Integrated single-cell and spatial transcriptomic profiling reveals higher intratumour heterogeneity and epithelial-fibroblast interactions in recurrent bladder cancer.

BACKGROUND: Recurrent bladder cancer is the most common type of urinary tract malignancy; nevertheless, the mechanistic basis for its recurrence is uncertain. Innovative technologies such as single-cell transcriptomics and spatial transcriptomics (ST) offer new avenues for studying recurrent tumour progression at the single-cell level while preserving spatial data. METHOD: This study integrated single-cell RNA (scRNA) sequencing and ST profiling to examine the tumour microenvironment (TME) of six bladder cancer tissues (three from primary tumours and three from recurrent tumours). FINDINGS: scRNA data-based ST deconvolution analysis revealed a much higher tumour heterogeneity along with TME in recurrent tumours than in primary tumours. High-resolution ST analysis further identified that while the overall natural killer/T cell and malignant cell count or the ratio of total cells was similar or even lower in the recurrent tumours, a higher interaction between epithelial and immune cells was detected. Moreover, the analysis of spatial communication reveals a marked increase in activity between cancer-associated fibroblasts (CAFs) and malignant cells, as well as other immune cells in recurrent tumours. INTERPRETATION: We observed an enhanced interplay between CAFs and malignant cells in bladder recurrent tumours. These findings were first observed at the spatial level.

Mucin 4 Gene Silencing Reduces Oxidative Stress and Calcium Oxalate Crystal Formation in Renal Tubular Epithelial Cells Through the Extracellular Signal-Regulated Kinase Signaling Pathway in Nephrolithiasis Rat Model.

BACKGROUND/AIMS: Nephrolithiasis plagues a great number of patients all over the world. Increasing evidence shows that the extracellular signal-regulated kinase (ERK) signaling pathway and renal tubular epithelial cell (RTEC) dysfunction and attrition are central to the pathogenesis of kidney diseases. Mucin 4 (MUC4) is reported as an activator of ERK signaling pathway in epithelial cells. In this study, using rat models of calcium oxalate (CaOx) nephrolithiasis, the present study aims to define the roles of MUC4 and ERK signaling pathway as contributors to oxidative stress and CaOx crystal formation in RTEC. METHODS: Data sets of nephrolithiasis were searched using GEO database and a heat flow map was drawn. Then MUC4 function was predicted. Wistar rats were prepared for the purpose of model establishment of ethylene glycol and ammonium chloride induced CaOx nephrolithiasis. In order to assess the detailed regulatory mechanism of MUC4 silencing on the ERK signaling pathway and RTEC, we used recombinant plasmid to downregulate MUC4 expression in Wistar rat-based models. Samples from rat urine, serum and kidney tissues were reviewed to identify oxalic acid and calcium contents, BUN, Cr, Ca2+ and P3+ levels, calcium crystal formation in renal tubules and MUC4 positive expression rate. Finally, RT-qPCR, Western blot analysis, and ELISA were employed to access oxidative stress state and CaOx crystal formation in RTEC. RESULTS: Initially, MUC4 was found to have an influence on the process of nephrolithiasis. MUC4 was upregulated in the CaOx nephrolithiasis model rats. We proved that the silencing of MUC4 triggered the inactivation of ERK signaling pathway. Following the silencing of MUC4 or the inhibition of ERK signaling pathway, the oxalic acid and calcium contents in rat urine, BUN, Cr, Ca2+ and P3+ levels in rat serum, p-ERK1/2, MCP-1 and OPN expressions in RTEC and H2O2 and MDA levels in the cultured supernatant were downregulated, but the GSH-Px, CAT and SOD levels in the cultured supernatant were increased. Moreover, MUC4 silencing or ERK signaling pathway inactivation may decrease the formation of CaOx crystals. CONCLUSION: Taken together, silencing of MUC4 can inactivate the ERK signaling pathway and further restrain oxidative stress and CaOx crystal formation in RTEC. Thus, MUC4 represents a potential investigative focus target in nephrolithiasis.

Clinical relevance of expression of B7-H1 and B7-H4 in ovarian cancer.

The aim of the present study was to investigate the expression of B7-H1 and B7-H4 in ovarian neoplasm tissues and to examine their clinical relevance. A total of 112 ovarian biopsies were collected from patients with epithelial ovarian cancer (EOC) and 10 were taken from ovarian benign neoplasms. The samples were processed in paraffin tissue chips, and subjected to immunohistochemical staining and analysis. Associations of B7-H1 and B7-H4 expression with patients' clinical parameters, such as histological typing, cell grading, International Federation of Gynecology and Obstetrics staging, tumor size, and metastatic status, were examined by statistical analysis. Survival curves were constructed using the Kaplan-Meier method and the log-rank test. Independent prognostic factors were evaluated using the Cox regression model. The results showed an extremely low or negative expression of B7-H1 and B7-H4 in the 10 benign ovarian neoplasm tissues (control): By contrast, a positive expression of B7-H1 and B7-H4 was observed in 55.4% (62/112) and 37.5% (42/112) of the EOC tissues, respectively. The differences between the two groups were significant. In addition, the co-expression of B7-H1 and B7-H4 was found in 31.3% (35/112) of the EOC cases. Furthermore, the progression-free survival and overall survival were significantly lower in EOC patients with a high expression of B7-H1 and B7-H4 (χ2=45.60 and 37.99, respectively). These results demonstrated that the expression of B7-H1 and B7-H4 in EOC tissues was significantly associated with poor prognosis and high relapse rate of EOC. The findings suggest that B7-H1 and B7-H4 is a negative prognostic marker for EOC and a potential immunotherapeutic target for patients with EOC.

Expressions of ER, PR, HER-2, COX-2, and VEGF in primary and relapsed/metastatic breast cancers.

In the present study, we evaluated expressions of estrogen receptor (ER), progestin receptor (PR), human epidermal growth factor receptor-2 (HER-2), cyclooxygenase-2 (COX-2), and vascular endothelial growth factor (VEGF) in primary and relapsed/metastatic breast cancers to elucidate the clinical significance of these markers. The markers were evaluated by immunohistochemistry in specimens of 50 patients with primary or metastatic breast cancer. Positive rates of ER were significantly (p = 0.002) higher in primary versus relapsed/metastatic breast cancer (70 vs. 38 %, respectively). The VEGF positive expression rates were also significantly higher in primary versus metastatic cancer (82 vs. 38 %, respectively; p < 0.001). By contrast, positive rates of HER-2 and COX-2 were not significantly different between different types of cancer. COX-2 correlated with HER-2 expression in both primary and relapsed/metastatic focuses of breast cancer. COX-2 also correlated with VEGF expression in primary breast cancer. Expressions of ER, PR, HER2, and COX-2 did not correlate between primary and relapsed/metastatic breast cancers, indicating that the treatment decision should be made according to the status of these markers in relapsed/metastatic focuses. The total change rates of ER, PR, HER-2, COX-2, and VEGF were 26, 18, 10, 30, and 58 %, respectively. In conclusion, HER-2 and COX-2, along with VEGF, appear to play a role in the development and progression of breast cancer. In addition, all of the studied markers may serve as indicators of prognosis.