The impacts of the tumor microenvironment (TME) on tumor evolvability remain unclear. A challenge for nearly all cancer types is spatial heterogeneity, providing substrates for the emergence and evolvability of drug resistance and leading to unfavorable prognosis. Understanding TME heterogeneity among different tumor sites would provide deeper insights into personalized therapy. We found 9,992 cell profiles of the TME in human lung adenocarcinoma (LUAD) samples at a single-cell resolution. By comparing different tumor sites, we discovered high TME heterogeneity. Single-sample gene set enrichment analysis (ssGSEA) was utilized to explore functional differences between cell subpopulations and between the core, middle and edge of tumors. We identified 8 main cell types and 27 cell subtypes of T cells, B cells, fibroblasts and myeloid cells. We revealed CD4+ naive T cells in the tumor core that express high levels of immune checkpoint molecules and have a higher activity of immune-exhaustion signaling. CD8+ T cell subpopulations in the tumor core correlate with the upregulated activity of transforming growth factor-β (TGF-β) and fibroblast growth factor receptor (FGFR) signaling and downregulated T cell activity. B cell subtypes in the tumor core downregulate cytokine production. In this study, we revealed that there was immunological heterogeneity in the TME of patients with LUAD that have different ratios of immune cells and stromal cells, different functions, and various degrees of activation of immune-related pathways in different tumor parts. Therefore, clarifying the spatial heterogeneity of the tumor in the immune microenvironment can help clinicians design personalized treatments.

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignancies worldwide which originates from the malignant transformation of esophageal epithelial cells. Dysregulated expression of Keratin17 (KRT17) has been claimed in a variety of malignancies, while its role in ESCC remains unclear. Therefore, our study aimed to explore the potential function and underlying molecular mechanism of KRT17 in ESCC.MethodsData-independent acquisition-mass spectrometry (DIA-MS) workflow was used to analysis KRT17 expression between ESCC and adjacent non-cancerous esophageal tissues. The online database gene expression profiling interactive analysis (GEPIA) was used to further determine the differential expression of KRT17 in tissues. The function of KRT17 in ESCC was tested on two human esophageal cancer cell lines (EC9706 and ECA109). Small interfering RNA (siRNA) was used to inhibit KRT17 expression. Cell proliferation was examined by cell counting kit 8 (CCK8) reagent, colony formation assay, cell cycle distribution analysis and apoptosis. Cell migration was examined by transwell and wound healing assay. Cell invasion was also examined by transwell assay. Western blot and quantitative real-time PCR (qRT-PCR) was used to evaluate protein and mRNA levels, respectively.ResultsKRT17 expression was higher in cancer tissues compared with normal tissues. Transfected with siKRT17 attenuated protein and mRNA levels of KRT17, inhibited proliferation, migration and invasion, and decreased mTOR/S6K1 phosphorylation levels in EC9706 and ECA109.ConclusionKRT17 facilitates proliferation, migration and invasion in ESCC cells, and these cell viability functions were mediated by mTOR/S6K1 pathway.

Background: Blood based liquid biopsy has proved its potential in enormous clinical applications, such as cancer screening, diagnosis, treatment guidance, disease tracking and monitoring. In certain scenario (e.g., molecular residual disease), it requires the technique to be able to detect mutation with very low frequency (0.001% ~ 1%). The major hurdle of ultra-sensitive circulating tumor DNA sequencing is the high background noise of plasma cell-free DNA (cfDNA) and clonal hematopoiesis (CH). Here in this study, we investigated the prevalence of CH in lung cancer patients and its interference with liquid biopsy. Methods: We retrospectively analyzed cfDNA and blood cell genomic DNA (gDNA) sequencing data sets (n=1261) from a group of Chinese lung cancer patients. Threshold (1%) and subthreshold (0.2%) for variant allele frequency were set and compared. We focused on 23 clonal hematopoiesis genes that were selected based on previous publications. Results: CH mutations were detected in 27.68% of all the patients at the threshold and 62.01% at the subthreshold, and the detection rate increased with age. DNMT3A was the most frequently mutated CH gene, accounted for more than half of the CH mutations. The CH mutations had a higher detection rate in smokers (72%) than non-smokers (59.4%) at subthreshold. VAFs of CH mutations in cell-free DNA strongly correlated with their VAFs in gDNA (Pearson’s R =0.92, p<2.2x10-16), while tumor derived somatic mutations didn’t have such correlation. Conclusion: Our study showed that clonal hematopoiesis is very common in lung cancer patients, especially when examining low frequency mutations. Sequencing of gDNA at equivalent depth is very important to filter out CH mutation in cancer liquid biopsy.