Anti-SSPN monoclonal antibody Made to order

Background Duchenne muscular dystrophy (DMD) is caused by loss of sarcolemma connection to the extracellular matrix. Transgenic overexpression of the transmembrane protein sarcospan (SSPN) in the DMD mdx mouse model significantly reduces disease pathology by restoring membrane adhesion. Identifying SSPN-based therapies has the potential to benefit patients with DMD and other forms of muscular dystrophies caused by deficits in muscle cell adhesion. Methods Standard cloning methods were used to generate C2C12 myoblasts stably transfected with a fluorescence reporter for human SSPN promoter activity. Assay development and screening were performed in a core facility using liquid handlers and imaging systems specialized for use with a 384-well microplate format. Drug-treated cells were analyzed for target gene expression using quantitative PCR and target protein expression using immunoblotting. Results We investigated the gene expression profiles of SSPN and its associated proteins during myoblast differentiation into myotubes, revealing an increase in expression after 3 days of differentiation. We created C2C12 muscle cells expressing an EGFP reporter for SSPN promoter activity and observed a comparable increase in reporter levels during differentiation. Assay conditions for high-throughput screening were optimized for a 384-well microplate format and a high-content imager for the visualization of reporter levels. We conducted a screen of 3200 compounds and identified seven hits, which include an overrepresentation of L-type calcium channel antagonists, suggesting that SSPN gene activity is sensitive to calcium. Further validation of a select hit revealed that the calcium channel inhibitor felodipine increased SSPN transcript and protein levels in both wild-type and dystrophin-deficient myotubes, without increasing differentiation. Conclusions We developed a stable muscle cell line containing the promoter region of the human SSPN protein fused to a fluorescent reporter. Using the reporter cells, we created and validated a scalable, cell-based assay that is able to identify compounds that increase SSPN promoter reporter, transcript, and protein levels in wild-type and dystrophin-deficient muscle cells.

Synovial sarcoma of peripheral nerve (SSPN) is rare with only 26 cases reported in English. SSPN is often mistaken for a benign or malignant peripheral nerve sheath tumor (PNST) by clinicians and pathologists. Fifteen cases of SSPN were retrieved from the pathology files of 3 institutions. All tumors arose in a nerve based on imaging and/or operative findings and the diagnoses were histologically confirmed. Neuropathic symptoms predominated in 11 women and 4 men, 19- to 62-year-old (median, 39 years) with tumors involving the ulnar (5), median (3), peroneal (3) or sciatic (2) nerve, or the L4 or T1 nerve root (2). Tumor sizes ranged from 2 to 13 cm (median, 3.8 cm). The leading clinical diagnosis was PNST (9). Treatment was surgical (14) supplemented with radiation therapy (8) and chemotherapy (6). Fourteen tumors were monophasic and 1 was biphasic; 4 had poorly differentiated (PD) foci (1 rhabdoid). Diagnoses in 12 cases were verified by fluorescence in situ hybridization, reverse transcription polymerase chain reaction or both methods. Follow-up in 14 patients (median, 32 mo) revealed that 2/4 patients with PD tumors died with pulmonary metastases; another was alive with no current evidence of disease (NED) following 2 local recurrences, while the fourth had NED. In contrast, 9/10 patients without PD tumors were alive (7 NED) and 1 died at 12 months with pulmonary infiltrates. SSPN is under-recognized clinically and histologically as it mimics benign and malignant PNST. Molecular analysis is recommended to confirm the diagnosis. PD foci, including rhabdoid areas, may portend a worse outcome, similar to non-neural-based tumors.