0374 - Proteoglycan-4 Inhibits TGF-beta Induced Differentiation of Synovial Fibroblasts into Myofibroblasts in a CD44-Dependent Manner
Year: 2019
Type: other
Abstract: Introduction: Chondrosarcoma (CS) is the most common primary skeletal sarcoma in adults. Treatment of CS is currently limited to surgery. An ideal therapy for CS would be a minimally-invasive local or systemic treatment modality to halt growth within the lesion and kill the tumor cells. At present this is a far reaching goal as there has not been a major breakthrough or improvement in survival of CS for over 30 years. Previous research on retinoic acid (RA) has demonstrated that this molecule alters chondrocyte growth in vitro and can cause regression of CS in animal studies, but RA has potential to cause severe adverse effects. Palovarotene, an agonist of nuclear retinoic acid receptor gamma (RARu03b3), has been studied in a Phase-III human clinical trial for treatment of heterotopic ossification in patients with fibrodysplasia ossificans progressiva without reports of critical side effects (NCT03312634). Furthermore, a clinical trial on Palovarotene has been started for Multiple Osteochondroma (NCT03442985). These prior findings as well as the effects of Palovarotene on the biology of human cartilage lead us to the idea that Palovarotene and other RARu03b3 agonists may be applicable to chondrosarcoma. We hypothesized that human CS specimens retain RARu03b3 signaling and that both systemic and local administration of palovarotene results in decreased growth in an in vitro and in vivo animal model. The aims of our study were (1) to determine if RARu03b3 is expressed in human chondrosarcoma specimens and (2) to determine the in vitro and in vivo therapeutic action of selective RARu03b3 agonists in a human CS cell line.Materials and Methods: All animal experiments were performed following institutional guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) at UMB. Human CS specimens were obtained from the University of Maryland Pathology Biorepository Shared Service (PBBS) and immunostained with anti-RARu03b3 antibody (Sigma). Tumor sections were graded according to the American Joint Committee on Cancer with support from PBBS. A human CS cell line (HCS2.8) established from grade II chondrosarcoma (72yr, male) was used. Pellet cultures (300,000/tube) were treated with 100nM Palovarotene or the same volume of ethanol (vehicle) for 7 days (n=3, 3 times). Protein and glycosaminoglycan (GAG) content of the pellets were measured. Gene expression of cartilage matrix-related genes were evaluated by qPCR. For systemic palovarotene treatment, cells (1 million cells in 100ml) were inoculated subcutaneously in NOD/Shi-scid IL2rgamma(null) (NOG) mice. Mice were treated with Palovarotene (5 mg/kg) or the same volume of vehicle (1:9, DMSO:corn oil) via gavage 3 times/week starting 1 week after tumor inoculation demonstrated a viable cell mass (n=10). Tumor volume (WxWxLx1/2) was calculated by measurement of the tumor width and length weekly. The histology of treated and control tumors were analyzed by light microscopy after hematoxylin and eosin staining. For local in vivo Palovarotene treatment with nanoparticle drug delivery, cells (1 million cells in 100ml) were inoculated subcutaneously in NOD/Shi-scid IL2rgamma(null) (NOG) mice. Mice received subcutaneous injections (every 3-4 days) of nanoparticles tagged with Palovarotene (5 mg/kg) or the same volume of untagged nanoparticles (n=5) after tumor inoculation demonstrated a viable cell mass. Tumor volume (WxWxLx1/2) was calculated by measurement of the tumor width and length weekly. Histology of treated and control tumors were analyzed by light microscopy after hematoxylin and eosin staining. Statistical analysis was performed using one-way analysis of variance (ANOVA) and a p-value < 0.05 was considered to be statistically significant when compared to control.Results: Human chondrosarcomas were found to retain RARu03b3 expression. For in vitro analysis, Palovarotene decreased GAG and protein content and reduced cartilage matrix gene expression in HCS2/8 cells. In vivo results demonstrated that systemic treatment with Palovarotene inhibited growth of the tumor mass and local treatment using nanoparticle delivery of Palovarotene inhibited tumor growth, reduced the size of the mass, and stimulated surrounding fibrous connective tissue.Conclusions: All human CS specimens expressed RARu03b3. Palovarotene treatment reduced the mass size of pellet cultures (HCS2/8 human chondrosarcoma cell line cells), significantly decreased total GAG and total protein levels, and strongly reduced gene expression of cartilage matrix molecules compared to control. HCS2/8 cells responded to RARu03b3 agonists and reduced tumor mass size when subcutaneously implanted in mice. This finding was more pronounced when nanoparticle drug delivery was utilized. To our knowledge, this is the first evaluation of effects of Palovarotene on a human chondrosarcoma cell line. Future studies will focus on multiple cell lines and the effect of Palovarotene not only on primary tumor growth but also on metastasis. This data supports further investigations on the role of Palovarotene as a possible adjuvant therapy or treatment for chondrosarcoma in the future as current treatment modalities are limited.
Authors: Khaled Elsaid, Gregory Jay
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4391553315