processed as shown in (A). ITF = intratumoral fluid. ITF was isolated from a known mass of tumor by resuspending freshly harvested tumors in equivolume, unsupplemented (serum-free) tissue culture medium, finely mincing tumor, and removing the remaining tumor debris by centrifugation. Measurements of soluble MSLN (B-C) and MPF (D-E) in serum, ITF, total tumor lysate, and lysate from remnant tumor following ITF extraction as measured by ELISA are shown.

Fig. 4

Measuring transit of MSLN between compartments. A) Immunoblot showing MSLN expression for MSLN knock-out (KO) cell lines transduced with a doxycycline (dox)-inducible MSLN expression plasmid (KB+, KLM1+, T3M4+). GAPDH is loading control. B-E) Nude mice were inoculated subcutaneously with the KB+, KLM1+ or T3M4+ cell lines. Dox treatment was administered by IP drug injection to initiate MSLN expression (time 0) once tumors had reached at least 100mm³, and dox-containing chow was provided ad lib to mice for the remainder of the experiment. Mice (at least 3 for each timepoint) were euthanized at the indicated times and serum, ITF, total tumor and lysate from remnant (RMNT) tumor were assessed by ELISA for MSLN concentration. B) Snapshot of MSLN concentrations at steady state (36 h) for each cell line in each compartment as indicated. C-D) MSLN concentration in indicated compartment as measured over time. E) Rate of MSLN secretion into the serum was calculated using semi-mechanistic modeling as described in Materials and Methods.

Fig. 5

Low MSLN secretion efficiency is associated with physical properties of tumor. A) Subcutaneous tumors grown in nude mice were stained with Maisson Trichrome to assess collagen density. Representative images are shown. B) Subcutaneous tumors were grown in nude mice as described previously. Once tumors reached ∼300mm³ (left) trypsin or control (trypsin inactivated by boiling for >30 min), or (right) collagen/dispase solution versus control (collagen/dispase inactivated by boiling for >30 min) was injected directly into tumors. Serum MSLN was examined by ELISA pre- and 24 h post-trypsin treatment. C) Immunoblot of tumor lysates showing expression of MSLN binding partner MUC16. GAPDH is used as a loading control. D) Indicated KO cells were incubated with MSLN-containing conditioned medium (red) or control conditioned medium lacking MSLN (blue). Surface MSLN on the KO cells was then measured by flow cytometry. Tracing for MSLN-expressing KLM1 cells is shown as positive control (gray).