\u3ci\u3ePTHR1/SOX9\u3c/i\u3e and \u3ci\u3eIDH1/IDH2\u3c/i\u3e Relative Expression in Primary Chondrocyte and Chondrosarcoma Cells Under the Synergistic Influence of Inducible Hypoxia and Extracellular Acidosis

Cartilage cells (Chondrocytes) grow in rather unique environmental conditions in the human body. Cartilage is avascular tissue and lacks innervation. Its main source of nutrients is derived from the synovial fluid and/or perichondrium. Consequently, these cells must survive and thrive under hypoxic and acidic stressors. Published data suggests that there are a multitude of genes affected from either one of these two stressors or both. However, these factors are frequently overlooked in cartilage research, and results are reported in either normoxia/pH=7.0 conditions, or they only account for one of the conditions. The scope of this study is to examine how these stressors affect gene expression in primary chondrocytes and chondrosarcomas. In this study, one primary chondrocyte cell line (CON5) and two chondrosarcoma grade II cell lines, JJ012-IDH1 mutant and SW1353- IDH2 mutant, were grown in four experimental conditions: hypoxia (5% O2), acidosis (pH=5.5), hypoxia and acidosis, and normoxia/(pH=7). Four genes of interest were analyzed via RT-qPCR relative to the ACTB housekeeping gene: parathyroid hormone receptor-1 (PTHR1), SRY-box transcription factor 9 (SOX9), and isocitrate dehydrogenase 1 and 2 (IDH1/IDH2). PTHR1 and SOX9 keep chondrocytes in a proliferative state and delay their hypertrophy. On the other hand, IDH1 and IDH2 are metabolic enzymes that convert isocitrate to α-ketoglutarate (α-KG). Their mutant counterparts further convert α-KG into a competitive oncometabolite D-2-Hydroxygluterate (D-2-HG). Our colorimetric assay data suggest that D-2-HG concentration levels are 10.5-fold and 6-fold more elevated in JJ012/SW1353 respectively than in the IDH wild type CON5. Our gene expression data indicates that both inducible hypoxia and extracellular acidosis alter gene expression not only separately but also when combined. This study further highlights the importance of these stressors in cartilage biology research.

Gene Expression Under Combined Hypoxia And Acidosis In Chondrosarcoma

Chondrosarcomas are the second most common cause of bone cancer and are removed surgically with wide margins. On recurrence, they are resistant to chemo and radiation therapy and new treatment options are critically required. This tumor type produces hyaline cartilage, a cartilage normally formed under hypoxic and acidic environment due to lack of vasculature in cartilage. Paradoxically, chondrosarcomas arise in the well vascularized, oxygen rich environment of the bone. Hypoxia and acidosis are two stressors where the cellular effects are typically reported separately even though cells experience combined effects of hypoxia and acidosis. Given the mechanistic links between hypoxia and acidosis we hypothesized that gene expression profiles will be differentially changed when chondrosarcoma cells were exposed to individual compared to combined stressors. We investigated expression of four genes expressed during cartilage and cartilage tumor formation in primary chondrocytes and two grade II chondrosarcoma cell lines, SW1353 and JJ012. Two genes, PTH1R and SOX9 are known to respond to hypoxia and acidosis separately. Two genes, IDH1 and IDH2, are mutated in chondrosarcoma cell lines JJ012 and SW1353 respectively. These mutations confer a condition of false hypoxia on the cells through stabilization of HIF-1α. The result is chondrosarcoma cells metabolize glycolytically through aerobic glycolysis. How the cells respond to hypoxia and acidosis is of considerable interest as metabolically the cells are molecularly predisposed to these conditions. Our gene expression data found that combined hypoxia and extracellular acidosis alter gene expression compared to either stressor alone. Cells showed gene specific responses to stressors that were cell type specific likely indicating influence on gene expression regulatory sequences. The importance of this work is highlighting that conditions under which cells are investigated is crucial and should be considered when measuring cell response to in vitro treatment exposures

Application of In vitro transcytosis models to brain targeted biologics.

The blood brain barrier (BBB) efficiently limits the penetration of biologics drugs from blood to brain. Establishment of an in vitro BBB model can facilitate screening of central nervous system (CNS) drug candidates and accelerate CNS drug development. Despite many established in vitro models, their application to biologics drug selection has been limited. Here, we report the evaluation of in vitro transcytosis of anti-human transferrin receptor (TfR) antibodies across human, cynomolgus and mouse species. We first evaluated human models including human cerebral microvascular endothelial cell line hCMEC/D3 and human colon epithelial cell line Caco-2 models. hCMEC/D3 model displayed low trans-epithelial electrical resistance (TEER), strong paracellular transport, and similar transcytosis of anti-TfR and control antibodies. In contrast, the Caco-2 model displayed high TEER value and low paracellular transport. Anti-hTfR antibodies demonstrated up to 70-fold better transcytosis compared to control IgG. Transcytosis of anti-hTfR.B1 antibody in Caco-2 model was dose-dependent and saturated at 3 μg/mL. Enhanced transcytosis of anti-hTfR.B1 was also observed in a monkey brain endothelial cell based (MBT) model. Importantly, anti-hTfR.B1 showed relatively high brain radioactivity concentration in a non-human primate positron emission tomography study indicating that the in vitro transcytosis from both Caco-2 and MBT models aligns with in vivo brain exposure. Typically, brain exposure of CNS targeted biologics is evaluated in mice. However, antibodies, such as the anti-human TfR antibodies, do not cross-react with the mouse target. Therefore, validation of a mouse in vitro transcytosis model is needed to better understand the in vitro in vivo correlation. Here, we performed transcytosis of anti-mouse TfR antibodies in mouse brain endothelial cell-based models, bEnd3 and the murine intestinal epithelial cell line mIEC. There is a good correlation between in vitro transcytosis of anti-mTfR antibodies and bispecifics in mIEC model and their mouse brain uptake. These data strengthen our confidence in the predictive power of the in vitro transcytosis models. Both mouse and human in vitro models will serve as important screening assays for brain targeted biologics selection in CNS drug development