Candidate tumor-specific CD8+ T cell subsets identified in the malignant pleural effusion of advanced lung cancer patients by single-cell analysis.

Isolation of tumor-specific T cells and their antigen receptors (TCRs) from malignant pleural effusions (MPE) may facilitate the development of TCR-transduced adoptive cellular immunotherapy products for advanced lung cancer patients. However, the characteristics and markers of tumor-specific T-cells in MPE are largely undefined. To this end, to establish the phenotypes and antigen specificities of CD8+ T cells, we performed single-cell RNA and TCR sequencing of samples from three advanced lung cancer patients. Dimensionality reduction on a total of 4,983 CD8+ T cells revealed 10 clusters including naïve, memory, and exhausted phenotypes. We focused particularly on exhausted T cell clusters and tested their TCR reactivity against neoantigens predicted from autologous cancer cell lines. Four different TCRs specific for the same neoantigen and one orphan TCR specific for the autologous cell line were identified from one of the patients. Differential gene expression analysis in tumor-specific T cells relative to the other T cells identified CXCL13, as a candidate gene expressed by tumor-specific T cells. In addition to expressing CXCL13, tumor-specific T cells were present in a higher proportion of T cells co-expressing PDCD1(PD-1)/TNFRSF9(4-1BB). Furthermore, flow cytometric analyses in advanced lung cancer patients with MPE documented that those with high PD-1/4-1BB expression have a better prognosis in the subset of 57 adenocarcinoma patients (p = .039). These data suggest that PD-1/4-1BB co-expression might identify tumor-specific CD8+ T cells in MPE, which are associated with patients' prognosis. (233 words).

Single-cell sequencing on CD8+ TILs revealed the nature of exhausted T cells recognizing neoantigen and cancer/testis antigen in non-small cell lung cancer.

BACKGROUND: CD8+tumor infiltrating lymphocytes (TILs) are often observed in non-small cell lung cancers (NSCLC). However, the characteristics of CD8+ TILs, especially T-cell populations specific for tumor antigens, remain poorly understood. METHODS: High throughput single-cell RNA sequencing and single-cell T-cell receptor (TCR) sequencing were performed on CD8+ TILs from three surgically-resected lung cancer specimens. Dimensional reduction for clustering was performed using Uniform Manifold Approximation and Projection. CD8+ TIL TCR specific for the cancer/testis antigen KK-LC-1 and for predicted neoantigens were investigated. Differentially-expressed gene analysis, Gene Set Enrichment Analysis (GSEA) and single sample GSEA was performed to characterize antigen-specific T cells. RESULTS: A total of 6998 CD8+ T cells was analyzed, divided into 10 clusters according to their gene expression profile. An exhausted T-cell (exhausted T (Tex)) cluster characterized by the expression of ENTPD1 (CD39), TOX, PDCD1 (PD1), HAVCR2 (TIM3) and other genes, and by T-cell oligoclonality, was identified. The Tex TCR repertoire (Tex-TCRs) contained nine different TCR clonotypes recognizing five tumor antigens including a KK-LC-1 antigen and four neoantigens. By re-clustering the tumor antigen-specific T cells (n=140), it could be seen that the individual T-cell clonotypes were present on cells at different stages of differentiation and functional states even within the same Tex cluster. Stimulating these T cells with predicted cognate peptide indicated that TCR signal strength and subsequent T-cell proliferation and cytokine production was variable but always higher for neoantigens than KK-LC-1. CONCLUSIONS: Our approach focusing on T cells with an exhausted phenotype among CD8+ TILs may facilitate the identification of tumor antigens and clarify the nature of the antigen-specific T cells to specify the promising immunotherapeutic targets in patients with NSCLC.

Effect of apple polyphenol extract on hepatoma proliferation and invasion in culture and on tumor growth, metastasis, and abnormal lipoprotein profiles in hepatoma-bearing rats.

The effect of apple polyphenol extract (APE) on the proliferation and invasion of a rat ascites hepatoma cell line of AH109A was examined in vitro. APE suppressed both the hepatoma proliferation and invasion in a dose-dependent manner up to 200 mug/ml. Serum obtained from rats orally given APE also inhibited hepatoma proliferation and invasion when added to the culture medium. Subsequently, the effect of dietary APE on growth and the metastasis of AH109A hepatomas were investigated in vivo. APE reduced the growth and metastasis of solid hepatomas and significantly suppressed the serum lipid peroxide level in rats transplanted with AH109A. APE also suppressed the serum very-low-density lipoprotein + low-density lipoprotein (VLDL + LDL)-cholesterol level. These in vitro and in vivo findings suggest that APE has anti-hepatoma activities.

Hydroxymatairesinol and its mammalian metabolite enterolactone reduce the growth and metastasis of subcutaneous AH109A hepatomas in rats.

Both epidemiological and experimental evidence is accumulating to show that a lignan-rich diet may reduce the risk of human breast cancer. Possible anti-cancer effects of dietary lignans on hepatomas or hepatoma cells have not been the topic of earlier studies. In the present study, we examined the effect of 7-hydroxymatairesinol (HMR) and its mammalian metabolite, enterolactone (ENL), on AH109A hepatoma cell proliferation and invasion in vitro. HMR and ENL inhibited the proliferation and invasion of AH109A hepatoma cells in vitro. The 50% inhibitory concentration (IC50) of hepatoma cell proliferation was lower for ENL (10 microM) than HMR (> 200 microM). Likewise, IC50 of hepatoma cell invasion was lower for ENL (9 microM) than HMR (144 microM). ENL suppressed hepatoma cell proliferation by accumulating cells in G1 phase and elongating doubling time of these cells, and by increasing the rate of apoptosis. Subsequently, we investigated in vivo the effect of dietary HMR and ENL on growth and metastasis of AH109A hepatomas in rats. Both of these compounds reduced the growth and metastasis of solid AH109A hepatomas in rats. These in vitro and in vivo findings suggest that HMR has inhibitory activities on tumor growth and metastasis in the hepatoma-bearing rats, and that this anti-tumor effect is mediated at least partially by ENL, a metabolite of HMR.

Hypocholesterolemic action of pre-germinated brown rice in hepatoma-bearing rats.

The effect of pre-germinated brown rice (PGBR) on cholesterol metabolism was studied in Donryu rats subcutaneously implanted with the ascites hepatoma cell line AH109A and compared with that of white rice (WR). The effect of brown rice (BR), the source of PGBR, was also studied. Hepatoma-bearing rats fed a WR diet exhibited hypercholesterolemia compared with normal rats fed the same diet. Feeding hepatoma-bearing rats a PGBR or BR diet suppressed hepatoma-induced hypercholesterolemia, and enhanced fecal bile acid excretion and the activity of cholesterol 7alpha-hydroxylase, the rate-limiting enzyme of bile acid biosynthesis, in the microsomal fraction of the liver without affecting cholesterol synthesis in the host liver of hepatoma-bearing rats. These results suggest that PGBR as well as BR suppresses hypercholesterolemia induced by hepatoma growth by up-regulating cholesterol catabolism.

Resveratrol inhibits hepatoma cell invasion by suppressing gene expression of hepatocyte growth factor via its reactive oxygen species-scavenging property.

Resveratrol, present in grapes and other plants, is a polyphenolic compound with strong antioxidative activity. In our previous studies, we found that reactive oxygen species (ROS) accelerated the invasive capacity of a rat ascites hepatoma cell line of AH109A in culture and that resveratrol and resveratrol-loaded rat sera suppressed the ROS-potentiated invasion of the hepatoma cells. To study mechanisms by which resveratrol and its in vivo metabolite(s) suppress the invasion, we estimated intracellular peroxide level and expression of hepatocyte growth factor (HGF), a known cell motility factor, in AH109A cells. Exogenously added ROS promoted the intracellular peroxide level and the expression of HGF. Resveratrol and resveratrol-loaded rat sera canceled the rise in the peroxide level and HGF expression in ROS-stimulated tumor cells. These results suggest an involvement of the antioxidative property of resveratrol and sera from rats orally given resveratrol in their suppressive effects on ROS-potentiated invasion of AH109A cells.

Hypolipidemic action of dietary resveratrol, a phytoalexin in grapes and red wine, in hepatoma-bearing rats.

Resveratrol is an antioxidant present in grapes and their related products. We investigated whether dietary resveratrol could inhibit the proliferation and metastasis of tumors and hyperlipidemia in Donryu rats subcutaneously implanted with an ascites hepatoma cell line of AH109A. By feeding 10 or 50 ppm resveratrol in the diet to hepatoma-bearing rats for 20 days, solid tumor growth and metastasis tended to be suppressed dose-dependently. Resveratrol (50 ppm) significantly suppressed the serum lipid peroxide level, indicating its antioxidative properties or those of its metabolite(s) in vivo. Resveratrol dose-dependently suppressed both the serum triglyceride and very-low-density lipoprotein + low-density lipoprotein (VLDL + LDL)-cholesterol levels. The hypocholesterolemic action of resveratrol is attributed, at least in part, to an increased excretion of neutral sterols and bile acids into feces. These results suggest that dietary resveratrol is hypolipidemic with a tendency for anti-tumor-growth and anti-metastasis effects in hepatoma-bearing rats.

Restoration by Prostaglandins E(2) and F (2) (alpha) of Resveratrol-Induced Suppression of Hepatoma Cell Invasion in Culture.

In our previous study, resveratrol, a polyphenolic compound in grapes with antioxidative property, and resveratol-loaded rat serum (RS) were found to suppress the invasion of AH109A cells, an ascite hepatoma cell line. The aim of the present study was to investigate whether and which prostaglandins (PGs) would be involved in the invasion of AH109A cells and its suppression by resveratrol and resveratrol-loaded RS, using an in vitro invasion assay system. Not only PGE(2) but also PGF(2) (alpha) stimulated the spontaneous invasion of AH109A cells.They also canceled the resveratrol-induced suppression of hepatoma cell invasion. Results obtained suggest an involvement of PGs, especially PGE(2), in the invasion of hepatoma cells.