Unlocking the potential of HHLA2: identifying functional immune infiltrating cells in the tumor microenvironment and predicting clinical outcomes in laryngeal squamous cell carcinoma.

BACKGROUND: HHLA2 (human endogenous retrovirus-H long terminal repeat-associating protein 2) represents a recently identified member of the B7 immune checkpoint family, characterized by limited expression in normal tissues but notable overexpression in various cancer types. Nevertheless, the precise function and interaction with immune cells remain poorly understood, particularly in laryngeal squamous cell carcinoma (LSCC). This investigation endeavored to elucidate the biological significance of HHLA2 within the tumor microenvironment of human LSCC tissues and delineate the clinical relevance and functional roles of HHLA2 in LSCC pathogenesis. METHODS: Through multiplexed immunohistochemistry analyses conducted on tissue microarrays sourced from LSCC patients (n = 72), the analysis was executed to assess the expression levels of HHLA2, density and spatial patterns of CD68+HLA-DR+CD163- (M1 macrophages), CTLA-4+CD4+FoxP3+ (CTLA-4+Treg cells), CTLA-4+CD4+FoxP3- (CTLA-4+Tcon cells), exhausted CD8+T cells, and terminally exhausted CD8+T cells in LSCC tissues. Survival analysis was conducted to evaluate the prognostic significance of HHLA2 and these immune checkpoints or immune cell populations, employing COX regression analysis to identify independent prognostic factors. RESULTS: Kaplan-Meier (K-M) survival curves revealed a significant association between HHLA2 expression and overall survival (OS) in LSCC. Elevated levels of HHLA2 were linked to reduced patient survival, indicating its potential as a prognostic marker (HR: 3.230, 95%CI 0.9205-11.34, P = 0.0067). Notably, increased infiltration of CD68+ cells (total macrophages), STING+CD68+HLA-DR+CD163- (STING+M1 macrophages), CTLA-4+CD4+FoxP3+, CTLA-4+CD4+FoxP3-, PD-1+LAG-3+CD8+T cells, and PD-1+LAG-3+TIM-3+CD8+T cells strongly linked to poorer survival outcomes (P < 0.05). A discernible trend was observed between the levels of these immune cell populations, STING+CD68+ (STING+ total macrophages), CD68+HLA-DR+CD163-, STING+CD68+CD163+HLA-DR- (STING+M2 macrophages), PD-1+LAG-3-CD8+T cells, PD-1+TIM-3+CD8+T cells, and PD-1+LAG-3+TIM-3-CD8+T cells and prognosis. Importantly, multivariate COX analysis identified HHLA2 as an independent predictive factor for OS in LSCC patients (HR = 3.86, 95% CI 1.08-13.80, P = 0.038). This underscored the potential of HHLA2 as a critical marker for predicting patient outcomes in LSCC. CONCLUSIONS: HHLA2 emerged as a detrimental prognostic biomarker for assessing OS in LSCC patients. Relative to other immune checkpoints, HHLA2 exhibited heightened predictive efficacy for the prognosis of LSCC patients.

Prognostic value of lactate dehydrogenase to absolute lymphocyte count ratio and albumin to fibrinogen ratio in diffuse large B-cell lymphoma.

With the continuous improvement of treatment strategy, the prognostic value of international prognostic index (IPI) alone is limited for diffuse large B-cell lymphoma (DLBCL). Our study aims to explore the effect of lactate dehydrogenase (LDH)to absolute lymphocyte count (ALC) ratio (LAR) and albumin to fibrinogen ratio (AFR) on the prognosis of patients with DLBCL. The venous blood LDH, ALC, albumin and fibrinogen within 1 week before the first chemotherapy in 74 DLBCL patients were collected to calculate the LAR and AFR values. The impact of LAR and AFR on the progression-free survival (PFS) of patients with DLBCL was studied by the survival analysis. The area under the receiver operating characteristic curve (AUC) and concordance index (C-index) were used to analyze the predictive efficiency of each model for the PFS of DLBCL patients. Cox univariate analysis suggested that elevated LAR (P < .001) and decreased AFR (P < .001) were risk factors for PFS in DLBCL patients. Multivariate analysis revealed that LAR (P < .001) and AFR (P = .004) were 2 independent prognostic parameters. The AUC values of IPI, AFR + IPI, LAR + IPI and AFR + LAR + IPI to predict the PFS of DLBCL patients were 0.806 (95%CI 0.707-0.905, P < .001), 0.839 (95%CI 0.747-0.932, P < .001), 0.851 (95%CI 0.764-0.938, P < .001), and 0.869 (95%CI 0.787-0.952, P < .001), respectively. The C-index values of above 4 models were 0.802 (95%CI 0.629-0.975, P < .001), 0.842 (95% CI 0.735-0.949, P < .001), 0.846 (95%CI 0.716-0.976, P < .001), and 0.864 (95%CI 0.781-0.941, P < .001), respectively. The results suggest that both LAR and AFR are independent prognostic factors for PFS in DLBCL patients. Furthermore, their combination with IPI has better predictive efficiency for the prognosis of DLBCL patients.

CircHIF1A induces cetuximab resistance in colorectal cancer by promoting HIF1α-mediated glycometabolism alteration.

Epidermal growth factor receptor (EGFR)-targeted therapy is an important treatment for RAS wild-type metastatic colorectal cancer (mCRC), but the resistance mechanism remains unclear. Here, the differential expression of circRNAs between Cetuximab sensitive and resistant cell lines was analyzed using whole-transcriptome sequencing. We identified that the expression of circHIF1A was significantly higher in LIM1215-R than in LIM1215. When treated with Cetuximab, downregulation of circHIF1A level weakened the proliferation and clonal formation ability of LIM1215-R, caused more cells to enter G0-G1 phase, and significantly reduced the basal respiration, ATP production, and maximal respiration, as well as the glycolytic capacity and glycolytic reserve. The response rate and prognosis of circHIF1A-positive patients were inferior to those of negative patients. Mechanistically, circHIF1A can upregulate the level of hypoxia-inducible factor 1 A (HIF1A) by competitively binding to miR-361-5p, inducing the overexpression of enzymes such as glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA). In a xenograft model, inhibition of circHIF1A expression increased the sensitivity to Cetuximab treatment. In conclusion, circHIF1A can promote HIF1α-mediated glycometabolism alteration to induce Cetuximab resistance in CRC. It has the potential to become a screening indicator for the Cetuximab beneficial population in mCRC and a new therapeutic target for enhancing treatment efficacy.

The m6A demethylase FTO targets POLQ to promote ccRCC cell proliferation and genome stability maintenance.

BACKGROUND AND AIM: As the first identified m6A demethylase, FTO has been implicated in the progression of various cancers. However, the specific mechanism of FTO in clear cell renal cell carcinoma (ccRCC) remains incompletely understood. In this study, we aimed to explore the potential molecular mechanisms influencing the progression of ccRCC. METHODS: We initially assessed the expression of FTO in tumor and adjacent tissues using TCGA database, RT-qPCR, and Western blot. We then conducted CCK-8, cell cycle analysis, and colony formation assay to investigate the impact of FTO on ccRCC cell proliferation. MeRIP-seq and RNA-seq were employed to identify potential downstream targets of FTO in ccRCC, and these findings were further validated through dual-luciferase reporter assays and MeRIP-qPCR. Then, DNA damage and cell death were assessed separately through gammaH2AX immunofluorescence detection and the LIVE/DEAD Fixable Dead Cell Stain assay, respectively. Subsequently, we identified downstream pathways influenced by FTO's regulation of POLQ through TCGA database analysis and GSEA enrichment analysis. Validation was carried out through Western blot. RESULTS: FTO is highly expressed in ccRCC tissues and cell lines. Furthermore, ROC curve demonstrates that FTO contributes to the diagnosis of ccRCC. FTO modulates m6A modification, consequently influencing the expression of POLQ, thus facilitating cell proliferation and maintaining genome stability in ccRCC. CONCLUSION: FTO could potentially serve as a diagnostic marker for ccRCC. FTO promotes the progression of ccRCC by regulating m6A modification, making the inhibition of FTO a potential novel therapeutic strategy in ccRCC.

TET2 inhibits the proliferation and metastasis of lung adenocarcinoma cells via activation of the cGAS-STING signalling pathway.

BACKGROUND: Effective identification and development of new molecular methods for the diagnosis, treatment and prognosis of lung adenocarcinoma (LUAD) remains an urgent clinical need. DNA methylation patterns at cytosine bases in the genome are closely related to gene expression, and abnormal DNA methylation is frequently observed in various cancers. The ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) and promote locus-specific DNA methylation reversal. This study aimed to explore the role of the TET2 protein and its downstream effector, 5-hmC/5-mC DNA modification, in LUAD progression. METHODS: The expression of TET2 was analysed by real-time PCR, Western blotting and immunohistochemistry. The 5-hmC DNA content was determined by a colorimetric kit. Activation of the cGAS-STING signalling pathway was evaluated by Western blotting. CCK-8, wound healing and Transwell assays were performed to evaluate the effect of TET2 on cell proliferation, migration and invasion abilities. A xenograft model was used to analyse the effect of TET2 on the tumorigenic ability of A549 cells. RESULTS: TET2 overexpression decreased proliferation and metastasis of A549 and H1975 cells in vitro and in vivo. However, TET2 knockdown dramatically enhanced the proliferation, migration and invasion of A549 and H1975 cells. Mechanistically, activation of the cGAS-STING signalling pathway is critical for the TET2-mediated suppression of LUAD cell tumorigenesis and metastasis. CONCLUSION: In this study, we demonstrate a tumour suppressor role of TET2 in LUAD, providing new potential molecular therapeutic targets and clinical therapies for patients with non-small cell lung cancer.

Prognostic values of tissue-resident CD8+T cells in human hepatocellular carcinoma and intrahepatic cholangiocarcinoma.

BACKGROUND: Tissue-resident CD8+T cells (CD103+CD8+T cells) are the essential effector cell population of anti-tumor immune response in tissue regional immunity. And we have reported that IL-33 can promote the proliferation and effector function of tissue-resident CD103+CD8+T cells. As of now, the immunolocalization and the prognostic values of tissue-resident CD8+T cells in human hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) still remain to be illustrated. METHODS: In our present study, we used the tissue microarrays of HCC and ICC, the multicolor immunohistochemistry (mIHC), and imaging analysis to characterize the tissue-resident CD8+T cells in HCC and ICC tissues. The prognostic values and clinical associations were also analyzed. We also studied the biological functions and the cell-cell communication between tumor-infiltrating CD103+CD8+T cells and other cell types in HCC and ICC based on the published single-cell RNA sequencing (scRNA-seq) data. RESULTS: Our work unveiled the expressions of CD8 and CD103 and immunolocalization of tissue-resident CD8+T cells in human HCC and ICC. Elevated CD8+T cells indicated a better overall survival (OS) rate, implying that tumor-infiltrating CD8+T cells in HCC and ICC could serve as an independent prognostic factor. Moreover, the number of CD103+CD8+T cells was increased in HCC and ICC tissues compared with adjacent normal tissues. HCC patients defined as CD8highCD103high had a better OS, and the CD8lowCD103low group tended to have a poorer prognosis in ICC. Evaluation of the CD103+CD8+T-cell ratio in CD8+T cells could also be a prognostic predictor for HCC and ICC patients. A higher ratio of CD103+CD8+T cells over total CD8+T cells in HCC tissues was negatively and significantly associated with the advanced pathological stage. The percentage of higher numbers of CD103+CD8+T cells in ICC tissues was negatively and significantly associated with the advanced pathological stage. In contrast, the higher ratio of CD103+CD8+T cells over total CD8+T cells in ICC tissues was negatively and significantly associated with the advanced pathological stage. In addition, single-cell transcriptomics revealed that CD103+CD8+T cells were enriched in genes associated with T-cell activation, proliferation, cytokine function, and T-cell exhaustion. CONCLUSION: The CD103+ tumor-specific T cells signified an important prognostic marker with improved OS, and the evaluation of the tissue-resident CD103+CD8+T cells might be helpful in assessing the on-treatment response of liver cancer.

N-acetyltransferase 10 promotes colon cancer progression by inhibiting ferroptosis through N4-acetylation and stabilization of ferroptosis suppressor protein 1 (FSP1) mRNA.

BACKGROUND: N-acetyltransferase 10 (NAT10) is the only enzyme known to mediate the N4-acetylcytidine (ac4C) modification of mRNA and is crucial for mRNA stability and translation efficiency. However, its role in cancer development and prognosis has not yet been explored. This study aimed to examine the possible role of NAT10 in colon cancer. METHODS: The expression levels of NAT10 were evaluated by immunohistochemical analyses with a colon cancer tissue microarray, and its prognostic value in patients was further analyzed. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were performed to analyze NAT10 expression in harvested colon cancer tissues and cell lines. Stable NAT10-knockdown and NAT10-overexpressing colon cancer cell lines were constructed using lentivirus. The biological functions of NAT10 in colon cancer cell lines were analyzed in vitro by Cell Counting Kit-8 (CCK-8), wound healing, Transwell, cell cycle, and ferroptosis assays. Xenograft models were used to analyze the effect of NAT10 on the tumorigenesis and metastasis of colon cancer cells in vivo. Dot blotting, acetylated RNA immunoprecipitation-qPCR, and RNA stability analyses were performed to explore the mechanism by which NAT10 functions in colon cancer progression. RESULTS: NAT10 was upregulated in colon cancer tissues and various colon cancer cell lines. This increased NAT10 expression was associated with shorter patient survival. Knockdown of NAT10 in two colon cancer cell lines (HT-29 and LoVo) impaired the proliferation, migration, invasion, tumor formation and metastasis of these cells, whereas overexpression of NAT10 promoted these abilities. Further analysis revealed that NAT10 exerted a strong effect on the mRNA stability and expression of ferroptosis suppressor protein 1 (FSP1) in HT-29 and LoVo cells. In these cells, FSP1 mRNA was found to be modified by ac4C acetylation, and this epigenetic modification was associated with the inhibition of ferroptosis. CONCLUSIONS: Our study revealed that NAT10 plays a critical role in colon cancer development by affecting FSP1 mRNA stability and ferroptosis, suggesting that NAT10 could be a novel prognostic and therapeutic target in colon cancer.

Aberrant lncRNA-mRNA expression profile and function networks during the adipogenesis of mesenchymal stem cells from patients with ankylosing spondylitis.

Objective: We have already demonstrated that mesenchymal stem cells from patients with ankylosing spondylitis (ASMSCs) exhibited greater adipogenic differentiation potential than those from healthy donors (HDMSCs). Here, we further investigated the expression profile of long noncoding RNA (lncRNA) and mRNA, aiming to explore the underlying mechanism of abnormal adipogenic differentiation in ASMSCs. Methods: HDMSCs and ASMSCs were separately isolated and induced with adipogenic differentiation medium for 10 days. Thereafter, lncRNAs and mRNAs that were differentially expressed (DE) between HDMSCs and ASMSCs were identified via high-throughput sequencing and confirmed by quantitative real-time PCR (qRT-PCR) assays. Then, the DE genes were annotated and enriched by GO analysis. In addition, protein interaction network was constructed to evaluate the interactions between DE mRNAs and to find hub nodes and study cliques. Besides, co-expression network analysis was carried out to assess the co-expressions between DE mRNA and DE lncRNAs, and competing endogenous RNA (ceRNA) network analysis were conducted to predict the relationships among lncRNAs, mRNAs and miRNAs. The signaling pathways based on the DE genes and the predicted DE genes were enriched by KEGG analysis. Results: A total of 263 DE lncRNAs and 1376 DE mRNAs were found during adipogenesis in ASMSCs. qRT-PCR indicated that the expression of the top 20 mRNAs and the top 10 lncRNAs was consistent with the high-throughput sequencing data. Several lncRNAs (NR_125386.1, NR_046473.1 and NR_038937.1) and their target genes (SPN and OR1AIP2), together with the significantly co-expressed pairs of DE lncRNAs and DE mRNAs (SLC38A5-ENST00000429588.1, TMEM61-ENST00000400755.3 and C5orf46-ENST00000512300.1), were closely related to the enhanced adipogenesis of ASMSCs by modulating the PPAR signaling pathway. Conclusion: Our study analyzed the expression profiles of DE lncRNAs and DE mRNAs during adipogenesis in ASMSCs and HDMSCs. Several DE lncRNAs, DE mRNAs and signaling pathways that probably participate in the aberrant adipogenesis of ASMSCs were selected for future study. These results will likely provide potential targets for our intervention on fat metaplasia and subsequent new bone formation in patients with AS in the future.

TIGIT Blockade Exerts Synergistic Effects on Microwave Ablation Against Cancer.

Background: Combination immunotherapy based on immune checkpoint inhibitors (ICIs) has shown great success in the treatment of many types of cancers and has become the mainstream in the comprehensive treatment of cancers. Ablation in combination with immunotherapy has achieved tremendous efficacy in some preclinical and clinical studies. To date, our team proved that ablation in combination with ICIs was a promising antitumor therapeutic strategy for the liver metastasis of colorectal cancer (CRC). Moreover, we found that the expression of T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) expression was up-regulated after microwave ablation (MWA), indicating that TIGIT was involved in immunosuppression, and the combination of MWA and TIGIT blockade represented a potential clinical treatment strategy. Methods: In the present study, we examined the expression of TIGIT using a preclinical mouse model treated with MWA. Moreover, we evaluated the antitumor functions of MWA alone or in combination with TIGIT blockade by monitoring tumor growth and survival of the mice. Besides, we also detected the numbers of tumor-infiltrating lymphocytes (TILs), and effector molecules of CD8+ T cells using flow cytometry. Finally, we analyzed the single-cell RNA sequencing (scRNA-seq) data from the MWA and MWA plus anti-TIGIT groups. Results: The expression of TIGIT in various immune cells was up-regulated after MWA, and the addition of TIGIT blockade to MWA prolonged survival and delayed tumor growth in the MC38 tumor model. Taken together, our findings showed that TIGIT blockade in combination with MWA significantly promoted the expansion and functions of CD8+ TILs and reshaped myeloid cells in the tumor microenvironment (TME) using flow cytometry and scRNA-seq analysis. Conclusions: TIGIT blockade in combination with MWA was a novel treatment strategy for the liver metastasis of CRC, and this combination therapy could reprogram the TME toward an antitumor environment.

Would foot arch development in children characterize a body maturation process? A prospective longitudinal study.

BACKGROUND: Flatfoot (Pes Planus), often regarded as a physiological deviation in children, is of concern to parents because there is no test to predict the development of foot arch. This study aimed to use a new diagnostic flatfoot criterion to determine 1) how the footprint index changes during the development of foot arches, 2) what factors can predict a foot arch development, and 3) whether foot arch development could be a process of body growth. METHODS: 572 children were enrolled in a prospective longitudinal study of anthropometrical parameters and physical fitness twice at age of 6.7 and 8.2 years. The bimodal frequency distribution of the Chippaux-Smirak index (CSI) of the footprint was used to define flatfoot as CSI <0.58 and non-flatfoot as CSI >0.61. Body measurements and physical fitness tests were compared between children with flatfeet who developed foot arches and children who did not. RESULTS: Of 263 children with flatfeet, the CSI significantly changed from 0.72 to 0.46 in 70 children who developed foot arches over 1.5 years and the others had minimal change in the index. Children with foot arch development had a lower initial CSI, improved boys' performance in one-leg balance, and less increase in girls' body height than children who remained flatfooted, whereas sex and weight were similar in both groups. CONCLUSION: This longitudinal study with the bimodal distribution of the CSI investigated how the development of foot arch advances in children around age 7. A significant and unique pattern in change of the CSI suggests involvement of a maturational stage in foot arch development. Along with the improved performance in one-leg balance, the unidirectional transition from flatfoot to non-flatfoot is associated with improvement in motor control of the ankle. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR-OCS-14004300).