Evaluation of single-cell classifiers for single-cell RNA sequencing data sets.

Single-cell RNA sequencing (scRNA-seq) has been rapidly developing and widely applied in biological and medical research. Identification of cell types in scRNA-seq data sets is an essential step before in-depth investigations of their functional and pathological roles. However, the conventional workflow based on clustering and marker genes is not scalable for an increasingly large number of scRNA-seq data sets due to complicated procedures and manual annotation. Therefore, a number of tools have been developed recently to predict cell types in new data sets using reference data sets. These methods have not been generally adapted due to a lack of tool benchmarking and user guidance. In this article, we performed a comprehensive and impartial evaluation of nine classification software tools specifically designed for scRNA-seq data sets. Results showed that Seurat based on random forest, SingleR based on correlation analysis and CaSTLe based on XGBoost performed better than others. A simple ensemble voting of all tools can improve the predictive accuracy. Under nonideal situations, such as small-sized and class-imbalanced reference data sets, tools based on cluster-level similarities have superior performance. However, even with the function of assigning 'unassigned' labels, it is still challenging to catch novel cell types by solely using any of the single-cell classifiers. This article provides a guideline for researchers to select and apply suitable classification tools in their analysis workflows and sheds some lights on potential direction of future improvement on classification tools.

Modulation of hippocampal neuronal resilience during aging by the Hsp70/Hsp90 co-chaperone STI1.

Chaperone networks are dysregulated with aging, but whether compromised Hsp70/Hsp90 chaperone function disturbs neuronal resilience is unknown. Stress-inducible phosphoprotein 1 (STI1; STIP1; HOP) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. We combined in-depth analysis of chaperone genes in human datasets, analysis of a neuronal cell line lacking STI1 and of a mouse line with a hypomorphic Stip1 allele to investigate the requirement for STI1 in aging. Our experiments revealed that dysfunctional STI1 activity compromised Hsp70/Hsp90 chaperone network and neuronal resilience. The levels of a set of Hsp90 co-chaperones and client proteins were selectively affected by reduced levels of STI1, suggesting that their stability depends on functional Hsp70/Hsp90 machinery. Analysis of human databases revealed a subset of co-chaperones, including STI1, whose loss of function is incompatible with life in mammals, albeit they are not essential in yeast. Importantly, mice expressing a hypomorphic STI1 allele presented spontaneous age-dependent hippocampal neurodegeneration and reduced hippocampal volume, with consequent spatial memory deficit. We suggest that impaired STI1 function compromises Hsp70/Hsp90 chaperone activity in mammals and can by itself cause age-dependent hippocampal neurodegeneration in mice. Cover Image for this issue: doi: 10.1111/jnc.14749.

Single Cell Sequencing: A New Dimension in Cancer Diagnosis and Treatment.

Cancer is one of the leading causes of death worldwide and well known for its complexity. Cancer cells within the same tumor or from different tumors are highly heterogeneous. Furthermore, stromal and immune cells within tumor microenvironment interact with cancer cells to play important roles in how tumors progress and respond to different treatments. Recent advances in single cell technologies, especially massively parallel single cell sequencing, have made it possible to analyze cancer cells and cells in its tumor microenvironment in parallel with unprecedented high resolution. In this chapter, we will review recent developments in single cell sequencing technologies and their applications in cancer research. We will also explain how insights generated from single cell sequencing can be used to develop novel diagnostic and therapeutic approaches to conquer cancer.

Unraveling epigenomic abnormality in azoospermic human males by WGBS, RNA-Seq, and transcriptome profiling analyses.

PURPOSE: To determine associations between genomic DNA methylation in testicular cells and azoospermia in human males. METHODS: This was a case-control study investigating the differences and conservations in DNA methylation, genome-wide DNA methylation, and bulk RNA-Seq for transcriptome profiling using testicular biopsy tissues from NOA and OA patients. Differential methylation and different conserved methylation regions associated with azoospermia were identified by comparing genomic DNA methylation of testicular seminiferous cells derived from NOA and OA patients. RESULTS: The genome methylation modification of testicular cells from NOA patients was disordered, and the reproductive-related gene expression was significantly different. CONCLUSION: Our findings not only provide valuable knowledge of human spermatogenesis but also paved the way for the identification of genes/proteins involved in male germ cell development. The approach presented in this report provides a powerful tool to identify responsible biomolecules, and/or cellular changes (e.g., epigenetic abnormality) that induce male reproductive dysfunction such as OA and NOA.

An interpretable framework for clustering single-cell RNA-Seq datasets.

BACKGROUND: With the recent proliferation of single-cell RNA-Seq experiments, several methods have been developed for unsupervised analysis of the resulting datasets. These methods often rely on unintuitive hyperparameters and do not explicitly address the subjectivity associated with clustering. RESULTS: In this work, we present DendroSplit, an interpretable framework for analyzing single-cell RNA-Seq datasets that addresses both the clustering interpretability and clustering subjectivity issues. DendroSplit offers a novel perspective on the single-cell RNA-Seq clustering problem motivated by the definition of "cell type", allowing us to cluster using feature selection to uncover multiple levels of biologically meaningful populations in the data. We analyze several landmark single-cell datasets, demonstrating both the method's efficacy and computational efficiency. CONCLUSION: DendroSplit offers a clustering framework that is comparable to existing methods in terms of accuracy and speed but is novel in its emphasis on interpretabilty. We provide the full DendroSplit software package at https://github.com/jessemzhang/dendrosplit .

Mechanisms of neuroprotection against ischemic insult by stress-inducible phosphoprotein-1/prion protein complex.

Stress-inducible phosphoprotein 1 (STI1) acts as a neuroprotective factor in the ischemic brain and its levels are increased following ischemia. Previous work has suggested that some of these STI1 actions in a stroke model depend on the recruitment of bone marrow-derived stem cells to improve outcomes after ischemic insult. However, STI1 can directly increase neuroprotective signaling in neurons by engaging with the cellular prion protein (PrPC ) and activating α7 nicotinic acetylcholine receptors (α7nAChR). Given that α7nAChR activation has also been involved in neuroprotection in stroke, it is possible that STI1 can have direct actions on neurons to prevent deleterious consequences of ischemic insults. Here, we tested this hypothesis by exposing primary neuronal cultures to 1-h oxygen-glucose deprivation (OGD) and reperfusion and assessing signaling pathways activated by STI1/PrPC . Our results demonstrated that STI1 treatment significantly decreased apoptosis and cell death in mouse neurons submitted to OGD in a manner that was dependent on PrPC and α7nAChR, but also on the activin A receptor 1 (ALK2), which has emerged as a signaling partner of STI1. Interestingly, pharmacological inhibition of the ALK2 receptor prevented neuroprotection by STI1, while activation of ALK2 receptors by bone morphogenetic protein 4 (BMP4) either before or after OGD was effective in decreasing neuronal death induced by ischemia. We conclude that PrPC /STI1 engagement and its subsequent downstream signaling cascades involving α7nAChR as well as the ALK2 receptor may be activated in neurons by increased levels of STI1. This signaling pathway protects neurons from ischemic insults.

Systematic discovery of novel eukaryotic transcriptional regulators using sequence homology independent prediction.

BACKGROUND: The molecular function of a gene is most commonly inferred by sequence similarity. Therefore, genes that lack sufficient sequence similarity to characterized genes (such as certain classes of transcriptional regulators) are difficult to classify using most function prediction algorithms and have remained uncharacterized. RESULTS: To identify novel transcriptional regulators systematically, we used a feature-based pipeline to screen protein families of unknown function. This method predicted 43 transcriptional regulator families in Arabidopsis thaliana, 7 families in Drosophila melanogaster, and 9 families in Homo sapiens. Literature curation validated 12 of the predicted families to be involved in transcriptional regulation. We tested 33 out of the 195 Arabidopsis putative transcriptional regulators for their ability to activate transcription of a reporter gene in planta and found twelve coactivators, five of which had no prior literature support. To investigate mechanisms of action in which the predicted regulators might work, we looked for interactors of an Arabidopsis candidate that did not show transactivation activity in planta and found that it might work with other members of its own family and a subunit of the Polycomb Repressive Complex 2 to regulate transcription. CONCLUSIONS: Our results demonstrate the feasibility of assigning molecular function to proteins of unknown function without depending on sequence similarity. In particular, we identified novel transcriptional regulators using biological features enriched in transcription factors. The predictions reported here should accelerate the characterization of novel regulators.

The Transient Receptor Potential Melastatin 2 (TRPM2) Channel Contributes to ß-Amyloid Oligomer-Related Neurotoxicity and Memory Impairment.

In Alzheimer's disease, accumulation of soluble oligomers of ß-amyloid peptide is known to be highly toxic, causing disturbances in synaptic activity and neuronal death. Multiple studies relate these effects to increased oxidative stress and aberrant activity of calcium-permeable cation channels leading to calcium imbalance. The transient receptor potential melastatin 2 (TRPM2) channel, a Ca(2+)-permeable nonselective cation channel activated by oxidative stress, has been implicated in neurodegenerative diseases, and more recently in amyloid-induced toxicity. Here we show that the function of TRPM2 is augmented by treatment of cultured neurons with ß-amyloid oligomers. Aged APP/PS1 Alzheimer's mouse model showed increased levels of endoplasmic reticulum stress markers, protein disulfide isomerase and phosphorylated eukaryotic initiation factor 2α, as well as decreased levels of the presynaptic marker synaptophysin. Elimination of TRPM2 in APP/PS1 mice corrected these abnormal responses without affecting plaque burden. These effects of TRPM2 seem to be selective for ß-amyloid toxicity, as ER stress responses to thapsigargin or tunicamycin in TRPM2(-/-) neurons was identical to that of wild-type neurons. Moreover, reduced microglial activation was observed in TRPM2(-/-)/APP/PS1 hippocampus compared with APP/PS1 mice. In addition, age-dependent spatial memory deficits in APP/PS1 mice were reversed in TRPM2(-/-)/APP/PS1 mice. These results reveal the importance of TRPM2 for ß-amyloid neuronal toxicity, suggesting that TRPM2 activity could be potentially targeted to improve outcomes in Alzheimer's disease. SIGNIFICANCE STATEMENT: Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress sensing calcium-permeable channel that is thought to contribute to calcium dysregulation associated with neurodegenerative diseases, including Alzheimer's disease. Here we show that oligomeric ß-amyloid, the toxic peptide in Alzheimer's disease, facilitates TRPM2 channel activation. In mice designed to model Alzheimer's disease, genetic elimination of TRPM2 normalized deficits in synaptic markers in aged mice. Moreover, the absence of TRPM2 improved age-dependent spatial memory deficits observed in Alzheimer's mice. Our results reveal the importance of TRPM2 for neuronal toxicity and memory impairments in an Alzheimer's mouse model and suggest that TRPM2 could be targeted for the development of therapeutic agents effective in the treatment of dementia.

Inherent structure versus geometric metric for state space discretization.

Inherent structure (IS) and geometry-based clustering methods are commonly used for analyzing molecular dynamics trajectories. ISs are obtained by minimizing the sampled conformations into local minima on potential/effective energy surface. The conformations that are minimized into the same energy basin belong to one cluster. We investigate the influence of the applications of these two methods of trajectory decomposition on our understanding of the thermodynamics and kinetics of alanine tetrapeptide. We find that at the microcluster level, the IS approach and root-mean-square deviation (RMSD)-based clustering method give totally different results. Depending on the local features of energy landscape, the conformations with close RMSDs can be minimized into different minima, while the conformations with large RMSDs could be minimized into the same basin. However, the relaxation timescales calculated based on the transition matrices built from the microclusters are similar. The discrepancy at the microcluster level leads to different macroclusters. Although the dynamic models established through both clustering methods are validated approximately Markovian, the IS approach seems to give a meaningful state space discretization at the macrocluster level in terms of conformational features and kinetics.

ChAT-ChR2-EYFP mice have enhanced motor endurance but show deficits in attention and several additional cognitive domains.

Acetylcholine (ACh) is an important neuromodulator in the nervous system implicated in many forms of cognitive and motor processing. Recent studies have used bacterial artificial chromosome (BAC) transgenic mice expressing channelrhodopsin-2 (ChR2) protein under the control of the choline acetyltransferase (ChAT) promoter (ChAT-ChR2-EYFP) to dissect cholinergic circuit connectivity and function using optogenetic approaches. We report that a mouse line used for this purpose also carries several copies of the vesicular acetylcholine transporter gene (VAChT), which leads to overexpression of functional VAChT and consequently increased cholinergic tone. We demonstrate that these mice have marked improvement in motor endurance. However, they also present severe cognitive deficits, including attention deficits and dysfunction in working memory and spatial memory. These results suggest that increased VAChT expression may disrupt critical steps in information processing. Our studies demonstrate that ChAT-ChR2-EYFP mice show altered cholinergic tone that fundamentally differentiates them from wild-type mice.

Stress-inducible phosphoprotein 1 has unique cochaperone activity during development and regulates cellular response to ischemia via the prion protein.

Stress-inducible phosphoprotein 1 (STI1) is part of the chaperone machinery, but it also functions as an extracellular ligand for the prion protein. However, the physiological relevance of these STI1 activities in vivo is unknown. Here, we show that in the absence of embryonic STI1, several Hsp90 client proteins are decreased by 50%, although Hsp90 levels are unaffected. Mutant STI1 mice showed increased caspase-3 activation and 50% impairment in cellular proliferation. Moreover, placental disruption and lack of cellular viability were linked to embryonic death by E10.5 in STI1-mutant mice. Rescue of embryonic lethality in these mutants, by transgenic expression of the STI1 gene, supported a unique role for STI1 during embryonic development. The response of STI1 haploinsufficient mice to cellular stress seemed compromised, and mutant mice showed increased vulnerability to ischemic insult. At the cellular level, ischemia increased the secretion of STI1 from wild-type astrocytes by 3-fold, whereas STI1 haploinsufficient mice secreted half as much STI1. Interesting, extracellular STI1 prevented ischemia-mediated neuronal death in a prion protein-dependent way. Our study reveals essential roles for intracellular and extracellular STI1 in cellular resilience.

Longitudinal associations of social jetlag with obesity indicators among adolescents - Shanghai adolescent cohort.

OBJECTIVE: To explore the longitudinal association between social-jetlag (SJL) and obesity development among adolescents, sex-difference and related modifying factors in the association. METHODS: Based on Shanghai-Adolescent-Cohort during 2017-2021, a total of 609 students were investigated. In grade 6, 7 and 9, the information on SJL was collected using questionnaires, and anthropometric measures were conducted. The fingernail cortisol and progesterone levels in grade 6 (using LC-MS/MS) and body composition in grade 9 (using Inbody-S10) were measured. By the latent-class-mixture-modeling, two trajectories for SJL (high-level vs. low-level) throughout 4 years were developed. The prospective associations of SJL trajectories and weight/fat gains were analyzed by sex and under different (high/moderate/low) cortisol/progesterone stratifications. RESULTS: In grades 6-9, 39.00%-44.50 % of adolescents experienced at least 1 h of SJL. Compared with the low-level SJL trajectory, the high-level SJL trajectory was associated with greater differences in body-mass-index Z-scores and waist-to-height ratios across 4 years, higher levels of body-fat-percentage and fat-mass-index in grade 9 (P-values<0.05), and such associations were stronger among girls and under moderate-to-high (vs. low) baseline cortisol and progesterone levels. However, no significant associations among boys were observed. CONCLUSIONS: High-level SJL in adolescents may be associated with the development of obesity, especially among adolescent girls and under relatively high baseline cortisol and progesterone levels.

Increased prion protein processing and expression of metabotropic glutamate receptor 1 in a mouse model of Alzheimer's disease.

Prion protein (PrP(C) ), a glycosylphosphatidylinositol-anchored protein corrupted in prion diseases, has been shown recently to interact with group I metabotropic glutamate receptors (mGluRs). Moreover, both PrP(C) and mGluRs were proposed to function as putative receptors for ß-amyloid in Alzheimer's disease. PrP(C) can be processed in neurons via α or ß-cleavage to produce PrP(C) fragments that are neuroprotective or toxic, respectively. We found PrP(C) α-cleavage to be 2-3 times higher in the cortex of APPswe/PS1dE9 mice, a mouse model of Alzheimer's disease. A similar age-dependent increase was observed for PrP(C) ß-cleavage. Moreover, we observed considerable age-dependent increase in cortical expression of mGluR1, but not mGluR5. Exposure of cortical neuronal cultures to ß-amyloid oligomers upregulated mGluR1 and PrP(C) α-cleavage, while activation of group I mGluRs increased PrP(C) shedding from the membrane, likely due to increased levels of a disintegrin and metalloprotease10, a key disintegrin for PrP(C) shedding. Interestingly, a similar increase in a disintegrin and metalloprotease10 was detected in the cortex of 9-month-old APPswe/PS1dE9 animals. Our experiments reveal novel and complex processing of PrP(C) in connection with mGluR overexpression that seems to be triggered by ß-amyloid peptides. Prion protein (PrP(C) ) and metabotropic glutamate receptors (mGluR) are implicated in Alzheimer's disease (AD). We found age-dependent increase in PrP(C) processing, ADAM10 and mGluR1 levels in AD mouse model. These changes could be reproduced in cultured cortical neurons treated with Aß peptide. Our findings suggest that increased levels of Aß can trigger compensatory responses that may affect neuronal toxicity.

Graph representation of protein free energy landscape.

The thermodynamics and kinetics of protein folding and protein conformational changes are governed by the underlying free energy landscape. However, the multidimensional nature of the free energy landscape makes it difficult to describe. We propose to use a weighted-graph approach to depict the free energy landscape with the nodes on the graph representing the conformational states and the edge weights reflecting the free energy barriers between the states. Our graph is constructed from a molecular dynamics trajectory and does not involve projecting the multi-dimensional free energy landscape onto a low-dimensional space defined by a few order parameters. The calculation of free energy barriers was based on transition-path theory using the MSMBuilder2 package. We compare our graph with the widely used transition disconnectivity graph (TRDG) which is constructed from the same trajectory and show that our approach gives more accurate description of the free energy landscape than the TRDG approach even though the latter can be organized into a simple tree representation. The weighted-graph is a general approach and can be used on any complex system.

Homework, sleep insufficiency and adolescent neurobehavioral problems: Shanghai Adolescent Cohort.

BACKGROUND: The prospective associations between homework burdens and adolescent neurobehavioral problems, and whether sleep-durations mediated and sex modified such associations remained unclear. METHODS: Using Shanghai-Adolescent-Cohort study, 609 middle-school students were recruited and investigations took place at Grade 6, 7 and 9. Information on homework burdens (defined by homework completion-time and self-perceived homework difficulty), bedtime/wake-up-time and neurobehavioral problems was collected. Two patterns of comprehensive homework burdens ('high' vs. 'low') were identified by latent-class-analysis and two distinct neurobehavioral trajectories ('increased-risk' vs. 'low-risk') were formed by latent-class-mixture-modeling. RESULTS: Among the 6th-9th graders, the prevalence-rates of sleep-insufficiency and late-bedtime ranged from 44.0 %-55.0 % and 40.3 %-91.6 %, respectively. High homework burdens were concurrently associated with increased-risks of neurobehavioral problems (IRRs: 1.345-1.688, P < 0.05) at each grade, and such associations were mediated by reduced sleep durations (IRRs for indirect-effects: 1.105-1.251, P < 0.05). High homework burden at the 6th-grade (ORs: 2.014-2.168, P < 0.05) or high long-term (grade 6-9) homework burden (ORs: 1.876-1.925, P < 0.05) significantly predicted increased-risk trajectories of anxiety/depression and total-problems, with stronger associations among girls than among boys. The longitudinal associations between long-term homework burdens and increased-risk trajectories of neurobehavioral problems were mediated by reduced sleep-durations (ORs for indirect-effects: 1.189-1.278, P < 0.05), with stronger mediation-effects among girls. LIMITATIONS: This study was restricted to Shanghai adolescents. CONCLUSIONS: High homework burden had both short-term and long-term associations with adolescent neurobehavioral problems, with stronger associations among girls, and sleep-insufficiency may mediate such associations in a sex-specific manner. Approaches targeting appropriate homework-load/difficulty and sleep restoration may help prevent adolescent neurobehavioral problems.

Rapid increases in BMI waist to height ratio during adolescence and subsequent neurobehavioral deficits.

OBJECTIVE: The aim of this study was to explore prospective relationships between changing patterns of BMI/waist to height ratio (WHtR) during adolescence and subsequent neurobehavioral development. METHODS: In this prospective cohort study, randomized stratified sampling was used to recruit six middle schools and 609 students in Shanghai, China. In Grades 6, 7, and 9, the Youth Self Report scale was used to assess student neurobehavioral status and anthropometric measurements were conducted to calculate BMI z scores and WHtRs. Longitudinal data were analyzed using latent class mixture modeling to delineate trajectories of BMI z scores ("stable," "decreasing," "rapidly increasing") and WHtRs ("stable," "rapidly increasing"), and their associations with neurobehavioral status in Grade 9 were assessed. RESULTS: In Grades 6 through 9 (ages 11-15 years), the prevalence of overall obesity and abdominal obesity ranged from 10.7% to 13.0% and 13.0% to 19.8%, respectively. Compared with the stable BMI z score trajectory, the rapidly increasing BMI z score trajectory was longitudinally associated with delinquent behavior, aggressive behavior, and externalizing problems (incidence rate ratio: 1.564-1.613, adjusted p < 0.05). Compared with the stable WHtR trajectory, the rapidly increasing WHtR trajectory significantly predicted increased risks of social problems and delinquent behavior (incidence rate ratios: 1.776-1.967, adjusted p < 0.05). Significant associations of the rapidly increasing BMI z score/WHtR trajectories with subsequent neurobehavioral deficits were observed among girls (adjusted p < 0.05) but not among boys (adjusted p > 0.05). CONCLUSIONS: Rapid increases in BMI or WHtR during adolescence could predict subsequent neurobehavioral deficits, especially for externalizing behaviors. Timely intervention for weight control may be considered to promote adolescent mental health.

Screening for a single-chain variable-fragment antibody that can effectively neutralize the cytotoxicity of the Vibrio parahaemolyticus thermolabile hemolysin.

Vibrio parahaemolyticus is a halophilic bacterium that is widely distributed in water resources. The bacterium causes lethal food-borne diseases and poses a serious threat to human and animal health all over the world. The major pathogenic factor of V. parahaemolyticus is thermolabile hemolysin (TLH), encoded by the tlh gene, but its toxicity mechanisms are unknown. A high-affinity antibody that can neutralize TLH activity effectively is not available. In this study, we successfully expressed and purified the TLH antigen and discovered a high-affinity antibody to TLH, named scFv-LA3, by phage display screening. Cytotoxicity analysis showed that scFv-LA3 has strong neutralization effects on TLH-induced cell toxicity.

The potential effects and mechanisms of breast inflammatory lesions on the occurrence and development of breast cancer.

Breast cancer as the most common cancer in women has become the leading cause of cancer death for women. Although many inflammatory factors increase the risk of breast cancer, there are very few studies on the mechanisms by which inflammation affects the initiation and progression of breast cancer. Here, we profiled and compared the transcriptome of normal tissues, inflammatory breast tissues, benign breast tumors, and malignant breast tumors. To find key regulatory factors, a protein interaction network between characteristic modules in inflammatory lesions and ER-negative (ER-) breast cancer was constructed and inflammation-cancer interface genes were identified. We found that the transcriptional profile of inflammatory breast tissues was similar with ER- malignant tumors, featured with low ER expression levels and similar immune signaling pathway activation. Through comprehensive protein network analysis, we identified the interface genes and chemokine signaling pathway that have the potential to promote inflammatory cancer transformation. These interface genes could be used as a risk factor to provide a certain basis for the clinical early detection and treatment of breast cancer. This is the first study to explore the association between breast inflammatory lesions and breast cancer at the transcriptome level. Our inflammation data and research results provide a basis for future inflammation-cancer transformation analysis.

Human obstructive (postvasectomy) and nonobstructive azoospermia - Insights from scRNA-Seq and transcriptome analysis.

A substantial number of male infertility is caused by azoospermia. However, the underlying etiology and the molecular basis remain largely unknown. Through single-cell (sc)RNA sequencing, we had analyzed testis biopsy samples from two patients with obstructive azoospermia (OA) and nonobstructive azoospermia (NOA). We found only somatic cells in the NOA samples and explored the transcriptional changes in Sertoli cells in response to a loss of interactions with germ cells. Moreover, we observed a germ cell population discrepancy between an OA (postvasectomy) patient and a healthy individual. We confirmed this observation in a secondary study with two datasets at GSM3526588 and GSE124263 for detailed analysis wherein the regulatory mechanisms at the transcriptional level were identified. These findings thus provide valuable information on human spermatogenesis, and we also identified insightful information for further research on reproduction-related diseases.

Immune checkpoint inhibitors plus chemotherapy in patients with locally advanced or metastatic pulmonary sarcomatoid carcinoma: a multicentric real-world study.

Introduction: Immune checkpoint inhibitors (ICIs) have demonstrated promising efficacy as monotherapy in patients with pulmonary sarcomatoid carcinoma (PSC). We performed the current multi-institutional, real-world study to assess the efficacy of ICIs plus chemotherapy in patients with PSC. Methods: All consecutive patients with locally advanced or metastatic PSC from three centers treated with ICIs between January 2018 and July 2021 were enrolled. Programmed death ligand 1 (PD-L1) expression was stained and evaluated using immunohistochemical with 22C3. Single-cell RNA sequencing (scRNA-seq) was performed in two patients with PSC and two patients with adenocarcinoma to understand the cell-type-specific transcriptome landscape of cancer cells and tumor microenvironment (TME) of PSC. Results: A cohort of 42 PSC patients was identified. In the overall population, the objective response rate (ORR) was 73.8%, median progression-free survival (mPFS) was 10.3 months and median overall survival was not reached and 2-year survival rate was 51.2%. For 34 treatment-naïve patients who received first-line ICIs plus chemotherapy, the ORR was 70.6%, mPFS was 10.3 months and 2-year survival rate was 57.8%. In patients with PD-L1 tumor proportion score (TPS) < 1%, 1-49%, and ⩾50%, the ORR was 33.3%, 72.7%, and 85.7% and mPFS was 6.0, 6.7, and 10.3 months, respectively. Notably, two patients with transformed PSC from lung adenocarcinoma after epidermal growth factor receptor-tyrosine kinase inhibitor treatment also responded well to ICIs plus chemotherapy. scRNA-seq revealed immune-cell-inflamed TME, lower intratumoral heterogeneity, and activated immune response pathway in PSC. Conclusions: Our study demonstrated remarkable efficacy of ICIs plus chemotherapy as first-line therapy for patient with locally advanced or metastatic PSC.