Landscapes and mechanisms of CD8+ T cell exhaustion in gastrointestinal cancer.

CD8+ T cells, a cytotoxic T lymphocyte, are a key component of the tumor immune system, but they enter a hyporeactive T cell state in long-term chronic inflammation, and how to rescue this depleted state is a key direction of research. Current studies on CD8+ T cell exhaustion have found that the mechanisms responsible for their heterogeneity and differential kinetics may be closely related to transcription factors and epigenetic regulation, which may serve as biomarkers and potential immunotherapeutic targets to guide treatment. Although the importance of T cell exhaustion in tumor immunotherapy cannot be overstated, studies have pointed out that gastric cancer tissues have a better anti-tumor T cell composition compared to other cancer tissues, which may indicate that gastrointestinal cancers have more promising prospects for the development of precision-targeted immunotherapy. Therefore, the present study will focus on the mechanisms involved in the development of CD8+ T cell exhaustion, and then review the landscapes and mechanisms of T cell exhaustion in gastrointestinal cancer as well as clinical applications, which will provide a clear vision for the development of future immunotherapies.

The TGF-ß/Smad2/3 signaling pathway is involved in Musashi2-induced invasion and metastasis of colorectal cancer.

To identify Musashi2 as an effective biomarker regulated by the TGF-ß/Smad2/3 signaling pathway for the precise diagnosis and treatment of colorectal cancer (CRC) through bioinformatic tools and experimental verification. The Cancer Genome Atlas, Timer, and Kaplan-Meier analyses were performed to clarify the expression of Musashi2 and its influence on the prognosis of CRC. Transforming growth factor beta 1 (TGF-ß1) was used to activate the TGF-ß/Smad2/3 signaling pathway to identify whether it could regulate the expression and function of Musashi2. Western blot analysis and quantitative PCR analyses were conducted to verify the expression of Musashi2. Cell counting kit-8 (CCK8), EdU, wound healing, and Transwell assays were conducted to reveal the role of Musashi2 in the proliferation, migration, and invasion of CRC. Musashi2 was upregulated in CRC and promoted proliferation and metastasis. TGF-ß1 increased the expression of Musashi2, while the antagonist inducer of type II TGF-ß receptor degradation-1 (ITD-1) decreased the expression. CCK8 and EdU assays demonstrated that inhibition of Musashi2 or use of ITD-1 lowered proliferation ability. The Transwell and wound healing assays showed that the migration and invasion abilities of CRC cells could be regulated by Musashi2. The above functions could be enhanced by TGF-ß1 by activating the TGF-ß/Smad2/3 signaling pathway and reversed by ITD-1. A positive correlation was found between Musashi2 and the TGF-ß/Smad2/3 signaling pathway. TGF-ß1 activates the TGF-ß/Smad2/3 pathway to stimulate the expression of Musashi2, which promotes the progression of CRC. Musashi2 might become a target gene for the development of new antitumor drugs.

TGF-ß Pathways Stratify Colorectal Cancer into Two Subtypes with Distinct Cartilage Oligomeric Matrix Protein (COMP) Expression-Related Characteristics.

BACKGROUND: Colorectal cancers (CRCs) continue to be the leading cause of cancer-related deaths worldwide. The exact landscape of the molecular features of TGF-ß pathway-inducing CRCs remains uncharacterized. METHODS: Unsupervised hierarchical clustering was performed to stratify samples into two clusters based on the differences in TGF-ß pathways. Weighted gene co-expression network analysis was applied to identify the key gene modules mediating the different characteristics between two subtypes. An algorithm integrating the least absolute shrinkage and selection operator (LASSO), XGBoost, and random forest regression was performed to narrow down the candidate genes. Further bioinformatic analyses were performed focusing on COMP-related immune infiltration and functions. RESULTS: The integrated machine learning algorithm identified COMP as the hub gene, which exhibited a significant predictive value for two subtypes with an area under the curve (AUC) value equaling 0.91. Further bioinformatic analysis revealed that COMP was significantly upregulated in various cancers, especially in advanced CRCs, and regulated the immune infiltration, especially M2 macrophages and cancer-associated fibroblasts in CRCs. CONCLUSIONS: Comprehensive immune analysis and experimental validation demonstrate that COMP is a reliable signature for subtype prediction. Our results could provide a new point for TGFß-targeted anticancer drugs and contribute to guiding clinical decision making for CRC patients.

Microarray analysis of altered long non-coding RNA expression profile in liver cancer cells treated by ginsenoside Rh2.

Microarray expression profiles of lncRNAs and mRNAs were investigated in HepG2 cells treated with 20 µg/ml ginsenoside Rh2 as well as in ginsenoside Rh2-untreated cells. Microarray analysis showed 618 upregulated lncRNAs and 161 downregulated lncRNAs in HepG2 cells treated with ginsenoside Rh2 compared with the control group. Moreover, three differentially expressed lncRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). This may be beneficial to patients as an anti-cancer treatment and potentially provide novel targets for HCC (hepatocellular carcinoma) therapy.

Up-regulation of the tumor promoter Glyoxalase-1 indicates poor prognosis in breast cancer.

Glyoxalase 1 (Glo1) is an enzyme that plays a role to metabolize and inactivate methylglyoxal. Previous studies also have confirmed that Glo1 is closely related with tumorigenesis, metastasis, and drug-resistant, but its prognostic value in breast cancer has never been explored. In this study, we investigated the expression of Glo1 in breast cancer cell lines and tissues using real-time PCR, western blot and immunohistochemical analysis. We found Glo1 was frequently up-regulated in human breast cancer cells and tissues, and high expression of Glo1 was associated with positive lymph node, lymphovascular invasion, and TNM stage (all P<0.05). The Kaplan-Meier survival curve demonstrated that patients with high Glo1 expression had a shorter overall survival (OS) and recurrence-free survival (RFS) (Both P<0.001) than those with low Glo1 expression. Moreover, the univariate and further multivariate analysis revealed that Glo1 expression was an independent prognostic factor for both OS and RFS of breast cancer patients. Next, with CCK-8 assay, cell apoptosis analysis, colony formation assay, transwell invasion/migration assay, and wound-healing assay, we validated knock-down of Glo1 suppressed invasion and migration and promoted apoptosis of breast cancer cells. Taken together, we demonstrated the tumor-promoter Glo1 may serve as a prognostic biomarker for breast cancer.

[Full sequence analysis for a null allele of MICA gene (MICA*063N)].

OBJECTIVE: To analyze the full nucleotide sequence of a null allele of major histocompatibility complex class I chain-related gene (MICA). METHODS: A sequence-based typing method was used to determine the nucleotide sequence of the MICA gene. Potential alleles were identified with a computer program. RESULTS: The identified allele has possessed a sequence similar to that of MICA*027 except for a C→T substitution at position 184 in codon 62 (CAG→TAG) of exon 2. As a stop codon, this may result in a truncated protein. CONCLUSION: A null allele of MICA gene has been identified. The sequence has been submitted to the Genbank nucleotide sequence database (submission No. HWS10011131), which was officially named as MICA*063N by the WHO Nomenclature Committee in October 2010.

[Preparation of doxorubicin encapsulated in amphiphilic polysaccharide nanoparticles and anti-hepatocarcinoma effect thereof].

OBJECTIVE: To investigate the sustained release rule of doxorubicin/polylactide-grafted dextran copolymer (DOX/DEX-PLA) nanoparticles and the effect thereof in killing hepatocarcinoma cells. METHODS: DOX/DEX-PLA nanoparticles were prepared by method of emulsification & evaporation of organic solvent. Its morphology was observed by transmission electron microscopy and the encapsulating efficiency of DOX was determined by ultraviolet spectrophotometry. DOX/DEX-PLA was put in a dialysis bag to observe the releasing characteristics of DOX from DOX/DEX-PLA nanoparticles in vitro. Human liver carcinoma cells of the line HepG2 were cultured with DOX/DEX-PLA of different concentrations or the original drug of DOX as control group for 24, 36, or 48 h. MTT method was used to observe the cancer inhibition rate. BALB/c nude mice underwent subcutaneous injection of HepG2 cells at the right scapula and then randomly divided into 4 groups to undergo intravenous injection of DOX/DEX-PLA (excremental group), original drug of DOX (naked drug group), DEX-PLA (nanovector group), or normal saline (blank control group) once every 5 days for 3 times. Twenty-one days later the mice were killed with the tumors taken out to measure the weight to analyze the inhibitory effect against hepatocarcinoma cells. RESULTS: The DOX/DEX-PLA nanoparticles were of round or elliptical shape with the diameter of about 83 nm, and the DOX entrapment efficiency was about 67.1%. The releasing test in vitro manifested a sustained release of over 50% of DOX encapsulated in DOX/DEX-PLA nanoparticles for about 7 days. Both the DOX/DEX-PLA nanoparticles and naked drug DOX inhibited the growth of HepG2 cells with a similar inhibitory rate (51.3% vs 50.7%, P > 0.05), meanwhile the DEX-PLA nanovector failed to inhibit the HepG2 cells. In-vivo experiment showed an inhibitory rate of DOX/DEX-PLA nanoparticles on hepatocellular carcinoma xenografts of 68.56%, significantly higher than that of the naked drug DOX (48.17%). CONCLUSION: DOX/DEX-PLA nanoparticles can effectively inhibit the growth of hepatocarcinoma cells.

Genetic recombinant expression and characterization of human augmenter of liver regeneration.

AIMS: To establish a highly effective prokaryotic recombinant expression system for human augmenter of liver regeneration (hALR) and to characterize the recombinant hALR both in vitro and in vivo. METHODS: ALR cDNA was synthesized and inserted into expression vector pET28a+, the recombinant plasmid was transformed into BL21, and expression of hALR was induced by IPTG. Recombinant hALR (rhALR) was purified by sequential detergent wash, enterokinase (EK) digestion, gel-filtration, and chelating chromatography. The rhALR was identified by SDS-PAGE, immunoblotting, MALDI-TOF-MS, and N-terminal sequencer. Cell proliferative effect of rhALR on human hepatocytes was analyzed by MTT. The protective effect of rhALR on liver function was observed on CCl(4)-induced intoxicated mice. RESULTS: Recombinant expression plasmid of ALR [pET28(a+)-hALR] was confirmed by restriction enzyme digestion and DNA sequencing. The expressed rhALR constituted 30% of total bacterial protein. Molecular weight was 15,029 for monomer and 30,136 for dimer by mass determination. N-terminal was M-R-T-Q-Q, exactly the same as anticipated for hALR. The purified protein migrating at about 15 KD showed excellent antigenicity in immunoblotting. The rhALR also showed a strong stimulative effect on hepatocyte proliferation. ALT and AST levels, liver histological structure, as well as the survival rate of CCl(4)-intoxicated mice were significantly improved when rALR was administrated at 40 microg/kg or 200 microg/kg. CONCLUSIONS: The rhALR is successfully expressed highly effectively with anticipated MW, N-terminal, and antigenicity. It could play an important role in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and improving liver function in CCl(4)-intoxicated mice.