High-throughput microscopy reveals the impact of multifactorial environmental perturbations on colorectal cancer cell growth.

BACKGROUND: Colorectal cancer (CRC) mortality is principally due to metastatic disease, with the most frequent organ of metastasis being the liver. Biochemical and mechanical factors residing in the tumor microenvironment are considered to play a pivotal role in metastatic growth and response to therapy. However, it is difficult to study the tumor microenvironment systematically owing to a lack of fully controlled model systems that can be investigated in rigorous detail. RESULTS: We present a quantitative imaging dataset of CRC cell growth dynamics influenced by in vivo-mimicking conditions. They consist of tumor cells grown in various biochemical and biomechanical microenvironmental contexts. These contexts include varying oxygen and drug concentrations, and growth on conventional stiff plastic, softer matrices, and bioengineered acellular liver extracellular matrix. Growth rate analyses under these conditions were performed via the cell phenotype digitizer (CellPD). CONCLUSIONS: Our data indicate that the growth of highly aggressive HCT116 cells is affected by oxygen, substrate stiffness, and liver extracellular matrix. In addition, hypoxia has a protective effect against oxaliplatin-induced cytotoxicity on plastic and liver extracellular matrix. This expansive dataset of CRC cell growth measurements under in situ relevant environmental perturbations provides insights into critical tumor microenvironment features contributing to metastatic seeding and tumor growth. Such insights are essential to dynamical modeling and understanding the multicellular tumor-stroma dynamics that contribute to metastatic colonization. It also establishes a benchmark dataset for training and testing data-driven dynamical models of cancer cell lines and therapeutic response in a variety of microenvironmental conditions.

mTORC2 contributes to the metabolic reprogramming in EGFR tyrosine-kinase inhibitor resistant cells in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations are often successfully treated with EGFR tyrosine kinase inhibitor (TKI) such as erlotinib; however, treatment resistance inevitably occurs. Given tumor metabolism of glucose and therapeutic response are intimately linked, we explored the metabolic differences between isogenic erlotinib-sensitive and -resistant NSCLC cell lines. We discovered that the growth of erlotinib-resistant cells is more sensitive to glucose deprivation. Seahorse metabolic assay revealed erlotinib-resistant cells have lower spare respiratory capacity (SRC), an indicator of metabolic flexibility, compared to erlotinib-sensitive cells. Additionally, we found downstream components of mTORC2 signaling to be phosphorylated in erlotinib-resistant cells. Knockdown of an mTORC2 component, Rictor, enhanced the SRC and rescued the growth rate of erlotinib-resistant cells during glucose deprivation. Among NSCLCs with activating EGFR mutations, gene sets involved in glucose metabolism were enriched in patients with high expression of p-NDGR1, a readout of mTORC2 activity. Furthermore, overall survival was negatively correlated with p-NDRG1. Our work uncovers a link between mTORC2 and metabolic reprogramming in EGFR TKI-resistant cells and highlights the significance of mTORC2 in the progression of EGFR-mutated NSCLC.

A high-content image-based method for quantitatively studying context-dependent cell population dynamics.

Tumor progression results from a complex interplay between cellular heterogeneity, treatment response, microenvironment and heterocellular interactions. Existing approaches to characterize this interplay suffer from an inability to distinguish between multiple cell types, often lack environmental context, and are unable to perform multiplex phenotypic profiling of cell populations. Here we present a high-throughput platform for characterizing, with single-cell resolution, the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations both during standard growth and in response to multiple, co-occurring selective pressures. The speed of this platform enables a thorough investigation of the impacts of diverse selective pressures including genetic alterations, therapeutic interventions, heterocellular components and microenvironmental factors. The platform has been applied to both 2D and 3D culture systems and readily distinguishes between (1) cytotoxic versus cytostatic cellular responses; and (2) changes in morphological features over time and in response to perturbation. These important features can directly influence tumor evolution and clinical outcome. Our image-based approach provides a deeper insight into the cellular dynamics and heterogeneity of tumors (or other complex systems), with reduced reagents and time, offering advantages over traditional biological assays.

Photodynamic Therapy Synergizes with Irinotecan to Overcome Compensatory Mechanisms and Improve Treatment Outcomes in Pancreatic Cancer.

The ability of tumor cells to adapt to therapeutic regimens by activating alternative survival and growth pathways remains a major challenge in cancer therapy. Therefore, the most effective treatments will involve interactive strategies that target multiple nonoverlapping pathways while eliciting synergistic outcomes and minimizing systemic toxicities. Nanoliposomal irinotecan is approved by the FDA for gemcitabine-refractory metastatic pancreatic cancer. However, the full potential of irinotecan treatment is hindered by several cancer cell survival mechanisms, including ATP-binding cassette G2 (ABCG2) transporter-mediated irinotecan efflux from cells. Here, we demonstrate that benzoporphyrin derivative-based photodynamic therapy (PDT), a photochemical cytotoxic modality that activates the apoptotic pathway, reduced ABCG2 expression to increase intracellular irinotecan levels in pancreatic cancer. Moreover, we show that PDT inhibited survivin expression. Although PDT potentiated irinotecan treatment, we also demonstrate that irinotecan reduced the tumoral expression of monocarboxylate transporter 4, which was upregulated by PDT. Notably, using orthotopic xenograft models, we demonstrate that combination of single low-dose PDT and a subclinical dose of nanoliposomal irinotecan synergistically inhibited tumor growth by 70% for 3 weeks compared with 25% reduction after either monotherapies. Our findings offer new opportunities for the clinical translation of PDT and irinotecan combination therapy for effective pancreatic cancer treatment.

Rapamycin downregulates thymidylate synthase and potentiates the activity of pemetrexed in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) accounts for 80-85% of lung cancer cases, and almost half of newly diagnosed patients have metastatic disease. Pemetrexed is a widely used drug for NSCLC and inhibits several folate-dependent enzymes including thymidylate synthase (TS). Increased expression of TS confers resistance to pemetrexed in vitro and predicts poor response to pemetrexed. Rapamycin is an mTOR inhibitor and suppresses cap-dependent synthesis of specific mRNA species. Here, we show that the combination of rapamycin and pemetrexed synergistically inhibits proliferation of NSCLC cells. Although pemetrexed as a single agent induced TS, pretreatment with rapamycin suppressed pemetrexed-induced TS expression. In vivo, the combination of rapamycin and pemetrexed inhibited growth of NSCLC xenografts, which correlated with decreased mTOR activity and suppression of pemetrexed-induced TS expression. The ability of rapamycin to enhance the efficacy of pemetrexed and prevent TS expression has implications for the design of Phase I and/or Phase II NSCLC clinical trials with mTOR inhibitors in combination with pemetrexed.

Combinations of mTORC1 inhibitor RAD001 with gemcitabine and paclitaxel for treating non-Hodgkin lymphoma.

Single-agent mammalian target of rapamycin complex 1 (mTORC1) inhibitors have recently been reported as effective salvage treatment in non-Hodgkin lymphoma (NHL). The combined effect of mTORC1 inhibitor, RAD001, with chemotherapeutic agents used for relapsed or refractory NHL was examined. Synergistic interactions were observed for RAD001 plus gemcitabine or paclitaxel in six NHL cell lines; enhanced gemcitabine- and paclitaxel-induced caspase-dependent apoptosis associated with down-regulation of mTOR signaling was detected. Synergistic interactions were also observed with RAD001 plus gemcitabine and paclitaxel. In conclusion, synergistic cytotoxicity was observed with RAD001 plus gemcitabine and paclitaxel in NHL cells. Combination therapy with these three drugs should be examined in patients with refractory or relapsed NHL.

Diosgenin, a naturally occurring steroid, suppresses fatty acid synthase expression in HER2-overexpressing breast cancer cells through modulating Akt, mTOR and JNK phosphorylation.

Fatty acid synthase (FAS) expression is markedly elevated in HER2-overexpressing breast cancer cells. In this study, diosgenin, a plant-derived steroid, was found to be effective in suppressing FAS expression in HER2-overexpressing breast cancer cells. Diosgenin preferentially inhibited proliferation and induced apoptosis in HER2-overexpressing cancer cells. Furthermore, diosgenin inhibited the phosphorylation of Akt and mTOR, and enhanced phosphorylation of JNK. The use of pharmacological inhibitors revealed that the modulation of Akt, mTOR and JNK phosphorylation was required for diosgenin-induced FAS suppression. Finally, we showed that diosgenin could enhance paclitaxel-induced cytotoxicity in HER2-overexpressing cancer cells. These results suggested that diosgenin has the potential to advance as chemopreventive or chemotherapeutic agent for cancers that overexpress HER2.

Sensitizing HER2-overexpressing cancer cells to luteolin-induced apoptosis through suppressing p21(WAF1/CIP1) expression with rapamycin.

HER2 overexpression, which confers resistance to various therapeutic regimens, correlates with a poor clinical prognosis. In this study, we showed that luteolin, a naturally occurring flavonoid, is a potent stimulator of HER2 degradation. Luteolin effectively inhibited cell proliferation and induced apoptosis in HER2-overexpressing cancer cells. Furthermore, we found that low doses of luteolin up-regulated p21 expression and high doses of luteolin down-regulated its expression. Examination of the Akt/mammalian target of rapamycin (mTOR) signaling revealed that this signaling was only transiently inhibited by low doses of luteolin, which suggested that the inability to cause sustained Akt/mTOR inhibition may contribute to p21 induction and provide a survival advantage to HER2-overexpressing cancer cells. To test this hypothesis, we showed that the combined use of luteolin and mTOR inhibitor rapamycin prevented low doses of luteolin from inducing p21 expression, and HER2-overexpressing cancer cells would be sensitized toward luteolin-induced apoptosis. In addition, p21 small interfering RNA also increased the luteolin-induced cell death. In nude mice with xenografted SKOV3.ip1-induced tumors, luteolin significantly inhibited HER2 expression and tumor growth in a dose-dependent manner, and rapamycin further enhanced the effect of luteolin with a concomitant p21 inhibition. These results reveal an intriguing finding that suppressing p21 expression might have therapeutic implications and further suggest that combination of mTOR inhibitors may be a promising strategy to help increase the efficacy of preventive or therapeutic compounds against HER2-overexpressing tumors.

Comparative studies on the hypolipidemic and growth suppressive effects of oolong, black, pu-erh, and green tea leaves in rats.

The four major commercial teas, oolong, black, pu-erh, and green teas, have been manufactured in southeast Asia. In this study, we evaluated the growth suppressive and hypolipidemic effect of these four different tea leaves by oral feeding to male Sprague-Dawley rats for 30 weeks. The results showed that the suppression of body weights of tea leaves-fed groups were in the order: oolong tea > pu-erh tea > black tea > green tea. Pu-erh tea and oolong tea could lower the levels of triglyceride more significantly than that of green tea and black tea, but pu-erh tea and green tea were more efficient than oolong tea and black tea in lowering the level of total cholesterol. In lipoprotein, 4% pu-erh tea could increase the level of HDL-C and decrease the level of LDL-C, but other teas simply decrease the levels of both. The activity of antioxidant enzyme SOD is increased in all tea-fed groups as compared to the basal diet-fed group. Finally, relative weight ratios of liver to epididylmal adipose tissue were lower in feeding oolong tea and pu-erh tea groups. On the basis of these findings, it seemed that the fully fermented pu-erh and black tea leaves and partially fermented oolong tea leaves were more effective on their growth suppressive and hypolipidemic effects as compared to the nonfermented green tea leaves.

Pu-erh tea supplementation suppresses fatty acid synthase expression in the rat liver through downregulating Akt and JNK signalings as demonstrated in human hepatoma HepG2 cells.

Fatty acid synthase (FAS) is a key enzyme of lipogenesis. Overexpression of FAS is dominant in cancer cells and proliferative tissues. The expression of FAS in the livers of rats fed pu-erh tea leaves was significantly suppressed. The gains in body weight, levels of triacylglycerol, and total cholesterol were also suppressed in the tea-treated rats. FAS expression in hepatoma HepG2 cells was suppressed by the extracts of pu-erh tea at both the protein and mRNA levels. FAS expression in HepG2 cells was strongly inhibited by PI3K inhibitor LY294002 and JNK inhibitor II and slightly inhibited by p38 inhibitor SB203580 and MEK inhibitor PD98059, separately. Based on these findings, we suggest that the suppression of FAS in the livers of rats fed pu-erh tea leaves may occur through downregulation of the PI3K/AKt and JNK signaling pathways. The major components of tea that have been demonstrated to be responsible for the antiobesity and hypolipidemic effects are catechins, caffeine, and theanine. The compositions of catechins, caffeine, and theanine varied dramatically in pu-erh, black, oolong, and green teas. The active principles and molecular mechanisms that exerted these biological effects in pu-erh tea deserve future exploration.