Background Immunotherapy for malignant tumors has made great progress, but many patients do not benefit from it. The complex intratumoral heterogeneity (ITH) hindered the in-depth exploration of immunotherapy. Conventional bulk sequencing has masked intratumor complexity, preventing a more detailed discovery of the impact of ITH on treatment efficacy. Hence, we initiated this study to explore ITH at the multi-omics spatial level and to seek prognostic biomarkers of immunotherapy efficacy considering the presence of ITH. Methods Using the segmentation strategy of digital spatial profiling (DSP), we obtained differential information on tumor and stromal regions at the proteomic and transcriptomic levels. Based on the consideration of ITH, signatures constructed by candidate proteins in different regions were used to predict the efficacy of immunotherapy. Results Eighteen patients treated with a bispecific antibody (bsAb)-KN046 were enrolled in this study. The tumor and stromal areas of the same samples exhibited distinct features. Signatures consisting of 11 and 18 differentially expressed DSP markers from the tumor and stromal areas, respectively, were associated with treatment response. Furthermore, the spatially resolved signature identified from the stromal areas showed greater predictive power for bsAb immunotherapy response (area under the curve=0.838). Subsequently, our stromal signature was validated in an independent cohort of patients with non-small cell lung cancer undergoing immunotherapy. Conclusion We deciphered ITH at the spatial level and demonstrated for the first time that genetic information in the stromal region can better predict the efficacy of bsAb treatment. Trial registration number NCT03838848.

Immune checkpoint inhibitors combined with chemotherapy as a neoadjuvant therapy have been applied to the treatment of esophageal squamous cell carcinoma (ESCC). However, the optimal regimen needs to be further explored, particularly for older patients, and the mechanisms by which the immune checkpoint inhibitor combined with chemotherapy modulates the evolution of ESCC are unknown. In this single-arm phase 2 trial, patients with resectable (stage II/III/IV without metastasis) ESCC were enrolled and received nanoparticle albumin-bound (nab) paclitaxel for two cycles and oral S-1 for 2 weeks, combined with intravenous toripalimab for two cycles before surgery. Combination postoperative adjuvant therapy was administered. The primary outcome was the major pathological response (MPR). Secondary outcomes included pathological complete response (pCR), overall response rate (ORR), disease control rate (DCR), disease-free survival (DFS), overall survival (OS), improvement in Stooler's dysphagia score and degree of daily living ability (dADL). Biopsies and plasma pre- and post-neoadjuvant therapy were performed using whole-exome sequencing, transcriptome sequencing, immunohistochemistry (IHC) for PD-L1, multiplex immunofluorescence (mIF) and proximity extension assay technology (PEA) for 92 proteins. From November 2019 to July 2021, 60 patients were enrolled. After neoadjuvant therapy, R0 resection was achieved in 55 (98.21%) patients. MPR was identified in 27 patients (49.09%), and 16 patients (29.09%) achieved pCR. Patients with PR, SD and PD were 37 (61.67%), 21 (35.00%) and 2 (3.33%), respectively. The overall staging, Stooler dysphagia scores and dADL were significantly decreased after treatment. 11 patients (18.3%) experienced grade ≥3 AEs. Compared to PD-L1-Low patients, PD-L1-High patients had a significantly higher ratio of PR. During therapy, the tumor mutation burden (TMB) and tumor neoantigen burden (TNB) were significantly decreased in patients with PR. Differential clonal evolution within tumors was demonstrated by analysis of intratumoral heterogeneity. Transcriptome analyses revealed that the infiltration of CD4+ T lymphocytes at baseline was associated with clinical outcome. During therapy, CD8+ T cells and CD4+ T cells were increased in all patients; however, exhausted cells, nTregs and iTregs were significantly increased in patients with non-MPR. Protein analyses revealed that the levels of IFN-γ, Gal.1 and LAMP3 can predict the clinical benefit. In addition, the expression of CD83, TNFRSF4, TNFSF14, VEGFR2, ADA, ARG1, and HO-1 was associated with serious AEs. More importantly, the integration of CD4+ T cells with plasma protein of IFN-γ, Gal.1 or LAMP3 could further distinguish responders from non-responders. In this study, neoadjuvant therapy with toripalimab, nab-paclitaxel and S-1 was less toxic and showed promising antitumor activity in patients with resectable ESCC. Changes in the genome, transcriptome, PD-L1 expression and serum proteins were comprehensively analyzed and correlated with clinical outcomes, which provides insight into the mechanism of action of toripalimab combined with nab-paclitaxel and S-1 in patients with ESCC. This study was funded by Major projects of the ministry of science and technology of the 13th five-year plan of China [grant number: 2018ZX09201013].

Lung adenocarcinoma is heterogeneous and hierarchically organized, with a subpopulation of stem-like cells (CSCs) that reside at the apex of the hierarchy, in which exosomes act as important mediators by transporting specific molecules among different cell populations. Although there have been numerous studies on tumor exosomes, the constituents and functional properties of CSC-derived exosomes are still poorly characterized. Here we present a detail transcriptome and proteome atlas of the exosomes released by human lung adenocarcinoma stem-like cells (LSLCs). The transcriptome analysis indicates the specific patterns of exosomal constituents, including the fragmentation of transcripts and the low-level presence of circular RNAs, and identifies multiple exosomal-enriched mRNAs and lncRNAs. Integrative analysis of transcriptome and proteome data reveals the diverse functions of exosomal-enriched RNAs and proteins, many of which are associated with tumorigenesis. Importantly, several LSLC markers we identified are highly expressed in LSLC-derived exosomes and associate with poor survival, which may serve as promising liquid biopsy biomarkers for lung adenocarcinoma diagnosis. Our study provides a resource for the future elucidation of the functions of tumor-derived exosomes and their regulatory mechanisms in mediating lung cancer development.

Multiple pulmonary lung cancers (MPLCs) are frequently encountered on computed tomography (CT) scanning of chest, yet their intrinsic characteristics associated with genomic features and radiological or pathological textures that may lead to distinct clinical outcomes remain largely unexplored. A total of 27 pulmonary nodules covering different radiological or pathological textures as well as matched adjacent normal tissues and blood samples were collected from patients diagnosed with MPLCs. Whole-exome sequencing (WES) and whole-transcriptome sequencing were performed. The molecular and immune features of MPLCs associated with distinct radiological or pathological textures were comprehensively investigated. Genomics analysis unveiled the distinct branches of pulmonary nodules originating independently within the same individual. EGFR and KRAS mutations were found to be prevalent in MPLCs, exhibiting mutual exclusivity. The group with KRAS mutations exhibited stronger immune signatures compared to the group with EGFR mutations. Additionally, MPLCs exhibited a pronounced immunosuppressive microenvironment, which was particularly distinct when compared with normal tissues. The expression of the FDSCP gene was specifically observed in MPLCs. When categorizing MPLCs based on radiological or pathological characteristics, a progressive increase in mutation accumulation was observed, accompanied by heightened chromatin-level instability as ground-glass opacity component declined or invasive progression occurred. A close association with the immunosuppressive microenvironment was also observed during the progression of pulmonary nodules. Notably, the upregulation of B cell and regulatory T cell marker genes occurred progressively. Immune cell abundance analysis further demonstrated a marked increase in exhausted cells and regulatory T cells during the progression of pulmonary nodules. These results were further validated by independent datasets including nCounter RNA profiling, single-cell RNA sequencing, and spatial transcriptomic datasets. Our study provided a comprehensive representation of the diverse landscape of MPLCs originating within the same individual and emphasized the significant influence of the immunosuppressive microenvironment in the occurrence and development of pulmonary nodules. These findings hold great potential for enhancing the clinical diagnosis and treatment strategies for MPLCs.

The development of new biomarkers or therapeutic targets for cancer immunotherapies requires deep understanding of T cells. To date, the complete landscape and systematic characterization of long noncoding RNAs (lncRNAs) in T cells in cancer immunity are lacking. Here, by systematically analyzing full-length single-cell RNA sequencing (scRNA-seq) data of more than 20,000 libraries of T cells across three cancer types, we provided the first comprehensive catalog and the functional repertoires of lncRNAs in human T cells. Specifically, we developed a custom pipeline for de novotranscriptome assembly and obtained a novel lncRNA catalog containing 9433 genes. This increased the number of current human lncRNA catalog by 16% and nearly doubled the number of lncRNAs expressed in T cells. We found that a portion of expressed genes in single T cells were lncRNAs which had been overlooked by the majority of previous studies. Based on metacell maps constructed by the MetaCell algorithm that partitions scRNA-seq datasets into disjointed and homogenous groups of cells (metacells), 154 signature lncRNA genes were identified. They were associated with effector, exhausted, and regulatory T cell states. Moreover, 84 of them were functionally annotated based on the co-expression networks, indicating that lncRNAs might broadly participate in the regulation of T cell functions. Our findings provide a new point of view and resource for investigating the mechanisms of T cell regulation in cancer immunity as well as for novel cancer-immune biomarker development and cancer immunotherapies

Non‑small cell lung cancer (NSCLC) accounts for ~80% of all types of lung cancer, which has the highest morbidity and mortality of all types of cancer worldwide. It is important to identify novel biomarkers and the molecular mechanism of NSCLC to improve current treatments of NSCLC. The present study aimed to investigate the effect of miR‑148b expression on the proliferation, epithelial‑mesenchymal transition (EMT) and radiosensitivity of NSCLC cells. It was demonstrated that miR‑148b expression was significantly decreased in NSCLC tissues and cell lines. A549 cells were then transfected with a miR‑148b mimic and a miR‑148b inhibitor. Transfection with the miR‑148b mimic decreased proliferation whereas transfection with the miR‑148b inhibitor increased the proliferation of A549 cells. Additionally, the miR‑148b mimic increased E‑cadherin expression and decreased N‑cadherin and vimentin expression. By contrast, transfection with the miR‑148b inhibitor decreased E‑cadherin expression and increased N‑cadherin and vimentin expression. Irradiation‑induced cell death was significantly promoted by the miR‑148b mimic but inhibited by the miR‑148b inhibitor. The miR‑148b mimic significantly decreased the expression of Rho‑associated protein kinase 1 (ROCK1) and it was demonstrated that overexpression of ROCK1 significantly inhibited the effects of miR‑148b on cell proliferation, the EMT and irradiation‑induced cell death. Therefore, the current study revealed that miR‑148b inhibited NSCLC cell proliferation and the EMT, and increased the radiosensitivity of NSCLC cells by inhibiting ROCK1 expression. Therefore, miR‑148b/ROCK1 signaling may be a novel therapeutic target to inhibit the growth of NSCLC cells and enhance the effects of radiotherapy to treat patients with NSCLC.