Metabolic Enzymes in Sarcomagenesis: Progress Toward Biology and Therapy.

Cellular metabolism reprogramming is an emerging hallmark of cancer, which provides tumor cells with not only necessary energy but also crucial materials to support growth. Exploiting the unique features of cancer metabolism is promising in cancer therapies. The growing interest in this field has led to numerous inhibitors being developed against key molecules in metabolic pathways, though most of them are still in preclinical development. Potential targeted cancer cell metabolic pathways under investigation include glycolysis, tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), glutaminolysis, pentose phosphate pathway (PPP), lipid synthesis, amino acid and nucleotide metabolism. Sarcoma is a type of cancer that arises from transformed cells of mesenchymal origin, in contrast to carcinoma which originates from epithelial cells. Compared with carcinoma, progress towards harnessing the therapeutic potential of targeting sarcoma cell metabolism has been relatively slow. Recently however, with the discovery of cancer-specific mutations in metabolic enzymes such as isocitrate dehydrogenase (IDH) and succinate dehydrogenase (SDH) in certain sarcoma types, cancer cellular metabolism has been considered more as a source of new targets for treating sarcoma. In this article, we review metabolic enzymes currently tested for cancer therapies and describe the therapeutic potential of targeting IDH mutations and SDH deficiency in sarcomas.

Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells.

Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers including chondrosarcomas. The IDH1 inhibitor AGI-5198 abrogates the ability of mutant IDH1 to produce the oncometabolite D-2 hydroxyglutarate (D-2HG) in gliomas. We sought to determine if treatment with AGI-5198 would similarly inhibit tumorigenic activity and D-2HG production in IDH1-mutant human chondrosarcoma cells. Two human chondrosarcoma cell lines, JJ012 and HT1080 with endogenous IDH1 mutations and a human chondrocyte cell line C28 with wild type IDH1 were employed in our study. Mutation analysis of IDH was performed by PCR-based DNA sequencing, and D-2HG was detected using tandem mass spectrometry. We confirmed that JJ012 and HT1080 harbor IDH1 R132G and R132C mutation, respectively, while C28 has no mutation. D-2HG was detectable in cell pellets and media of JJ012 and HT1080 cells, as well as plasma and urine from an IDH-mutant chondrosarcoma patient, which decreased after tumor resection. AGI-5198 treatment decreased D-2HG levels in JJ012 and HT1080 cells in a dose-dependent manner, and dramatically inhibited colony formation and migration, interrupted cell cycling, and induced apoptosis. In conclusion, our study demonstrates anti-tumor activity of a mutant IDH1 inhibitor in human chondrosarcoma cell lines, and suggests that D-2HG is a potential biomarker for IDH mutations in chondrosarcoma cells. Thus, clinical trials of mutant IDH inhibitors are warranted for patients with IDH-mutant chondrosarcomas.

Epigenetic regulation of embryonic stem cell marker miR302C in human chondrosarcoma as determinant of antiproliferative activity of proline-rich polypeptide 1.

Metastatic chondrosarcoma of mesenchymal origin is the second most common bone malignancy and does not respond either to chemotherapy or radiation; therefore, the search for new therapies is relevant and urgent. We described recently that tumor growth inhibiting cytostatic proline-rich polypeptide 1, (PRP-1) significantly upregulated tumor suppressor miRNAs, downregulated onco-miRNAs in human chondrosarcoma JJ012 cell line, compared to chondrocytes culture. In this study we hypothesized the existence and regulation of a functional marker in cancer stem cells, correlated to peptides antiproliferative activity. Experimental results indicated that among significantly downregulated miRNA after PRP-1treatment was miRNAs 302c*. This miRNA is a part of the cluster miR302­367, which is stemness regulator in human embryonic stem cells and in certain tumors, but is not expressed in adult hMSCs and normal tissues. PRP-1 had strong inhibitory effect on viability of chondrosarcoma and multilineage induced multipotent adult cells (embryonic primitive cell type). Unlike chondrosarcoma, in glioblastoma, PRP-1 does not have any inhibitory activity on cell proliferation, because in glioblastoma miR-302-367 cluster plays an opposite role, its expression is sufficient to suppress the stemness inducing properties. The observed correlation between the antiproliferative activity of PRP-1 and its action on downregulation of miR302c explains the peptides opposite effects on the upregulation of proliferation of adult mesenchymal stem cells, and the inhibition of the proliferation of human bone giant-cell tumor stromal cells, reported earlier. PRP-1 substantially downregulated the miR302c targets, the stemness markers Nanog, c-Myc and polycomb protein Bmi-1. miR302c expression is induced by JMJD2-mediated H3K9me2 demethylase activity in its promoter region. JMJD2 was reported to be a positive regulator for Nanog. Our experimental results proved that PRP-1 strongly inhibited H3K9 activity comprised of a pool of JMJD1 and JMJD2. We conclude that inhibition of H3K9 activity by PRP-1 leads to downregulation of miR302c and its targets, defining the PRP-1 antiproliferative role.

Micropatterning cells on permeable membrane filters.

Epithelium is abundantly present in the human body as it lines most major organs. Therefore, ensuring the proper function of epithelium is pivotal for successfully engineering whole organ replacements. An important characteristic of mature epithelium is apical-basal polarization which can be obtained using the air-liquid interface (ALI) culture system. Micropatterning is a widely used bioengineering strategy to spatially control the location and organization of cells on tissue culture substrates. Micropatterning is therefore an interesting method for generating patterned epithelium. Enabling micropatterning of epithelial cells however requires micropatterning methods that are designed to (i) be compatible with permeable membranes substrates and (ii) allow prolonged culture of patterned cells, both of which are required for appropriate epithelial apical-basal polarization. Here, we describe a number of methods we have developed for generating monoculture as well as coculture of epithelial cells that are compatible with ALI culture.

Challenges and opportunities for tissue-engineering polarized epithelium.

The epithelium is one of the most important tissue types in the body and the specific organization of the epithelial cells in these tissues is important for achieving appropriate function. Since many tissues contain an epithelial component, engineering functional epithelium and understanding the factors that control epithelial maturation and organization are important for generating whole artificial organ replacements. Furthermore, disruption of the cellular organization leads to tissue malfunction and disease; therefore, engineered epithelium could provide a valuable in vitro model to study disease phenotypes. Despite the importance of epithelial tissues, a surprisingly limited amount of effort has been focused on organizing epithelial cells into artificial polarized epithelium with an appropriate structure that resembles that seen in vivo. In this review, we provide an overview of epithelial tissue organization and highlight the importance of cell polarization to achieve appropriate epithelium function. We next describe the in vitro models that exist to create polarized epithelium and summarize attempts to engineer artificial epithelium for clinical use. Finally, we highlight the opportunities that exist to translate strategies from tissue engineering other tissues to generate polarized epithelium with a functional structure.

Tools for micropatterning epithelial cells into microcolonies on transwell filter substrates.

Despite the importance of epithelial tissue in most major organs there have been limited attempts to tissue engineer artificial epithelium. A key feature of mature epithelium is the presence of an apical-basal polarization, which develops over 7-20 days in culture. Currently, the most widely used 2D system to generate polarized epithelium in vitro involves the filter insert culture system, however this system is expensive, laborious and requires large numbers of cells per sample. We have developed a set of micropatterning techniques to spatially control the organization of epithelial cells into microsheets on filter inserts under the culture conditions necessary to induce epithelial cell polarization. Micropatterning improves cell uniformity within each microsheet, allows multiple sheet analysis on one filter insert, and reduced cell number requirements. We describe an agarose patterning method that allows maintenance of cell patterns for over 15 days, the time necessary to induce apical-basal polarization. We also describe a Parafilm™ patterning method that allows patterning for 5 to 15 days depending on cell type and only allows the generation of stripes and circular microsheets. The parafilm™ method however is extremely straightforward and could be easily adopted by any laboratory without the need of access to specialized microfabrication equipment. We also demonstrate that micropatterning epithelial cells does not alter the localization of the apical-basal marker ZO-1 or the formation of cilia, a marker of epithelium maturation. Our methods provide a novel tool for studying epithelial biology in polarized epithelium microsheets of controlled size.

Engineering functional islets from cultured cells.

OBJECTIVE: Although pancreatic islet transplantation can now be performed minimally invasively in patients with type 1 diabetes, the availability of functional islet donors remains the chief obstacle to widespread clinical application. Tissue engineering islet cells in vitro that function when implanted in vivo provides a solution to this problem. RESEARCH DESIGN AND METHODS: Rat pancreatic islets were enzymatically dissociated into a single-cell suspension and seeded onto a polyglycolic acid (PGA) scaffold. The cells were cultured in CMRL 1099 medium containing epidermal growth factor, nerve growth factor, and insulin-like growth factor for 5 days. The PGA and isolated cells were then suspended in a thermoreversible gelatin polymer (TGP) with insulin, transferring and selenous acid, in F-12 and Dulbecco's modified Eagle's medium, to proliferate over a 40-day period. After the degradation of the PGA fibers, the TGP was removed using cold temperature extraction. The tissue-engineered (TE) islets were then collected manually and transplanted beneath the kidney capsule of Streptozotocin (STZ)-induced diabetic nude mice. RESULTS: All mice that received the TE islets reverted from the induced hyperglycemic state to a state of normoglycemia (n = 6). The treated mice demonstrated normal oral glucose tolerance tests. Testing for the species-specific C-peptide allowed discrimination between the exogenous insulin secretions of the TE rat islets and the endogenous secretions of the nude mice. Immunohistochemistry confirmed the multilineage potential of these TE endocrine cells, showing them capable of secreting insulin, glucagon, and somatostatin. CONCLUSIONS: The ability to tissue engineer pancreatic islets in vitro, through use of PGA and TGP, that fully function in vivo to return diabetic-induced mice to state of normoglycemia has potential implications for the treatment type 1 diabetes.

Bioengineered three-layered robust and elastic artery using hemodynamically-equivalent pulsatile bioreactor.

BACKGROUND: There is an essential demand for tissue engineered autologous small-diameter vascular graft, which can function in arterial high pressure and flow circulation. We investigated the potential to engineer a three-layered robust and elastic artery using a novel hemodynamically-equivalent pulsatile bioreactor. METHODS AND RESULTS: Endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts were harvested from bovine aorta. A polyglycolic acid (PGA) sheet and a polycaprolactone sheet seeded with SMCs, and a PGA sheet seeded with fibroblast, were wrapped in turn on a 6-mm diameter silicone tube and incubated in culture medium for 30 days. The supporting tube was removed, and the lumen was seeded with ECs and incubated for another 2 days. The pulsatile bioreactor culture, under regulated gradual increase in flow and pressure from 0.2 (0.5/0) L/min and 20 (40/15) mm Hg to 0.6 (1.4/0.2) L/min and 100 (120/80) mm Hg, was performed for an additional 2 weeks (n=10). The engineered vessels acquired distinctly similar appearance and elasticity as native arteries. Scanning electron microscopic examination and Von Willebrand factor staining demonstrated the presence of ECs spread over the lumen. Elastica Van Gieson and Masson Tricrome Stain revealed ample production of elastin and collagen in the engineered grafts. Alpha-SMA and calponin staining showed the presence of SMCs. Tensile tests demonstrated that engineered vessels acquired equivalent ultimate strength and similar elastic characteristics as native arteries (Ultimate Strength of Native: 882+/-133 kPa, Engineered: 827+/-155 kPa, each n=8). CONCLUSIONS: A robust and elastic small-diameter artery was engineered from three types of vascular cells using the physiological pulsatile bioreactor.