Single-cell RNA sequencing reveals the lineage of malignant epithelial cells and upregulation of TAGLN2 promotes peritoneal metastasis in gastric cancer

Background Peritoneal metastasis (PM) is an important factor contributing to poor prognosis in patients with gastric cancer (GC). Transcriptomic sequencing has been used to explore the molecular changes in metastatic cancers, but comparing the bulk RNA-sequencing data between primary tumors and metastases in PM studies is unreasonable due to the small proportion of tumor cells in PM tissues. Methods We performed single-cell RNA-sequencing analysis on four gastric adenocarcinoma specimens, including one primary tumor sample (PT), one adjacent nontumoral sample (PN), one peritoneal metastatic sample (MT) and one normal peritoneum sample (MN), from the same patient. Pseudotime trajectory analysis was used to display the process by which nonmalignant epithelial cells transform into tumor cells and then metastasize to the peritoneum. Finally, in vitro and in vivo assays were used to validate one of the selected genes that promote peritoneal metastasis. Results Single-cell RNA sequencing showed that a development curve was found from normal mucosa to tumor tissues and then into metastatic sites on peritoneum. TAGLN2 was found to trigger this metastasis process. The migration and invasion capability of GC cells were changed by downregulating and upregulating TAGLN2 expression. Mechanistically, TAGLN2 might modulate tumor metastasis via alterations in cell morphology and several signaling pathways, thus promoting epithelial–mesenchymal transition (EMT). Conclusions In summary, we identified and validated TAGLN2 as a novel gene involved in GC peritoneal metastasis. This study provided valuable insight into the mechanisms of GC metastasis and developed a potential therapeutic target to prevent GC cell dissemination (AU)

Single-cell RNA sequencing reveals the lineage of malignant epithelial cells and upregulation of TAGLN2 promotes peritoneal metastasis in gastric cancer.

BACKGROUND: Peritoneal metastasis (PM) is an important factor contributing to poor prognosis in patients with gastric cancer (GC). Transcriptomic sequencing has been used to explore the molecular changes in metastatic cancers, but comparing the bulk RNA-sequencing data between primary tumors and metastases in PM studies is unreasonable due to the small proportion of tumor cells in PM tissues. METHODS: We performed single-cell RNA-sequencing analysis on four gastric adenocarcinoma specimens, including one primary tumor sample (PT), one adjacent nontumoral sample (PN), one peritoneal metastatic sample (MT) and one normal peritoneum sample (MN), from the same patient. Pseudotime trajectory analysis was used to display the process by which nonmalignant epithelial cells transform into tumor cells and then metastasize to the peritoneum. Finally, in vitro and in vivo assays were used to validate one of the selected genes that promote peritoneal metastasis. RESULTS: Single-cell RNA sequencing showed that a development curve was found from normal mucosa to tumor tissues and then into metastatic sites on peritoneum. TAGLN2 was found to trigger this metastasis process. The migration and invasion capability of GC cells were changed by downregulating and upregulating TAGLN2 expression. Mechanistically, TAGLN2 might modulate tumor metastasis via alterations in cell morphology and several signaling pathways, thus promoting epithelial-mesenchymal transition (EMT). CONCLUSIONS: In summary, we identified and validated TAGLN2 as a novel gene involved in GC peritoneal metastasis. This study provided valuable insight into the mechanisms of GC metastasis and developed a potential therapeutic target to prevent GC cell dissemination.

Intraoperative Pathological Evaluation of Suspicious Peritoneal Nodules for Surgical Decision-making in Gastric Cancer.

BACKGROUND: When frozen pathological results of suspicious peritoneal nodules found in gastric cancer (GC) patients are negative or indeterminant, whether to perform gastrectomy will always be a dilemma for surgeons. This study aimed to facilitate intraoperative surgical decision-making based on frozen section (FS) results and clinicopathological characteristics. METHODS: From January 2015 to July 2021, 318 GC patients were enrolled retrospectively. The correlations between frozen and paraffin pathology of peritoneal nodules were examined. Then, predictive factors of positive paraffin section (PS) results were identified, and a nomogram was constructed. The survival significance of gastrectomy was also explored. RESULTS: Of 70 FS-negative patients, 59 (84.3%) had concordant negative PS results, while the PS results of 11 (15.7%) were positive. Forty-six (93.9%) and 3 (6.1%) of 49 patients with indeterminant FS results had positive and negative PS results, respectively. The PS results of 95 FS-positive patients were all positive. A nomogram for predicting positive PS results was developed based on Lauren type, nodule distribution, and CA125. Gastrectomy for FS-negative patients improved survival compared to no gastrectomy (HR 0.26, 95% CI 0.11-0.62; P = 0.0012). Survival benefits for gastrectomy vs. no gastrectomy were not demonstrated in patients with indeterminant (HR 0.74, 95% CI 0.27-2.01; P = 0.53) and positive (HR 0.87, 95% CI 0.43-1.74; P = 0.69) FS results. CONCLUSIONS: Gastrectomy can be justified for the treatment of operable GC patients with negative frozen pathological results of peritoneal nodules. For patients with positive and indeterminant frozen pathological results, gastrectomy is not recommended unless it is performed as palliative surgery.

Identification of metabolism-related genes for predicting peritoneal metastasis in patients with gastric cancer.

OBJECTIVE: The reprogramming of metabolism is an important factor in the metastatic process of cancer. In our study, we intended to investigate the predictive value of metabolism-related genes (MRGs) in recurrent gastric cancer (GC) patients with peritoneal metastasis. METHODS: The sequencing data of mRNA of GC patients were obtained from Asian Cancer Research Group (ACRG) and the GEO databases (GSE53276). The differentially expressed MRGs (DE-MRGs) between a cell line without peritoneal metastasis (HSC60) and one with peritoneal metastasis (60As6) were analyzed with the Limma package. According to the LASSO regression, eight MRGs were identified as crucially related to peritoneal seeding recurrence in patients. Then, disease free survival related genes were screened using Cox regression, and a promising prognostic model was constructed based on 8 MRGs. We trained and verified it in two independent cohort. RESULTS: We confirmed 713 DE-MRGs and the enriched pathways. Pathway analysis found that the MRG-related pathways were related to tumor metabolism development. With the help of Kaplan-Meier analysis, we found that the group with higher risk scores had worse rates of peritoneal seeding recurrence than the group with lower scores in the cohorts. CONCLUSIONS: This study developed an eight-gene signature correlated with metabolism that could predict peritoneal seeding recurrence for GC patients. This signature could be a promising prognostic model, providing better strategy in treatment.

The kinase activity of integrin-linked kinase regulates cellular senescence in gastric cancer.

The activity of integrin-linked kinase (ILK) in cancerous cells is often oncogenic and associated with malignant properties, such as uncontrolled cell cycle progression and evasion from senescence. However, the role of ILK in cellular senescence in gastric cancer (GC) has not been previously examined. We generated single-cell clones of ILK knock-out using CRISPR-Cas9 in human GC lines with mesenchymal or epithelial histology. Cells with no residual ILK expression exhibited strong cellular senescence with diminished clathrin-mediated endocytosis, Surprisingly, ILK loss-induced cellular senescence appeared to be independent of its function in integrin signaling. The low dose of CPD22, a small molecule inhibitor of ILK activity-induced senescence in three GC cell lines with different histologies. Furthermore, senescent cells with ILK depletion transfected with N-terminal truncated ILK mutant remaining catalytic domains displayed the reduction of senescent phenotypes. RNA sequencing and cytokine array results revealed the enrichment of multiple pro-inflammatory signaling pathways in GC lines in the absence of ILK. Our study identified the important role and the potential mechanism of ILK in the cellular senescence of cancerous epithelial cells. The inhibition of ILK activity using small molecule compounds could have a pro-senescent effect as a therapeutic option for GC.

Bone marrow mesenchymal stem cells inhibit dendritic cells differentiation and maturation by microRNA-23b.

Research on regulation and its mechanism of bone marrow mesenchymal stem cells (BMSCs) on dendritic cells (DCs), which is the initiating factor in immune response has applicable clinical value. Although BMSCs have a significant regulatory effect on the maturation of DCs, its molecular mechanism is still unclear. BMSCs and DCs, were co-cultured by different concentration ratios. Flow cytometry was used to detect the expression of DC markers (CD83, CD11c). Quantitative reverse transcription PCR (qRT-PCR) was used to measure the expression of related genes in RNA level. Expression of the target proteins was detected with using Western blot assay. miRNA inhibitor and miRNA mimic were used to suppress and up-regulate the expression of the target gene. In this research, our results demonstrated that BMSCs notably inhibited maturation of DCs in the co-culture system of BMSCs and DCs and confirmed that this inhibition is due to overexpression of miR-23b Furthermore, this research found that miR-23b overexpression inhibited the expression of p50/p65, thus blocked the activation of the NF-κB pathway. In conclusion, BMSCs affected the activation of NF-κB pathway through miR-23b overexpression resulting in inhibition of the maturation and differentiation of DCs.