Modulation of Immune Checkpoints by Chemotherapy in Human Colorectal Liver Metastases.

Metastatic colorectal cancer (CRC) is a major cause of cancer-related death, and incidence is rising in younger populations (younger than 50 years). Current chemotherapies can achieve response rates above 50%, but immunotherapies have limited value for patients with microsatellite-stable (MSS) cancers. The present study investigates the impact of chemotherapy on the tumor immune microenvironment. We treat human liver metastases slices with 5-fluorouracil (5-FU) plus either irinotecan or oxaliplatin, then perform single-cell transcriptome analyses. Results from eight cases reveal two cellular subtypes with divergent responses to chemotherapy. Susceptible tumors are characterized by a stemness signature, an activated interferon pathway, and suppression of PD-1 ligands in response to 5-FU+irinotecan. Conversely, immune checkpoint TIM-3 ligands are maintained or upregulated by chemotherapy in CRC with an enterocyte-like signature, and combining chemotherapy with TIM-3 blockade leads to synergistic tumor killing. Our analyses highlight chemomodulation of the immune microenvironment and provide a framework for combined chemo-immunotherapies

Tumor slice culture as a biologic surrogate of human cancer.

Background: The tumor microenvironment (TME) is critical to every aspect of cancer biology. Organotypic tumor slice cultures (TSCs) preserve the original TME and have demonstrated utility in predicting drug sensitivity, but the association between clinicopathologic parameters and Methods: One hundred and eight fresh tumor specimens from liver resections at a tertiary academic center were procured and precisely cut with a Vibratome to create 250 μm × 6 mm slices. These fixed-dimension TSCs were grown on polytetrafluoroethylene inserts, and their metabolic activities were determined by a colorimetric assay. Correlation between baseline activities and clinicopathologic parameters was assessed. Tissue CEA mRNA expression was determined by RNAseq. Results: By standardizing the dimensions of a slice, we found that adjacent tumor slices have equivalent metabolic activities, while those derived from different tumors exhibit \u3e30-fold range in baseline MTS absorbances, which correlated significantly with the percentage of tumor necrosis based on histologic assessment. Extending this to individual cancers, we were able to detect intra-tumoral heterogeneity over a span of a few millimeters, which reflects differences in tumor cell density and Ki-67 positivity. For colorectal cancers, tissue CEA expression based on RNAseq of tumor slices was found to correlate with clinical response to chemotherapies. Conclusions: We report a standardized method to assess and compare human cancer growth ex vivo across a wide spectrum of tumor samples. TSC reflects the state of tumor behavior and heterogeneity, thus providing a simple approach to study of human cancers with an intact TME

Tuberous Sclerosis Complex-1 Deficiency Attenuates Diet-Induced Hepatic Lipid Accumulation

Non-alcoholic fatty liver disease (NAFLD) is causally linked to type 2 diabetes, insulin resistance and dyslipidemia. In a normal liver, insulin suppresses gluconeogenesis and promotes lipogenesis. In type 2 diabetes, the liver exhibits selective insulin resistance by failing to inhibit hepatic glucose production while maintaining triglyceride synthesis. Evidence suggests that the insulin pathway bifurcates downstream of Akt to regulate these two processes. Specifically, mTORC1 has been implicated in lipogenesis, but its role on hepatic steatosis has not been examined. Here, we generated mice with hepatocyte-specific deletion of Tsc1 to study the effects of constitutive mTORC1 activation in the liver. These mice developed normally but displayed mild hepatomegaly and insulin resistance without obesity. Unexpectedly, the Tsc1-null livers showed minimal signs of steatosis even under high-fat diet condition. This ‘resistant’ phenotype was reversed by rapamycin and could be overcome by the expression of Myr-Akt. Moreover, rapamycin failed to reduce hepatic triglyceride levels in models of steatosis secondary to Pten ablation in hepatocytes or high-fat diet in wild-type mice. These observations suggest that mTORC1 is neither necessary nor sufficient for steatosis. Instead, Akt and mTORC1 have opposing effects on hepatic lipid accumulation such that mTORC1 protects against diet-induced steatosis. Specifically, mTORC1 activity induces a metabolic shift towards fat utilization and glucose production in the liver. These findings provide novel insights into the role of mTORC1 in hepatic lipid metabolism

Glucose deprivation in tuberous sclerosis complex-related tumors

<p>Abstract</p> <p>Background</p> <p>Cancer cells possess unique metabolic phenotypes that are determined by their underlying oncogenic pathways. Activation of the PI3K/Akt/mTOR signaling cascade promotes glycolysis and leads to glucose-dependence in tumors. In particular, cells with constitutive mTORC1 activity secondary to the loss of TSC1/TSC2 function are prone to undergo apoptosis upon glucose withdrawal <it>in vitro</it>, but this concept has not been tested <it>in vivo</it>. This study examines the effects of restricting glucose metabolism by pharmacologic and dietary means in a tuberous sclerosis complex (TSC) tumor xenograft model.</p> <p>Results</p> <p>Tumor-bearing mice were randomly assigned to receive unrestricted carbohydrate-free ("Carb-free") or Western-style diet in the absence or presence of 2-deoxyglucose (2-DG) in one of four treatment groups. After 14 weeks, tumor sizes were significantly different among the four treatment groups with those receiving 2-DG having the smallest tumors. Unexpectedly, the "Carb-free" diet was associated with the largest tumors but they remained responsive to 2-DG. PET imaging showed significant treatment-related changes in tumor ¹⁸fluorodeoxyglucose-uptake but the standard uptake values did not correlate with tumor size. Alternative energy substrates such as ketone bodies and monounsaturated oleic acid supported the growth of the <it>Tsc2</it>-/- cells <it>in vitro</it>, whereas saturated palmitic acid was toxic. Correspondingly, tumors in the high-fat, "Carb-free" group showed greater necrosis and liquefaction that contributed to their larger sizes. In contrast, 2-DG treatment significantly reduced tumor cell proliferation, increased metabolic stress (i.e., ketonemia) and AMPK activity, whereas rapamycin primarily reduced cell size.</p> <p>Conclusions</p> <p>Our data support the concept of glycolytic inhibition as a therapeutic approach in TSC whereas dietary withdrawal of carbohydrates was not effective.</p

Latent Kaposi's Sarcoma-Associated Herpesvirus Infection of Endothelial Cells Activates Hypoxia-Induced Factors

Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) is the etiological agent of Kaposi's sarcoma, a highly vascularized, endothelial-derived tumor. A direct role for KSHV-mediated induction of angiogenesis has been proposed based upon the nature of the neoplasia and various KSHV gene overexpression and infection model systems. We have found that KSHV infection of endothelial cells induces mRNA of hypoxia-induced factor 1α (HIF1α) and HIF2α, two homologous alpha subunits of the heterodimeric transcription factor HIF. HIF is a master regulator of both developmental and pathological angiogenesis, composed of an oxygen-sensitive alpha subunit and a constitutively expressed beta subunit. HIF is classically activated posttranscriptionally with hypoxia, leading to increased protein stability of HIF1α and/or HIF2α. However, we demonstrate that both alpha subunits are up-regulated at the transcript level by KSHV infection. The transcriptional activation of HIF leads to a functional increase in HIF activity under normoxic conditions, as demonstrated by both luciferase reporter assay and the increased expression of vascular endothelial growth factor receptor 1 (VEGFR1), an HIF-responsive gene. KSHV infection synergizes with hypoxia mimics and induces higher expression levels of HIF1α and HIF2α protein, and HIF1α is increased in a significant proportion of the latently infected endothelial cells. Src family kinases are required for the activation of HIF and the downstream gene VEGFR1 by KSHV. We also show that KS lesions, in vivo, express elevated levels of HIF1α and HIF2α proteins. Thus, KSHV stimulates the HIF pathway via transcriptional up-regulation of both HIF alphas, and this activation may play a role in KS formation, localization, and progression

Metabolic response to rapamycin following HFD.

<p>Six-week old, wild-type mice were randomly assigned to one of 5 groups with n = 5 in each group (see text). At the end of 6 weeks, mice were fasted overnight and sacrificed. Shown are the results of body and liver weights, fasting serum glucose and insulin, plasma and hepatic triglyceride levels for each group. Values represent mean ±SEM. * associated with HFD indicates p<0.05 with respect to NCD group. * associated with HFD-rapamycin group indicates p<0.05 with respect to NCD-rapamycin group. NCD, normal chow diet; HFD, high-fat diet; Rapa, rapamycin.</p

Akt induces steatosis in the Tsc1−/− livers.

<p>A) Contrasting effects of <i>Pten</i>- and <i>Tsc1</i>-loss on Akt signaling in the liver. Immunoblot analyses of liver lysates from fasted 20 wk-old mice using indicated antibodies to highlight Akt and mTORC1 signaling. B) Effects of Akt on <i>Tsc1</i>−/− livers. <i>Tsc1</i>−/− mice were injected through the tail-vein with adenovirus (10⁷ PFUs) encoding genes for Myr-Akt1 or β–galactosidase control. After 96 hours, mice were fasted overnight and sacrificed for H&E histology and Oil Red “O” staining of the livers. Magnification, 400X. C) Expression of transgenes (HA-tagged Myr-Akt1 or β–gal) and components of the Akt/mTORC1 pathway in the <i>Tsc1</i>−/− livers following adenovirus injections. C, control for Tsc1 expression. Note up-regulation of Akt without significant alteration to mTORC1 signaling in the Myr-Akt1-treated liver.</p

Effects of rapamycin on Pten−/− livers.

<p>Hepatocyte-specific deletion of <i>Pten</i> was generated by crossing <i>Pten^fl/fl</i> with <i>Cre^Alb</i> mice. At 12 weeks of age, <i>Pten</i>−/− mice were randomly assigned to treatments with rapamycin (2 mg/kg IP daily, M-F) or DMSO as vehicle control (C) for 2 weeks and then sacrificed. A) Representative Western blot showing the effects of <i>Pten</i> loss (−/−) and rapamycin (Rapa) in the liver with respect to Akt and mTORC1 signaling. Liver lysates were subjected to immunoblot analyses with the indicated antibodies. B) Liver histology (H&E) and Oil Red “O” staining of <i>Pten</i>+/+ and <i>Pten</i>−/− mice treated with rapamycin or vehicle control. C) Quantification of liver triglyceride content of the corresponding groups. Values represent mean ±SEM. *, p<0.05 compared to <i>Pten</i>+/+ group.</p

Hepatocyte-specific deletion of Tsc1 leads to mild insulin resistance.

<p>A. Liver-specific ablation of <i>Tsc1</i>. <i>Tsc1^fl/fl</i> mice were crossed to <i>Cre^Alb</i> mice resulting in <i>Tsc1^fl/fl</i>; <i>Cre^+/+</i> (a.k.a. <i>Tsc1</i>+/+) and <i>Tsc1^fl/fl</i>; <i>Cre^Alb</i> (a.k.a. <i>Tsc1</i>−/−) littermates. Tissues from eight-week old <i>Tsc1</i>−/− and <i>Tsc1</i>+/+ animals were analyzed for the expression of Tsc1, Tsc2 and S6K by immunoblot analyses using the indicated antibodies. WAT, white adipose tissues. Note the reduced Tsc2 expression secondary to its diminished stability in the absence of Tsc1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018075#pone.0018075-Nellist1" target="_blank">[30]</a>. B. The loss of <i>Tsc1</i> in hepatocytes resulted in increased mTORC1 activity (based on the expression of phospho-S6K and phospho-S6) that was sensitive to rapamycin. <i>Tsc1</i>−/− mice were fasted and treated with or without rapamycin (2 mg/kg IP, 6 hrs). Liver lysates were analyzed by SDS-PAGE and blotted with the indicated antibodies. Note the effect of rapamycin on Akt phosphorylation in the <i>Tsc1</i>−/− liver. Actin, loading control. C. Systemic glucose tolerance (left) and insulin sensitivity (right) tests in 8-week old female (top) and male (bottom) mice. Following a 16-hr fast, glucose (1 mg/g) was given IP followed by serial blood glucose monitoring at indicated times. For insulin sensitivity test, 0.5 mU/g of insulin was injected IP after a 4-hr fast. *, p<0.05 between the <i>Tsc1</i>+/+ and <i>Tsc1</i>−/− groups. D. Fasting blood glucose and insulin levels in wild-type and mutant mice. Plasma insulin levels 30 minutes after glucose administration are also shown (filled boxes).</p