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The native hepatocellular cancer (HCC) microenvironment is characterized by more hypoxic, hypoglycemic, and acidic conditions than those used in standard cell culture. This study aimed to investigate whether HCC cells cultured in more native conditions have an altered phenotype and drug sensitivity compared to those cultured in standard conditions. Six HCC cell lines were cultured in "standard" (21% O2, 25 mM glucose) or more "native" (1% O2, 5 mM glucose, 10 mM lactate) conditions. Cells were assessed for growth rates, cell cycle distribution, relevant metabolite and protein levels, genome-wide gene expression, mitochondrial DNA sequence and sensitivity to relevant drugs. Many differences in cellular and molecular phenotypes and drug sensitivity were observed between the cells. HCC cells cultured in native conditions had slower doubling times, increased HK2 and GLUT, lower PHDA and ATP levels, and mutations in mitochondrial DNA. Thirty-one genes, including the hypoxia-associated NDRG1, were differentially expressed between the cells. HCC patients in The Cancer Genome Atlas (TCGA) with tumors with a high score based on these 31 genes had a poorer prognosis than those with a low score (p = 0.002). From 90 comparisons of drug sensitivity, increased resistance and sensitivity for cells cultured in native conditions was observed in 14 (16%) and 8 (9%) comparisons respectively. In conclusion, cells cultured in more native conditions can have a more glycolytic and aggressive phenotype and varied drug sensitivity to those cultured in standard conditions, and may provide new insights to understanding tumor biology and drug development.

Acquired resistance (AQR) to drug treatment occurs frequently in cancer patients and remains an impediment to successful therapy. The aim of this study was to gain insight into how AQR arises following the application of PI3K/mTOR inhibitors. H1975 lung cancer cells with EGFR T790M mutations that confer resistance to EGFR inhibitors underwent prolonged treatment with the PI3K/mTOR inhibitor, BEZ235. Monoclonal cells with stable and increased resistance to BEZ235 were obtained after 8 months treatment. These AQR clones showed class-specific resistance to PI3K/mTOR inhibitors, reduced G1 cell cycle arrest and impedance of migration following PI3K/mTOR inhibition, reduced PTEN expression and increased Akt and S6RP phosphorylation. Transcriptome analysis revealed the AQR clones had increased expression of the metabolite transporters SLC16A9 and SLC16A7, suggestive of altered cell metabolism. Subsequent experiments revealed that AQR clones possess features consistent with elevated glycolysis, including increased levels of glucose, lactate, glutamine, glucose dependence, GLUT1 expression, and rates of post-glucose extracellular acidification, and decreased levels of reactive oxygen species and rates of oxygen consumption. Combination treatment of BEZ235 with the glycolysis inhibitor 3-bromopyruvate was synergistic in AQR clones, but only additive in parental cells. DNA sequencing revealed the presence of a mitochondrial DNA (mtDNA) MT-C01 variant in AQR but not parental cells. Depletion of mitochondrial DNA in parental cells induced resistance to BEZ235 and other PI3K/mTOR inhibitors, and was accompanied by increased glycolysis. The results of this study provide the first evidence that a metabolic switch associated with mtDNA mutation can be an underlying mechanism for AQR.

Background: Hepatocellular carcinoma (HCC) is characterized by hypoxia, hypoglycemia and lactic acidosis within the microenvironment due a highly glycolytic phenotype. However, routine culture conditions of HCC cells employ supra-physiological glucose, pH-buffered media, and normoxia. The aim of the study was to simulate the microenvironment features of HCC in vitro by culturing HCC cells in their native (NAT) conditions (hypoglycemia, hypoxia and lactic acidosis) and examine phenotypic, transcriptomic and pharmacological differences compared to cells cultured in standard (ST) culture conditions. Methods: Six HCC cell lines were cultured in NAT or ST conditions. Pharmacological response to cytotoxic drugs and targeted agents were evaluated using the MTS assay. Phenotypic differences were examined using standard techniques. Transcriptomic analysis was carried out using the Illumina HumanHT-12 expression kit. Results: HCC cells cultured in NAT conditions have higher doubling time than cells cultured in ST conditions. However, no remarkable differences in the cell cycle profiles were observed when cultured in either NAT or ST conditions. Protein analysis revealed an increase in phosphorylation of AKT and decrease in the levels of AMPK in all cells cultured in NAT but not in ST conditions. Increase in the protein levels of GLUT1, HK2, LDHA, and a decrease in PDHA was observed only in cells cultured in NAT conditions. Furthermore, the HCC cells in NAT conditions exhibited lower levels of reactive oxygen species and ATP consistent with the elevated glycolysis and inefficient oxidative phosphorylation phenotype of HCC. Thirty-one genes were found to be aberrantly expressed by gene expression analysis, most notably NDRG1, a hypoxia-associated gene was upregulated in NAT cells, validated by qPCR. Upregulated NDRG1 was maintained even up reversal of the NAT condition. The panel of thirty-one genes were found to be associated with poor prognosis exclusively in HCC based on data available via TCGA supporting the validity of NAT culture condition. Differences in the IC50 of doxorubicin, sorafenib, PI3K, c-MET and HDAC inhibitors but not AKT, MEK, mTOR, Wnt inhibitors were observed in cells cultured in NAT compared with ST conditions suggesting microenvironment modelling can influence pharmacological response of certain class of compounds. Additional genomic and metabolomics analysis are currently being undertaken to characterize the HCC cells in NAT conditions. Conclusion: In conclusion, HCC cells when cultured in NAT condition exhibit more of the tumor characteristics than cells in ST condition and this can be utilized as an informative platform for better understanding of disease biology and pharmacology. Citation Format: Mohd Feroz Mohd Omar, Benny Tang, Sheng Chun Chang, Sarah Hong Hui Low, Gim Hwa Tan, Richie Soong, Bhaskar Bhattacharya. A novel in vitro microenvironment modeling platform for HCC therapeutics and biomarker development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 165. doi:10.1158/1538-7445.AM2017-165

Hepatocellular carcinoma (HCC) is an endemic disease globally, with a 5-year survival rate of 15%. Current treatment of late-stage HCC involves the use of chemotherapy and sorafenib, a multi-kinase inhibitor. Metabolic studies have indicated HCC has a high glycolysis rate, and is surrounded by a hypoglycemic microenvironment. Given the need for therapeutic options for HCC and its glycolytic nature, this study explored the activity of novel GLUT1 inhibitors in varied glucose concentrations. To assess target inhibition, [3H] deoxy-D-glucose (2DG) uptake assays were performed in HEK293 cells stably over-expressing GLUT1-4 individually. Six HCC cell lines were cultured in media with standard high glucose (HG:25mM) and low glucose/lactic acidosis (LGLA: 5mM glucose/20mM lactic acid) to mimic the hypoglycemic microenvironment. IC50s were assessed using the SRB assay, and other phenotypes using standard methods. Four novel GLUT1 inhibitors (IOM1-4) from the same chemical series were identified from diversity screening. The compounds exhibited differential potencies to GLUT proteins at the sub-μM level. The inhibitors reduced the uptake of 2DG, the extrusion of lactate, and increased apoptosis. In HCC cells, IC50s were lowest with IOM1 and 2 and highest with IOM4 (Table 1). HEP3B cells displayed exceptional sensitivity to the inhibitors. The ranking of potency according to the IC50s correlated with the 2DG uptake studies. There was no correlation between GLUT protein levels and IC50s. Significant differences in sensitivity to inhibitors were observed in cells cultured in HG and LGLA conditions. Cells cultured in LGLA had consistently lower glucose uptake compared those in HG conditions. In LGLA conditions, cells with low levels of reactive oxygen species (ROS) were more sensitive to GLUT1 inhibitors compared to cells with high ROS levels. In conclusion, GLUT1 inhibition can be influenced by microenvironment glucose levels and could be a strategy for HCC treatment. IC50 values (uM) for respective inhibitors in respective HCC cell linesCELL LINESInhibitorCultureC3AHEP3BHUH7PLCSKHEP1SNU449IOM1HG>5.00.8±0.040.3±0.06>5.01.4±0.451.7±0.38IOM1LGLA>5.00.2±0.134.0±1.20>5.00.1±0.021.6±0.28IOM2HG4.7±0.445.00.1±0.080.2±0.093.2±1.290.1±0.021.3±0.42IOM3HG>5.00.2±0.040.9±0.222.3±0.251.1±0.641.6±0.40IOM3LGLA>5.05.00.4±0.092.3±1.13IOM4HG>5.02.8±0.354.3±1.494.6±1.404.7±0.85>5.0IOM4LGLA>5.01.4±0.493.8±1.10>5.03.1±0.56>5.0 Citation Format: Bhaskar Bhattacharya, Sanamerjit S. Mann, Min Ji Han, Sarah HH Low, Gim Hwa Tan, Barry E. McGuinness, Sarah C. Trewick, Phillip M. Cowley, Alan Wise, Richie Soong. Identification and activity of novel GLUT1 inhibitors in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1283.