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Simple Summary: Circulating tumor cells (CTCs) derived from peripheral blood have a notable advantage in that they are noninvasive and real-time biomarkers and have attracted attention in monitoring therapeutic efficacy. Combination immunotherapy has promising outcomes in patients with unresectable hepatocellular carcinoma (HCC). However, there is no reliable biomarker for predicting disease progression. In this study, CTCs were serially collected from HCC patients undergoing atezolizumab plus bevacizumab (Atezo+Bev). Changes in CTC numbers during Atezo+Bev treatment reflected the tumor volume, and patients with elevated transforming growth factor (TGF)-β signaling molecules had a poorer response, whereas those with elevated apoptosis signaling molecules had a favorable response. This highlights that changes in the expression of TGF-β molecules of CTCs could serve as novel biomarkers for the early prediction of therapeutic response in patients with unresectable HCC undergoing Atezo+Bev. Immune checkpoint inhibitors have promising outcomes in patients with hepatocellular carcinoma (HCC); however, there is no reliable biomarker for predicting disease progression. Circulating tumor cells (CTCs) derived from peripheral blood have attracted attention in monitoring therapeutic efficacy. In this study, CTCs were serially collected from HCC patients undergoing atezolizumab plus bevacizumab (Atezo+Bev), and changes in molecular expression and CTC numbers were analyzed to identify effective biomarkers. Changes in CTC numbers during Atezo+Bev reflected the tumor volume. Targeted RNA sequencing with next-generation sequencing (NGS) revealed that patients with elevated transforming growth factor (TGF)-β signaling molecules had a poorer response, whereas those with elevated apoptosis signaling molecules had a favorable response. In addition, compared with changes in CTC counts, changes in TGF-β signaling molecule expression in CTCs accurately and promptly predicted treatment response. Overall, NGS analysis of CTC-derived RNA showed that changes in TGF-β signaling molecules predict treatment response earlier than changes in CTC counts. These findings suggest that changes in the expression of TGF-β molecules in CTCs could serve as novel biomarkers for the early prediction of therapeutic response in patients with unresectable HCC undergoing Atezo+Bev. [ABSTRACT FROM AUTHOR]

Simple Summary: Hepatocellular carcinoma (HCC) represents a significant health challenge, demanding innovative approaches for effective treatment. Despite advances in immune checkpoint inhibitor (ICI) therapy, reliable biomarkers to predict treatment response in HCC have yet to be found. Our study addressed this critical research gap by exploring the role of circulating tumor cells (CTCs) and their PD-L1 RNA expression in predicting response to atezolizumab and bevacizumab (Atezo/Bev) treatment. CTC-derived RNA collected serially during Atezo/Bev treatment suggests that patients with higher levels of PD-L1 expression in CTCs at baseline were more responsive to this treatment, but not lenvatinib treatment. Moreover, PD-L1 RNA levels in CTCs were an accurate response predictor and a monitorable biomarker that changed dynamically to reflect the response during Atezo/Bev treatment. The results of this study highlight the potential for individualized treatment decisions based on PD-L1 expression in CTCs and have implications for clinical practice. There remains no reliable biomarker of therapeutic efficacy in hepatocellular carcinoma (HCC) for the PD-L1 inhibitor atezolizumab and bevacizumab (Atezo/Bev). Circulating tumor cells (CTCs) enable the serial collection of living tumor cells. Pre-treatment and serial CTC gene expression changes and tumor histology were evaluated to identify predictors of response to Atezo/Bev. Peripheral blood from 22 patients with HCC treated with Atezo/Bev and 24 patients treated with lenvatinib was serially collected. The RNA expression in CTCs was analyzed using qRT-PCR. Higher PD-L1 expression in pre-treatment CTCs was associated with response and improved prognosis with Atezo/Bev treatment, but not with lenvatinib. There was no correlation between PD-L1 expression in CTCs and that in liver tumor biopsy specimens scored using imaging software. Furthermore, PD-L1 RNA expression in CTCs was dynamically altered by Atezo/Bev, decreasing during effective response and increasing upon progression. CTC-derived RNA collected during Atezo/Bev indicates that patients with higher PD-L1 expression in CTCs at baseline were 3.9 times more responsive to treatment. Therefore, PD-L1 RNA levels in CTCs are an accurate response predictor and may be a monitorable biomarker that changes dynamically to reflect the response during Atezo/Bev treatment. [ABSTRACT FROM AUTHOR]

Obese conditions increase the expression of adipocytokine monocyte chemoattractant protein-1 (MCP-1) in adipose tissue as well as MCP-1 plasma levels. To investigate the mechanism behind increased MCP-1, we used a model in which 3T3-L1 adipocytes were artificially hypertrophied by preloading with palmitate in vitro. As observed in obesity, under our model conditions, palmitate-preloaded ceils showed significantly increased oxidative stress and increased MCP-1 expression relative to control cells. This increased MCP-1 expression was enhanced by adding exogenous tumor necrosis factor-[alpha] (TNF-[alpha]; 17.8-fold vs. control cells, P < 0.01 ) rather than interleukin-1[beta] (IL-1[beta]; 2.6-fold vs. control cells, P < 0.01). However, endogenous TNF-[alpha] and IL-1[beta] release was not affected in hypertrophied cells, suggesting that these endogenous cytokines do not mediate hypertrophy-induced increase in MCP-1. MCP-1 secretion from hypertrophied cells was significantly decreased by treatment with antioxidant N-acetyl-cysteine, JNK inhibitors SP600125 and JIP-1 peptide, and I[kappa]B phosphorylation inhibitors BAY 11-7085 and BMS-345541 (P < 0.01). MCP-1 secretion was not affected by peroxisome proliferator-activated receptor-[gamma] (PPAR[gamma]) antagonists assayed. Adiponectin, another adipocytokine studied in parallel, also showed increased release in hypertrophy relative to control cells. But in contrast to MCP-1, adiponectin release was significantly suppressed by both exogenous TNF-[alpha] and IL-1[beta] as well as by PPAR[gamma] antagonists bisphenol A diglycidyl ether and T0070907 (P < 0.01). JNK inhibitors and I[kappa]B phosphorylation inhibitors showed no significant effect on adiponectin. We conclude that adipocyte hypertrophy through palmitate loading causes oxidative stress, which in turn increases MCP-1 expression and secretion through JNK and I[kappa]B signaling. In contrast, the parallel increase in adiponectin expression appears to be related to the PPAR[gamma] ligand properties of palmitate. c-Jun N[H.sub.2]-terminal kinase; monocyte chemoattractant protein-1; tumor necrosis factor-[alpha]; interleukin-1[beta]

To determine whether the increase in glucose uptake following AMP-activated protein kinase (AMPK) activation in adipocytes is mediated by accelerated GLUT4 translocation into plasma membrane, we constructed a chimera between GLUT4 and enhanced green fluorescent protein (GLUT4-eGFP) and transferred its cDNA into the nucleus of 3T3-L1 adipocytes. Then, the dynamics of GLUT4-eGFP translocation were visualized in living cells by means of laser scanning confocal microscopy. It was revealed that the stimulation with 5-aminoimidazole-4-carboxamide-1-[beta]-D-ribofuranoside (AICAR) and 2,4-dinitrophenol (DNP), known activators of AMPK, promptly accelerates its translocation within 4 min, as was found in the case of insulin stimulation. The insulin-induced GLUT4 translocation was markedly inhibited after addition of wortmannin (P < 0.01). However, the GLUT4 translocation through AMPK activators AICAR and DNP was not affected by wortmannin. Insulin- and AMPK-activated translocation of GLUT4 was not inhibited by SB-203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Glucose uptake was significantly increased after addition of AMPK activators AICAR and DNP (P < 0.05). AMPK- and insulin-stimulated glucose uptake were similarly suppressed by wortmannin (P < 0.05-0.01). In addition, SB-203580 also significantly prevented the enhancement of glucose uptake induced by AMPK and insulin (P < 0.05). These results suggest that AMPK-activated GLUT4 translocation in 3T3-L1 adipocytes is mediated through the insulin-signaling pathway distal to the site of activated phosphatidylinositol 3-kinase or through a signaling system distinct from that activated by insulin. On the other hand, the increase of glucose uptake dependent on AMPK activators AICAR and DNP would be additionally due to enhancement of the intrinsic activity in translocated GLUT4 protein, possibly through a p38 MAPK-dependent mechanism. glucose transporter 4; mitogen-activated protein kinase; phosphatidylinositol 3-kinase; enhanced green fluorescent protein