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. 2021 Oct 23;22(21):11452.
doi: 10.3390/ijms222111452.

The Collagen Receptor uPARAP in Malignant Mesothelioma: A Potential Diagnostic Marker and Therapeutic Target

Pınar Çakılkaya  1 , Rikke Raagaard Sørensen  2 , Henrik Jessen Jürgensen  1 , Oliver Krigslund  1 , Henrik Gårdsvoll  1 , Christoffer F Nielsen  3 , Eric Santoni-Rugiu  1   2 , Niels Behrendt  1 , Lars H Engelholm  1
Affiliations

The Collagen Receptor uPARAP in Malignant Mesothelioma: A Potential Diagnostic Marker and Therapeutic Target

Pınar Çakılkaya et al. Int J Mol Sci. .

Abstract

Malignant mesothelioma (MM) is a highly aggressive cancer with limited therapeutic options. We have previously shown that the endocytic collagen receptor, uPARAP, is upregulated in certain cancers and can be therapeutically targeted. Public RNA expression data display uPARAP overexpression in MM. Thus, to evaluate its potential use in diagnostics and therapy, we quantified uPARAP expression by immunohistochemical H-score in formalin-fixed paraffin-embedded bioptic/surgical human tissue samples and tissue microarrays. We detected pronounced upregulation of uPARAP in the three main MM subtypes compared to non-malignant reactive mesothelial proliferations, with higher expression in sarcomatoid and biphasic than in epithelioid MM. The upregulation appeared to be independent of patients' asbestos exposure and unaffected after chemotherapy. Using immunoblotting, we demonstrated high expression of uPARAP in MM cell lines and no expression in a non-malignant mesothelial cell line. Moreover, we showed the specific internalization of an anti-uPARAP monoclonal antibody by the MM cell lines using flow cytometry-based assays and confocal microscopy. Finally, we demonstrated the sensitivity of these cells towards sub-nanomolar concentrations of an antibody-drug conjugate formed with the uPARAP-directed antibody and a potent cytotoxin that led to efficient, uPARAP-specific eradication of the MM cells. Further studies on patient cohorts and functional preclinical models will fully reveal whether uPARAP could be exploited in diagnostics and therapeutic targeting of MM.

Keywords: ADC; CD280; Endo180; MRC2; antibody-drug conjugate; extracellular matrix; immunohistochemistry; mesothelioma; tumor microenvironment; uPARAP.

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Conflict of interest statement

C.F.N., N.B. and L.H.E. are cofounders of, and have shares in, the spin-out company Adcendo ApS that works to develop ADC-based cancer drugs. C.F.N. is an employee of Adcendo ApS. Other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The gene expression profile of MRC2. TCGA data have been adapted from the GEPIA web server [20,21]. Dot plots (mRNA levels) across tumor samples are shown in red. MESO represents malignant mesothelioma [18]. For most tumors (although not for MM), data for their normal tissue of origin are also shown in green. The median values are indicated with bars. For the abbreviations of the analyzed cancer types, see Table S1.
Figure 2
Figure 2
IHC analysis of uPARAP expression in tissue sections from MM and RMP. (a) Graphs indicate H-scores that were generated by uPARAP IHC analysis of FFPE tissue sections from biopsies taken from chemotherapy-naïve patients (upper graph: RMP (n = 3), EMM (n = 5), BMM (n = 7), SMM (n = 8)) or pleural resections from patients after neoadjuvant chemotherapy (lower graph: RMP (n = 10), EMM (n = 7), BMM (n = 10), SMM (n = 3)). Data are shown as mean ± SD for the individual MM subtypes and RMPs. Welch’s ANOVA test was used for the comparison of the groups. *: (p ≤ 0.05), ***: (p ≤ 0.0008), ****: (p ≤ 0.0001). (b) Representative examples of H&E, uPARAP and pan-cytokeratin (CK) staining performed on parallel tissue sections from a pleural RMP induced by spontaneous pneumothorax. The CK staining highlights the CK-positive layer of hyperplastic mesothelium covering the pleura affected by chronic inflammation, vessel proliferation and mild fibrosis. The inset in the image from the corresponding uPARAP immunostaining (left) is illustrated at higher magnification (right) to highlight the lack of uPARAP expression in the hyperplastic mesothelium and underlying pleural stroma. (c) Representative examples of H&E and uPARAP staining performed on parallel tissue sections from samples representing the three MM subtypes (EMM, BMM, and SMM). The insets in the images from uPARAP immunostainings taken at low magnification (middle row) are illustrated at higher magnification at the bottom. High magnification bars: 100 µm, low magnification bars: 50 µm.
Figure 3
Figure 3
IHC analysis of uPARAP expression in TMAs from MM and RMP. (a) The graph indicates H-scores obtained by uPARAP IHC analysis of cores representing RMP (n = 13), EMM (n = 73), BMM (n = 27) and SMM (n = 17). Data are shown as mean ± SD for the individual MM subtypes and RMPs. Welch’s ANOVA test was used for the comparison of the groups. **** (p ≤ 0.0001). (b) Representative examples of uPARAP staining in TMA cores from RMP (no staining in the mesothelium and fibrotic stroma, except for scattered stained macrophages), EMM, BMM, and SMM tissue (all three showing diffuse positive uPARAP expression). Note the stronger stain of the sarcomatoid vs. epithelioid tumor cells in the BMM example. Scale bars: 100 µm.
Figure 4
Figure 4
Expression of uPARAP in various cell lines. The relative expression was assessed by western blot in a non-malignant human mesothelial cell line (MeT-5A), human mesothelioma cell lines (ONE58, JL-1, H-Meso-1) and positive and negative control cells (osteosarcoma cell lines 143B +/+ and 143B -/-; see text). Bands of apparent 180 kD molecular mass indicate expression of uPARAP in all of the cancer cell lines except for the negative control. Very low expression levels were observed in MeT-5A. Comparable loading of total protein was confirmed by Coomassie® blue staining of residual protein in the SDS gel, left after electroblotting (Figure S5).
Figure 5
Figure 5
Specific uptake of anti-uPARAP mAb in MM cells. (a) Analysis of mAb uptake by flow cytometry. Cells were analyzed after uptake of Alexa Fluor 647 conjugated mAb 9b7 (AF647-9b7) against uPARAP. The AF647-9b7 uptake is shown as histograms (in pink) for all of the cell lines examined (MM cell lines ONE58, JL-1 and H-Meso-1 and osteosarcoma lines 143B +/+ and 143B -/-). Histograms for cells without antibody (orange) and with cells pre-incubated with competing anti-uPARAP mAb 5f4 before the addition of AF647-9b7 (light blue) are included as controls. The bar chart shows the mean fluorescence intensity of the analyzed samples (MFI ± SD). (b) Internalization of anti-uPARAP mAb in H-Meso-1, JL-1 and ONE58 cells, shown by confocal imaging. Cells were analyzed after uptake of AF647-9b7 against uPARAP or Alexa Fluor 647-conjugated mAb aTNP (AF647-aTNP) as a negative control. In the right panel, the merged images are displayed after incubation with AF647-9b7 (red) and AF647-aTNP (red); cells were stained with WGA Alexa Fluor 488-conjugate for surface staining (green) and Hoechst stain 33258 for visualization of the cell nuclei (blue). The internalized amount is displayed in greyscale representation (left panels). Scale bar: 20 μm.
Figure 6
Figure 6
Effect of uPARAP-directed ADCs on human cancer cell lines. Graphs depict the viability of uPARAP-expressing cells after 6 days of culture in the presence of the indicated concentrations of 9b7-PNU (blue) or aTNP-PNU (red). The green dotted line shows the viability of uPARAP negative (143B -/-) cells, treated with 9b7-PNU in the same manner (receptor negative control). The percentage of viable cells is shown relative to an untreated control population. Data are presented as mean ± SD.
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