Malignant mesothelioma-associated inflammatory microenvironment promotes tumor progression via GPNMB

Abstract

Background: Tumor-Associated Macrophages (TAMs) are the main immune component of the tumor stroma with heterogeneous functional activities, predominantly suppressing the immune response and promoting tumor progression, also via secretion of different factors. Among these, GPNMB (Glycoprotein non-metastatic B) is usually associated with disease progression in several tumor types. Malignant pleural mesothelioma (MPM) a severe neoplasia with poor prognosis, is characterized by an abundancy of TAMs, testifying the presence of a long-lasting inflammation which is pathogenetic of the disease. However, the role of GPNMB in MPM is unclear.

Methods: Clinical samples from patients with MPM were used to measure RNA and protein levels of GPNMB. The functional role of GPNMB in vivo was studied in an orthotopic mouse model of mesothelioma using the murine cell lines AB1 and AB22. Experiments included in vivo tumor growth in wild type and in GPNMB-deficient mice and blocking of GPNMB-induced signaling with anti-CD44 antibodies.

Results: We show that in human and murine MPM tissues the protein GPNMB is mainly produced by infiltrating TAMs. Gpnmb RNA levels in MPM patients from TCGA are significantly associated with lower survival. Using an orthotopic mouse model of mesothelioma we observed that in GPNMB-defective mice (DBA2/J mice) unable to produce the protein, tumors formed by AB1 and AB22 mesothelioma cells grow significantly less than in GPNMB-proficient mice (DBA2/J-Gpnmb+ mice), indicating that host GPNMB is involved in tumor progression. Likewise, the ectopic expression of GPNMB in AB1 and AB22 cells causes an acceleration of tumor growth in vivo, significantly different compared to mock-transduced cells. Treatment of tumor-bearing mice with blocking anti-CD44 (a major receptor for GPNMB) results in a significant reduction of tumor growth.

Conclusions: Overall, these results indicate that the protein GPNMB, a product and marker gene of TAMs, is a driver of mesothelioma progression and may constitute a promising therapeutic target.

Keywords: Anti-CD44; GPNMB; Malignant mesothelioma; Orthotopic model; Tumor-associated macrophages.

Fig. 1

Expression of GPNMB in human malignant mesothelioma tissues. A TCGA analysis in 85 MPM patients for mRNA levels of Gpnmb, CD44 and IL-33 genes, and B survival of patients classified according to high/low expression of the GPNMB and CD44 genes. P value by log-rank test. C Plasmatic levels of soluble GPNMB measured by ELISA in 72 patients with pathologically confirmed diagnosis of MPM and in 86 healthy donors. Statistical analysis Unpaired t-test (< 0.0001). D Representative images of immunohistochemistry of GPNMB and E of CD206 in two distinct cases of human MPM. F heat map quantification in 28 MPM patients. G RNAscope experiment in two distinct cases of human MPM: single staining of Gpnmb RNA (purple) or CD68 (brown); double staining (H) is shown for each corresponding sample

Fig. 2

Growth of murine MPM cells in DBA/2J-Gpnmb mutant mice. A Crossing of DBA/2J mice (GPNMB-deficient) and DBA/2J-GPNMB + mice (GPNMB-reconstituted) with BALB/c mice to generate a hybrid strain on a BALB/c background. BALB/c X DBA/2J strain (GPNMB KO) and the reconstituted strain BALB/c X DBA/2J-Gpnmb + (GPNMB WT) were used in the experiments. B Tumor progression (mean values of 5 mice) of AB1 cells in GPNMB KO mice (red lines) and in GPNMB WT mice (black lines), and C in each single mouse. Results are expressed as fold increase (F.I.) relative to the previous quantification. D Tumor progression (mean values of 5 mice) of AB22 cells in GPNMB KO mice (blue lines) and in GPNMB WT mice (black lines) and E in each single mouse. Statistical analysis Two-way ANOVA

Fig. 3

Effects of GPNMB on the growth of murine MPM cells in vitro. A ELISA of GPNMB in Mock and GPNMB-transduced AB1 and AB22 cells. B In vitro proliferation of Mock and GPNMB-transduced cells. C In vitro sphere formation of Mock and GPNMB-transduced tumors cells from AB1 and AB22 cell lines. D Quantification of spheres and phenotype analysis for stem cell markers in adherent Mock-AB1 cells (adh) and in dissociated spheres from GPNMB-AB22 cells (sph). E Same as in D for AB22 cells. Statistical analysis Two-way ANOVA for B, **** p < 0.01, Student t-test, Welch’s correction for D and E

Fig. 4

Effects of GPNMB on the growth of murine MPM cells in vivo. A Tumor progression of AB1 GPNMB-transduced and Mock cells in vivo (mean values of 5 mice) and, B in each single mouse. Results are expressed as fold increase (F.I.) relative to the previous quantification. C In vivo imaging by IVIS. D Tumor progression of AB22 GPNMB-transduced and Mock cells (mean values of 5 mice) and, E in each single mouse. F in vivo imaging by IVIS. G Representative images of histopathology of murine lungs of mice injected with AB1 GPNMB-transduced and Mock cells, and H of mice injected with AB22 GPNMB-transduced and Mock cells. I Histopathological quantification of tumor nodules. L Immuno-histochemistry of tumor sections stained with H&E, anti-Iba1 (macrophages), anti-GPNMB, anti-CD31 (vessels) and anti CD8 (T lymphocytes). M Quantification of the tumor area positive for GPNMB. N Quantification of the tumor area positive for Iba1. Statistical analysis: two-way ANOVA for A, B, D, E; Student t-test Welch’s correction for I (p < 0.01) and L (p < 0.05)

Fig. 5

Blockade of the CD44 receptor reduces tumor growth in vivo. Mice were treated with a neutralizing anti-CD44 mAb or an irrelevant Ab (10 mg/kg) at days 8, 11, 14, 17 and 20 post AB22 cell injection. A Tumor progression of AB22 cells in control and anti-CD44 treated animals (mean values of 5 mice, expressed as average radiance) and, B in each single mouse. C Results expressed as fold increase (F.I.) relative to the previous quantification (mean values of 5 mice, and D in each single mouse. E In vivo imaging by IVIS. F Immunohistochemistry of tumor nodules stained for the quantification of the leukocyte infiltrate and for the expression of GPNMB. Statistical analysis Mixed effect model for A–D; Student t-test Welch’s correction for F (*: < 0.05; ***: (*: < 0.01))