Characterization of Chronic and Intermittent Hypoxia in Canine Soft Tissue Sarcomas

Low oxygen levels (hypoxia) in tumors results in increased metastasis and failure to respond to treatment. Tumor cells experiencing hypoxia alter their gene expression and these changes likely contribute to the worsened outcome. It has been shown hypoxia associated gene expression predicts more about patient outcome than other clinical parameters routinely used to make treatment decisions. Previous work has shown that two phenotypes of hypoxia exist in cancer cells, ongoing (chronic) hypoxia and cycling (intermittent) hypoxia. Tumor cells experiencing intermittent hypoxia are more likely to have greater proliferation rates, increased angiogenesis, and metastasize as compared to oxygenated or chronically hypoxic cells.;The purpose of the studies described herein was to determine a gene, protein, and metabolic profile for different hypoxic subtypes and durations of hypoxic exposure. One goal of the research was to establish a signature for each subtype of hypoxia to be used by researchers and clinicians as a less invasive method for characterization of individual patient tumors. An additional goal was to better define the time needed to observe differential gene and protein expression in cells exposed to intermittent hypoxia. Genotypic knowledge of different subtypes of hypoxia, along with the temporal course of each, may elucidate molecular signaling pathways or functional groups for therapeutic targeting. One hurdle for canine soft tissue sarcoma research is the lack of readily available cell lines. Therefore, first we established a new canine cell line from a tumor biopsy taken from canine patient seen by the oncology service at the NCSU College of Veterinary medicine. The cell line is now established as MBSa1.;Three canine soft issue sarcoma cell lines, CoFSA, PSTS, and MBSa1 were used to profile the hypoxic subtypes described. Cells were exposed to chronic hypoxia, intermittent hypoxia, or reoxygenation following chronic hypoxia for varying amounts of time. Profiles of each subtype were generated by the use Affymetrix Canine GeneChip(c) microarray for gene expression, whole proteome mass spectrometry for protein expression, and the Biolog Phenotype Microarray(TM) system for metabolic profiling. The expression results were further analyzed with standard bioinformatic resources.;Gene and protein expression analysis of intermittent hypoxia did not reveal a distinct signature. Very few transcripts had robust changes in expression as compared to their chronic or non-hypoxic counterparts. However, the heat shock protein 70 (HSP70) transcript was found to have greater expression in cells exposed to intermittent hypoxia than those exposed to chronic hypoxia. HSP70 has been demonstrated to be a negative regulator of hypoxia inducible factor 1 (HIF1) The number of proteins for both chronic and intermittent hypoxia identified by mass spectrometry was limited. The initial gene expression profile revealed, among many differentially regulated transcripts, those related to cellular metabolism were highly expressed in cells exposed to chronic hypoxia and reoxygenation, but less so in cells exposed to intermittent hypoxia. Metabolic profiling revealed all three cell lines readily use lactate as an energy source irrespective of the type of hypoxic exposure and the use of other carbohydrate sources is cell line dependent.;The final study here describes the gene expression patterns of twenty-two canine patients with known tumor oxygenation levels enrolled in a clinical trial at the NCSU-College of Veterinary Medicine. Patients with defined hypoxic tumors were found to have very little changes in gene expression as compared to patients with tumors determined to be non-radiobiologically hypoxic. The lack of induction of hypoxia responsive genes was surprising considering the patients demonstrated hypoxic tumors. However, HSP70 had a greater than twofold increase in expression in the hypoxic patients. Future work needs to be done to explore the HSP70/intermittent hypoxia connection.