Examination of cells and acquisition of images were done using a Zeiss LSM 800 confocal microscope. == 4.8. 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:uPARAP, Endo180, CD280, MRC2, mesothelioma, antibody-drug conjugate, ADC, immunohistochemistry, tumor microenvironment, extracellular matrix == 1. Introduction == MM is a highly aggressive cancer type originating from the mesothelium lining the pleura or other serosal cavities, including the peritoneum, pericardium, and tunica vaginalis testis. Pleural MM is the most common of these, representing 90% of all cases. MM is a particularly challenging disease due to its heterogeneity, often delayed diagnosis with conventional methods and inadequate response to current treatment options. Consequently, the prognosis of pleural MM remains dismal, with median overall survival after diagnosis of approximately 12 months [1]. The MM mortality rates are increasing despite the protective measures taken to regulate asbestos, the most important causative agent of this disease [2]. There are three main histopathological subtypes: epithelioid MM (EMM), sarcomatoid MM (SMM) and biphasic MM (BMM), the latter being composed of both epithelioid and sarcomatoid components. EMM is associated with a somewhat better prognosis than the two other subtypes, while SMM has the poorest outcome in patients [1]. The treatment options for unresectable MM (75% of all cases) are very limited, given the poor efficacy of the current approved standard chemotherapy with the platin-pemetrexed doublet. These insufficient options and the hitherto disappointing attempts with targeted therapies against MM signaling pathways lately urged potential immunotherapy-based strategies to be developed against this malignancy [3,4,5]. However, there remains a crucial unmet need for identifying novel and effective therapeutic targets for MM. For the therapeutic utilization of novel protein targets on the cell surface, an appealing treatment option would be the antibody-drug conjugate (ADC) strategy. In principle, various cytotoxic payloads can be attached to the monoclonal antibodies (mAbs) that recognize tumor-associated proteins to achieve specific elimination. Although this strategy has indeed been studied for BMS-777607 certain potential target proteins in MM Rabbit Polyclonal to Dipeptidyl-peptidase 1 (H chain, Cleaved-Arg394) [6,7,8,9,10,11], none of these studies has led to clinically approved ADC so far [3,12]. Therefore, it is essential to identify targetable proteins with defined expression patterns BMS-777607 correlated with mesothelioma but with BMS-777607 restricted expression in normal tissue. In previous studies, the urokinase plasminogen activator receptor-associated protein (uPARAP/Endo180, in the following designated as uPARAP) has been thoroughly investigated in terms of its function in cancer, notably including collagen degradation and cancer invasion [13,14]. BMS-777607 In addition, however, uPARAP presents several properties that are advantageous for a potential ADC target. In particular, this protein is a recycling endocytic receptor that utilizes clathrin-associated internalization and delivers its bound cargo into the endosomal/lysosomal compartments, enabling intracellular toxin release [15]. It has a unique cellular expression pattern, including upregulation in a particular group of cancers in contrast to low expression in most noncancerous cells, thus potentially enabling targeted drug delivery with minimal side effects. So far, uPARAP has been found to be upregulated in non-epithelial cancers, including sarcomas and glioblastomas, although the receptor also has detectable expression in certain subsets of non-malignant fibroblasts and osteoblasts [16]. We recently created a uPARAP-targeted BMS-777607 ADC and used this reagent in a leukemia xenograft model, demonstrating a high anti-tumor effect with complete cure in all of the treated mice with no distinct side effects [17]. To survey additional cancer types with a potentially targetable expression of uPARAP, we performed data mining of publicly available mRNA datasets. This survey pointed to MM with an exceptionally high mRNA expression of the MRC2 gene that encodes uPARAP. Herein, we show that the uPARAP protein is highly and consistently expressed in all MM subtypes, that its expression.