Insights into the heterogeneity of the tumor microenvironment in lung adenocarcinoma and squamous carcinoma through single-cell transcriptomic analysis: Implications for distinct immunotherapy outcomes.

BACKGROUND: Immune checkpoint blockade has emerged as a key strategy to the therapy landscape of non-small cell lung cancer (NSCLC). However, notable differences in immunotherapeutic outcomes exist between the two primary NSCLC subtypes: lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). This disparity may stem from the tumor immune microenvironment's heterogeneity at the transcriptome level. METHODS: By integrative analysis of transcriptomic characterization of 38 NSCLC patients by single-cell RNA sequencing, the present study revealed a distinct tumor microenvironment (TME) between LUAD and LUSC, with relevant results further confirmed in bulk transcriptomic and multiplex immunofluorescence (mIF) validation cohort of neoadjuvant immunotherapy patients. RESULTS: LUAD exhibited a more active immune microenvironment compared to LUSC. This included highly expression of HLA I/II in cancer cells, reinforced antigen presentation potential of dendritic cells and enhanced cytotoxic activity observed in T/NK cells. In LUSC, cancer cells highly expressed genes belonging to the aldo-keto reductases, glutathione S-transferases and aldehyde dehydrogenase family, negatively correlating with immunotherapy outcomes in the validation cohort of our center. Further analysis revealed elevated infiltrated cancer-associated fibroblasts (CAFs) in LUSC, which was corroborated in The Cancer Genome Atlas cohort. Corresponding increased infiltration of ADH1B+ CAFs in major pathologic response (MPR) patients and the higher presence of FAP+ CAFs in non-MPR patients were demonstrated by multiplex mIF. Moreover, upregulating immunosuppressive extracellular matrix remodeling was identified in LUSC. CONCLUSIONS: These comprehensive analyses advance the understanding of the differences in TME between LUAD and LUSC, offering insights for patient selection and developing subtype-specific treatment strategies.

A protein sequence-based deep transfer learning framework for identifying human proteome-wide deubiquitinase-substrate interactions.

Protein ubiquitination regulates a wide range of cellular processes. The degree of protein ubiquitination is determined by the delicate balance between ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase (DUB)-mediated deubiquitination. In comparison to the E3-substrate interactions, the DUB-substrate interactions (DSIs) remain insufficiently investigated. To address this challenge, we introduce a protein sequence-based ab initio method, TransDSI, which transfers proteome-scale evolutionary information to predict unknown DSIs despite inadequate training datasets. An explainable module is integrated to suggest the critical protein regions for DSIs while predicting DSIs. TransDSI outperforms multiple machine learning strategies against both cross-validation and independent test. Two predicted DUBs (USP11 and USP20) for FOXP3 are validated by "wet lab" experiments, along with two predicted substrates (AR and p53) for USP22. TransDSI provides new functional perspective on proteins by identifying regulatory DSIs, and offers clues for potential tumor drug target discovery and precision drug application.

Tumor microenvironment remodeling after neoadjuvant immunotherapy in non-small cell lung cancer revealed by single-cell RNA sequencing.

BACKGROUND: Immunotherapy has revolutionized cancer treatment, but most patients are refractory to immunotherapy or acquire resistance, with the underlying mechanisms remaining to be explored. METHODS: We characterized the transcriptomes of ~92,000 single cells from 3 pre-treatment and 12 post-treatment patients with non-small cell lung cancer (NSCLC) who received neoadjuvant PD-1 blockade combined with chemotherapy. The 12 post-treatment samples were categorized into two groups based on pathologic response: major pathologic response (MPR; n = 4) and non-MPR (NMPR; n = 8). RESULTS: Distinct therapy-induced cancer cell transcriptomes were associated with clinical response. Cancer cells from MPR patients exhibited a signature of activated antigen presentation via major histocompatibility complex class II (MHC-II). Further, the transcriptional signatures of FCRL4+FCRL5+ memory B cells and CD16+CX3CR1+ monocytes were enriched in MPR patients and are predictors of immunotherapy response. Cancer cells from NMPR patients exhibited overexpression of estrogen metabolism enzymes and elevated serum estradiol. In all patients, therapy promoted expansion and activation of cytotoxic T cells and CD16+ NK cells, reduction of immunosuppressive Tregs, and activation of memory CD8+T cells into an effector phenotype. Tissue-resident macrophages were expanded after therapy, and tumor-associated macrophages (TAMs) were remodeled into a neutral instead of an anti-tumor phenotype. We revealed the heterogeneity of neutrophils during immunotherapy and identified an aged CCL3+ neutrophil subset was decreased in MPR patients. The aged CCL3+ neutrophils were predicted to interact with SPP1+ TAMs through a positive feedback loop to contribute to a poor therapy response. CONCLUSIONS: Neoadjuvant PD-1 blockade combined with chemotherapy led to distinct NSCLC tumor microenvironment transcriptomes that correlated with therapy response. Although limited by a small patient sample size subjected to combination therapy, this study provides novel biomarkers to predict therapy response and suggests potential strategies to overcome immunotherapy resistance.

Comparative profiling of single-cell transcriptome reveals heterogeneity of tumor microenvironment between solid and acinar lung adenocarcinoma.

BACKGROUND: The diversity of histologic composition reflects the inter- and intra-tumor heterogeneity of lung adenocarcinomas (LUADs) macroscopically. Insights into the oncological characteristics and tumor microenvironment (TME) of different histologic subtypes of LUAD at the single-cell level can help identify potential therapeutic vulnerabilities and combinational approaches to improve the survival of LUAD patients. METHODS: Through comparative profiling of cell communities defined by scRNA-seq data, we characterized the TME of LUAD samples of distinct histologic subtypes, with relevant results further confirmed in multiple bulk transcriptomic, proteomic datasets and an independent immunohistochemical validation cohort. RESULTS: We find that the hypoxic and acidic situation is the worst in the TME of solid LUADs compared to other histologic subtypes. Besides, the tumor metabolic preferences vary across histologic subtypes and may correspondingly impinge on the metabolism and function of immune cells. Remarkably, tumor cells from solid LUADs upregulate energy and substance metabolic activities, particularly the folate-mediated one-carbon metabolism and the key gene MTHFD2, which could serve as a potential therapeutic target. Additionally, ubiquitination modifications may also be involved in the progression of histologic patterns. Immunologically, solid LUADs are characterized by a predominance of exhausted T cells and immunosuppressive myeloid cells, where the hypoxic, acidified and nutrient-deprived TME has a non-negligible impact. Discrepancies in stromal cell function, evidenced by varying degrees of stromal remodeling and fibrosis, may also contribute to the specific immune phenotype of solid LUADs. CONCLUSIONS: Overall, our research proposes several potential entry points to improve the immunosuppressive TME of solid LUADs, thereby synergistically potentiating their immunotherapeutic efficacy, and may provide precise therapeutic strategies for LUAD patients of distinct histologic subtype constitution.

Sleeve resection after neoadjuvant chemoimmunotherapy in the treatment of locally advanced non-small cell lung cancer.

Background: We aimed to characterize the outcomes of sleeve resection after neoadjuvant chemoimmunotherapy for the treatment of non-small cell lung cancer (NSCLC), including perioperative and oncologic outcomes, and to identify any impact of operative approach on resultant findings. Methods: We identified patients with NSCLC who underwent sleeve resection after ≥2 cycles of neoadjuvant chemoimmunotherapy between May 2019 and April 2021 and retrospectively reviewed clinical records. Perioperative data were collected and compared between video-assisted thoracoscopic surgery (VATS) (n=8) and thoracotomy (n=15) groups. Immunohistochemistry (IHC) scores were compared between tumors with and without major pathological response (MPR). Results: Twenty-three patients met inclusion criteria, with clinical stages as follows: IB, 2 (8.7%); IIIA, 14 (60.9%); and IIIB, 7 (30.4%). Treatment-related adverse events (TRAE) were recorded in 17 patients (73.9%), including anemia and neutropenia, with no patients exhibiting serious TRAE. Radiological evaluation revealed 5 (21.7%) patients with complete response (CR), 14 (60.9%) with partial response (PR), and 4 (17.4%) with stable disease (SD). Complete resection was accomplished for all patients. One VATS procedure was converted to thoracotomy due to extensive pleural adhesions. There were no significant differences in intraoperative blood loss (87.5±51.8 vs. 193.9±145.3 mL), operative time (198.8±79.7 vs. 225.5±55.0 min), number of lymph node examined (16.9±6.6 vs. 18.2±6.5), and hospital stay (5.5±2.8 vs. 9.2±11.2 days) between the VATS and thoracotomy groups (all P>0.05). Postoperative complications occurred in 3 patients, and 1 patient died of bronchopleural fistula (BPF) in the thoracotomy group. Complete pathological response (CPR) and MPR were achieved in seven (30.4%) and 13 (56.5%) patients, respectively. Both preoperative histopathology (P=0.024) and radiological response (P=0.002) were significantly associated with MPR. In postoperative specimens with MPR, the IHC scores of cluster of differentiation (CD)4, CD8, and CD20 were modestly higher, while programmed cell death receptor 1 (PD-1), lymphocyte-activation gene 3 (LAG3) and T cell immunoglobulin and ITIM domain (TIGIT) were lower compared with non-MPR specimens, albeit insignificantly. Conclusions: Sleeve resection after neoadjuvant chemoimmunotherapy was feasible in patients with locally advanced NSCLC. Perioperative outcomes were comparable between the VATS and thoracotomy groups.

Single-Cell Transcriptome Identifies Drug-Resistance Signature and Immunosuppressive Microenvironment in Metastatic Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is a deadly neuroendocrine malignancy with high metastasis. However, the heterogeneity of metastatic SCLC at the single-cell level remains elusive. The single-cell transcriptome of a total of 24 081 cells in metastatic lymph node samples from seven SCLC patients via endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is examined. Genomic alterations are also examined by whole exome sequencing (WES) and the immune infiltration between SCLC and non-SCLC (NSCLC) is compared using public single-cell RNA sequencing (scRNA-seq) data. It is identified that malignant cells in lymph-node metastatic SCLC have inter-patient and intra-tumor heterogeneity characterized by distinct ASCL1 and NEUROD1 expression patterns. High expression of genes such as FZD8 in WNT pathway is associated with drug resistance in malignant cells. Compared to NSCLC, SCLC harbors a unique immunosuppressive tumor microenvironment. Malignant cells exhibit a pattern of attenuated MHC-I antigen presentation-related gene expression, which is associated with relatively low proportion of exhausted T cells. Natural killer (NK) cells display impaired antitumor function with high expression of TGFBR2. This work characterizes the inter-patient and intra-tumor heterogeneity of metastatic SCLC and uncovers the exhaustion signatures in NK cells, which may pave the way for novel treatments for SCLC including immune checkpoint blockade-based immunotherapy.

Single-Cell Transcriptome Identifies Drug-Resistance Signature and Immunosuppressive Microenvironment in Metastatic Small Cell Lung Cancer (Advanced Genetics 2/03).

Single-Cell RNA Sequencing This cover illustrates the work of Xujun Wang, Xianmin Zhu, Peng Zhang, and co-workers in article number 2100060 which reveals the drug-resistance signature and immunosuppressive microenvironment in small cell lung cancer (SCLC) by single-cell RNA-sequencing. "Wu Song Fought the Tiger" comes from the famous Chinese novel: Outlaws of the Marsh. In the cover, the warrior Wu Song stands for the doctors and researchers. The tiger bearing "SCLC" on its face is dangerous for its sharp teeth and claws (early metastasis and drug resistance) and the surrounding water bubbles (immune infiltration). In addition, 2022 is the Year of the Tiger.