Hepatocyte MMP14 mediates liver and inter-organ inflammatory responses to diet-induced liver injury.

The matrix metalloproteinase MMP14 is a ubiquitously expressed, membrane-bound, secreted endopeptidase that proteolyzes substrates to regulate development, signaling, and metabolism. However, the spatial and contextual events inciting MMP14 activation and its metabolic sequelae are not fully understood. Here, we introduce an inducible, hepatocyte-specific MMP14-deficient model (MMP14LKO mice) to elucidate cell-intrinsic and systemic MMP14 function. We show that hepatocyte MMP14 mediates diet-induced body weight gain, peripheral adiposity, and impaired glucose homeostasis and drives diet-induced liver triglyceride accumulation and induction of hepatic inflammatory and fibrotic gene expression. Single-nucleus RNA sequencing revealed that hepatocyte MMP14 mediates Kupffer cell and T-cell accumulation and promotes diet-induced hepatocellular subpopulation shifts toward protection against lipid absorption. MMP14 co-immunoprecipitation and proteomic analyses revealed MMP14 substrate binding across both inflammatory and cytokine signaling, as well as metabolic pathways. Strikingly, hepatocyte MMP14 loss-of-function suppressed skeletal muscle and adipose inflammation in vivo, and in a reductionist adipose-hepatocyte co-culture model. Finally, we reveal that trehalose-type glucose transporter inhibitors decrease hepatocyte MMP14 gene expression and nominate these inhibitors as translatable therapeutic metabolic agents. We conclude that hepatocyte MMP14 drives liver and inter-organ inflammatory and metabolic sequelae of obesogenic dietary insult. Modulating MMP14 activation and blockade thus represents a targetable node in the pathogenesis of hepatic inflammation.

Passivation of Sodium Benzenesulfonate at the Buried Interface of a High-Performance Wide-Bandgap Perovskite Solar Cell.

The phase segregation of wide-bandgap perovskite is detrimental to a device's performance. We find that Sodium Benzenesulfonate (SBS) can improve the interface passivation of PTAA, thus addressing the poor wettability issue of poly[bis(4-phenyl)(2,4,6-trimethylphenyl)amine](PTAA). This improvement helps mitigate interface defects caused by poor contact between the perovskite and PTAA, reducing non-radiative recombination. Additionally, enhanced interface contact improves the crystallinity of the perovskite, leading to higher-quality perovskite films. By synergistically controlling the crystallization and trap passivation to reduce the phase segregation, SBS-modified perovskite solar cells (PSCs) achieved a power conversion efficiency (PCE) of 20.27%, with an open-circuit voltage (Voc) of 1.18 V, short-circuit current density (Jsc) of 20.93 mA cm-2, and fill factor (FF) of 82.31%.

The level of serum retinol-binding protein is associated with diabetic mild cognitive impairment.

BACKGROUND: Several studies have shown that retinol-binding protein (RBP) is linked to diabetes and neurodegenerative diseases. However, no studies have elucidated the relationship between RBP and diabetic cognitive disorders. OBJECTIVE: To determine whether the change characteristics of serum RBP are associated with alterations in cognitive functioning in type 2 diabetes mellitus (T2DM). METHODS: In this study, 252 patients with T2DM and 34 people as healthy controls were included. According to the Montreal Cognitive Assessment (MoCA), the diabetic subjects were divided into the mild cognitive impairment (MCI) group and the Non-MCI group. Demographic characteristics and clinical indicators as well as serum RBP levels were analyzed. RESULTS: The serum RBP levels in the MCI group were lower compared with the Non-MCI group (P = 0.02). The level of RBP was higher in the diabetes without MCI group than in the healthy control (P < 0.001). Serum RBP levels were positively correlated with MoCA scores (r = 0.178, P = 0.003). Binary Logistic regression model analysis showed that low RBP [odds ratio (OR) = 0.936], old age (OR = 1.074), high fasting blood glucose (OR = 1.164), and low fasting C-peptide (OR = 0.722) may be independent risk factors for diabetic MCI. The ROC curve of serum RBP for predicting diabetic MCI showed that the area under the curve was 0.630. CONCLUSIONS: Our study revealed an association between serum RBP and diabetic MCI. Serum RBP levels in diabetic MCI are lower and correlated with cognitive function.

Short tandem repeat profiling via next-generation sequencing for cell line authentication.

Cell lines are indispensable models for modern biomedical research. A large part of their usefulness derives from the ability of a cell line to proliferate over multiple passages (often indefinitely), allowing multiple experiments to be performed. However, over time, cell line identity and purity can be compromised by human errors. Cross-contamination from other cell lines and complete misidentification are both possible. Routine cell line authentication is a necessary preventive measure and has become a requirement for many funding applications and publications. Short tandem repeat (STR) profiling is the most common method for cell line authentication and is usually carried out using standard polymerase chain reaction-capillary electrophoresis analysis (STR-CE). Here, we evaluated next-generation sequencing (NGS)-based STR profiling of human and mouse cell lines at 18 and 15 loci, respectively, in a high-throughput format. Using the Python program STRight, we demonstrate that NGS-based analysis (STR-NGS) is superior to standard STR-CE in terms of the ability to report the sequence context of repeat motifs, sensitivity and flexible multiplexing capability. STR-NGS is thus a valuable alternative for cell line authentication.

Lrp1 is essential for lethal Rift Valley fever hepatic disease in mice.

Rift Valley fever virus (RVFV) is an emerging arbovirus found in Africa. While RVFV is pantropic and infects many cells and tissues, viral replication and necrosis within the liver play a critical role in mediating severe disease. The low-density lipoprotein receptor-related protein 1 (Lrp1) is a recently identified host factor for cellular entry and infection by RVFV. The biological significance of Lrp1, including its role in hepatic disease in vivo, however, remains to be determined. Because Lrp1 has a high expression level in hepatocytes, we developed a mouse model in which Lrp1 is specifically deleted in hepatocytes to test how the absence of liver Lrp1 expression affects RVF pathogenesis. Mice lacking Lrp1 expression in hepatocytes showed minimal RVFV replication in the liver, longer time to death, and altered clinical signs toward neurological disease. In contrast, RVFV infection levels in other tissues showed no difference between the two genotypes. Therefore, Lrp1 is essential for RVF hepatic disease in mice.

Floxing by Electroporating Single-Cell Embryos with Two CRISPR RNPs and Two ssODNs.

Floxed alleles and Cre drivers are two components of most conditional knockout mouse models, which are not only important for studying a given gene in a tissue-specific manner, but also useful for functional analysis of various sized genomic regions. With the increased demand for floxed mouse models in biomedical research, reliable and economical creation of floxed alleles is clearly highly valuable yet remains challenging. Here we provide technical details on the method consisting of electroporating single-cell embryos with CRISPR RNPs and ssODNs, next-generation sequencing (NGS)-based genotyping, an in vitro Cre assay (recombination followed by PCR) for loxP phasing determination, and optional second round targeting of an indel in cis with one loxP insertion in embryos obtained via in vitro fertilization (IVF). As importantly, we present protocols for validation of gRNAs and ssODNs before electroporation of embryos, to confirm phasing of loxP and the indel to be retargeted in individual blastocysts and an alternative strategy to insert loxP sites sequentially. Together, we hope to help researchers reliably obtain floxed alleles in a predictable and timely manner.

Bi-allelic CAMSAP1 variants cause a clinically recognizable neuronal migration disorder.

Non-centrosomal microtubules are essential cytoskeletal filaments that are important for neurite formation, axonal transport, and neuronal migration. They require stabilization by microtubule minus-end-targeting proteins including the CAMSAP family of molecules. Using exome sequencing on samples from five unrelated families, we show that bi-allelic CAMSAP1 loss-of-function variants cause a clinically recognizable, syndromic neuronal migration disorder. The cardinal clinical features of the syndrome include a characteristic craniofacial appearance, primary microcephaly, severe neurodevelopmental delay, cortical visual impairment, and seizures. The neuroradiological phenotype comprises a highly recognizable combination of classic lissencephaly with a posterior more severe than anterior gradient similar to PAFAH1B1(LIS1)-related lissencephaly and severe hypoplasia or absence of the corpus callosum; dysplasia of the basal ganglia, hippocampus, and midbrain; and cerebellar hypodysplasia, similar to the tubulinopathies, a group of monogenic tubulin-associated disorders of cortical dysgenesis. Neural cell rosette lineages derived from affected individuals displayed findings consistent with these phenotypes, including abnormal morphology, decreased cell proliferation, and neuronal differentiation. Camsap1-null mice displayed increased perinatal mortality, and RNAScope studies identified high expression levels in the brain throughout neurogenesis and in facial structures, consistent with the mouse and human neurodevelopmental and craniofacial phenotypes. Together our findings confirm a fundamental role of CAMSAP1 in neuronal migration and brain development and define bi-allelic variants as a cause of a clinically distinct neurodevelopmental disorder in humans and mice.

Oropouche orthobunyavirus infection is mediated by the cellular host factor Lrp1.

Oropouche orthobunyavirus (OROV; Peribunyaviridae) is a mosquito-transmitted virus that causes widespread human febrile illness in South America, with occasional progression to neurologic effects. Host factors mediating the cellular entry of OROV are undefined. Here, we show that OROV uses the host protein low-density lipoprotein-related protein 1 (Lrp1) for efficient cellular infection. Cells from evolutionarily distinct species lacking Lrp1 were less permissive to OROV infection than cells with Lrp1. Treatment of cells with either the high-affinity Lrp1 ligand receptor-associated protein (RAP) or recombinant ectodomain truncations of Lrp1 significantly reduced OROV infection. In addition, chimeric vesicular stomatitis virus (VSV) expressing OROV glycoproteins (VSV-OROV) bound to the Lrp1 ectodomain in vitro. Furthermore, we demonstrate the biological relevance of the OROV-Lrp1 interaction in a proof-of-concept mouse study in which treatment of mice with RAP at the time of infection reduced tissue viral load and promoted survival from an otherwise lethal infection. These results with OROV, along with the recent finding of Lrp1 as an entry factor for Rift Valley fever virus, highlight the broader significance of Lrp1 in cellular infection by diverse bunyaviruses. Shared strategies for entry, such as the critical function of Lrp1 defined here, provide a foundation for the development of pan-bunyaviral therapeutics.

PDA modified NIR-II NaEr0.8Yb0.2F4nanoparticles with high photothermal effect.

Polydopamine (PDA)-modified NaEr0.8Yb0.2 F4nanoparticles were synthesized, with strong NIR-II emission, quantum yield of 29.63%, and excellent photothermal performance. Crystal phases and microstructures are characterized. Optical properties such as absorption, NIR-II emission, and light stability are studied, and the luminescence mechanism is discussed in detail. Key factors in NIR-II imaging were evaluated in fresh pork tissue, including penetration depth, spatial resolution, and signal-to-noise ratio (SNR). A high penetration depth of 5 mm and a high spatial resolution of 1 mm were detected. Mice are imaged in vivo afterintravenousinjection. Due to the accumulation of nanoparticles in the liver, high image quality with an SNR of 5.2 was detected in the abdomen of KM mice with hair. The photothermal conversion effect of PDA-modified NPs was twice that of the reported material. These NIR-II nanoparticles have superior optical properties, high photothermal efficiency and low cytotoxicity, and are potential fluorescent probes for further disease diagnosis and treatment.

Highly reliable creation of floxed alleles by electroporating single-cell embryos.

BACKGROUND: Floxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. However, an efficient and reliable strategy to flox genomic regions of any desired size is still lacking. RESULTS: Here, we demonstrate that the method combining electroporation of fertilized eggs with gRNA/Cas9 complexes and single-stranded oligodeoxynucleotides (ssODNs), assessing phasing of loxP insertions in founders using an in vitro Cre assay and an optional, highly specific and efficient second-round targeting ensures the generation of floxed F1 animals in roughly five months for a wide range of sequence lengths (448 bp to 160 kb reported here). CONCLUSIONS: Floxed alleles can be reliably obtained in a predictable timeline using the improved method of electroporation of two gRNA/Cas9 ribonucleoprotein particles (RNPs) and two ssODNs.

Expression of miR-92a in colon cancer tissues and its correlation with clinicopathologic features and prognosis.

OBJECTIVE: This study analyzed the expression of miR-92a in colon tumor tissues and its correlation with disease clinicopathologic features and prognosis. METHODS: 83 cases of colorectal cancer tissues and paracancerous normal tissues acquired from colon cancer resection surgery during January 2015-January 2017 were collected. We detected the expression of miR-92a in cancer tissues and paracancerous tissues by qRT-PCR, and analyzed the correlation between the relative expression of miR-92 in colon cancer tissues and clinicopathologic characteristics, progression-free survival (PFS), and overall survival (OS) of the patients accordingly. RESULTS: The relative expression level of miR-92a in colon cancer tissues was higher than of paracancerous tissues (P<0.05). Relative expression of miR-92a in cancer was correlated with the degree of differentiation, TNM stage, and lymph node metastasis (P<0.05), while uncorrelated with gender, age, tumor diameter, or invasion depth (P>0.05). Patients with low expression of miR-92a had superior PFS to the control group (P>0.05) and better OS (P<0.05). CONCLUSION: Abnormally high expression of miR-92a occurs in colon tumor tissues. Its expression is related to the occurrence, progression, and prognosis of patients with colon cancer. It may be a marker for diagnosis, treatment, and prognosis of disease.

Lrp1 is a host entry factor for Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAPD3) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAPD3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.

Displaying epitope B and epitope 7 of porcine reproductive and respiratory syndrome virus on virus like particles of porcine circovirus type 2 provides partial protection to pigs.

The Cap of porcine circovirus type 2 (PCV2) can be assembled into virus like particles (VLPs) in vitro that have multiple loops located on the particle surface. This would make it a good vehicle for displaying exogenous proteins or epitopes. We derived two epitopes, epitope B (EpB, S37HIQLIYNL45) and epitope 7 (Ep7, Q196WGRL200) from Gp5 of the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). We replaced the core region of Loop CD (L75PPGGGSN82) and the carboxyl terminus (K222DPPL226) of PCV2 Cap, respectively, to construct a bi-epitope chimeric PCV2 Cap. Its immunogenicity and protective effects were evaluated as one PRRSV subunit vaccine. The chimeric PCV2 Cap was soluble, efficiently expressed in an Escherichia coli expression system, and could be self-assembled into chimeric virus like particles (cVLPs) with a diameter of 12-15 nm. Western blotting confirmed that the cVLPs could be specifically recognized by anti-PCV2, anti-EpB and anti-Ep7 antibodies. The cVLPs vaccine could alleviate the clinical symptoms and reduce the viral loads after HP-PRRSV challenge in 100-120 days old pigs. These data suggest that the cVLPs vaccine could provide pigs with partial protection against homologous PRRSV strains, and it provides a new design for additional PRRSV subunit vaccines.

Adenoviral vectors for in vivo delivery of CRISPR-Cas gene editors.

Harnessing the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) system for genome editing in eukaryotes has revolutionized basic biomedical research and translational sciences. The ability to create targeted alterations of the genome through this easy to design system has presented unprecedented opportunities to treat inherited disorders and other diseases such as cancer through gene therapy. A major hurdle is the lack of an efficient and safe in vivo delivery system, limiting most of the current gene therapy efforts to ex vivo editing of extracted cells. Here we discuss the unique features of adenoviral vectors that enable tissue specific and efficient delivery of the CRISPR-Cas machinery for in vivo genome editing.

NLCIPS: Non-Small Cell Lung Cancer Immunotherapy Prognosis Score.

INTRODUCTION: Currently in China, many immune checkpoint inhibitors (ICIs) have been approved for the treatment of non-small cell lung cancer (NSCLC). Some patients can not benefit from ICIs, and approximately 50% of patients have immunotherapy-related toxicity. Therefore, it is necessary to monitor carefully the selection of immunotherapy population using biomarkers to maximize the benefit of patients with NSCLC. METHODS: A prospective analysis was performed on patients with advanced NSCLC who were treated with ICIS at our hospital from March 2018 to June 2019, up to the follow-up deadline of December 31, 2019. The primary end points were overall survival (OS) and progression-free survival (PFS), and the secondary end points were objective response rate and disease control rate. A lasso regression was used for the univariate analysis, and Cox regression analysis was used for the multivariate analysis. An efficacy prediction line chart was developed. RESULTS: A total of 63 patients were included in the study. The median PFS was 7.0 months (95% CI, 5.0-11.0) and did not reach the median OS. According to the lasso regression, significant univariate factors were smoking index, PD-ligand 1 expression, and neutrophil to lymphocyte ratio (NLR). According to the multivariate analysis, the Cox proportional hazards model showed that smoking index and NLR are independent predictors of PFS in immunotherapy. A model comprised of independent predictors was developed based on a multivariate logical analysis of the main cohort-non-small cell lung cancer immunotherapy prognosis score. This model is shown as a nomogram with a C-index of 0.801 (95% CI, 0.744, 0.858), which has high prediction accuracy. CONCLUSION: This predictive model, including NLR and smoking index, can achieve a 1-year PFS in immunotherapy of patients. PD-1 inhibitors have been demonstrated to be effective and safe in the clinical treatment of patients with NSCLC.

Meta-analysis of immune-related adverse events of immune checkpoint inhibitor therapy in cancer patients.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have significant clinical efficacy in the treatment of non-small cell lung cancer (NSCLC); however, the incidence of immune-related adverse events (irAEs) of up to 50% has prevented their widespread use. With the increase in the use of ICIs alone or as combination therapy, clinicians are required to have a better understanding of irAEs and be able to manage them systematically. In this study, we aimed to assess the incidence of irAEs associated with ICIs. METHODS: We searched PubMed, Embase, and the Web of Science databases, and also included relevant literature references to widen our search. The relevant data with inclusion criteria were performed using RevMan 3.6.0 for meta-analysis. We undertook a systematic literature search which included published data up to December 2019. RESULTS: Overall, 147 articles and 23 761 cancer patients with 11 different ICI treatment-related (grade 1-5 and 3-5) irAEs were included in the study. There were 46 articles on pembrolizumab (6598 patients), 27 on nivolumab (3576 patients), 13 on atezolizumab (2787 patients), 12 on avelumab (3213 patients), 10 on durvalumab (1780 patients), 22 on ipilimumab (4067 patients), eight on tremelimumab (1158 patients), three on JS001 (223 patients), four on camrelizumab (SHR-1210) (178 patients), one on sintilimab (96 patients), and one on cemiplimab (85 patients). Grade 1-5 irAEs were: cytotoxic T lymphocyte antigen 4 (CTLA-4) (82.87%), programmed cell death 1 (PD-1) (71.89%), and programmed cell death ligand-1 (PD-L1) (58.95%). Subgroup analysis was: Avelumab (44.53%), durvalumab (66.63%), pembrolizumab (67.25%), atezolizumab (68.77%), nivolumab (76.25%), Ipilimumab (82.18%), and tremelimumab (86.78%). Grade 3-5 irAEs were: CTLA-4 (27.22%), PD-1(17.29%), and PD-L1(17.29%). Subgroup analysis was: Avelumab (5.86%), durvalumab (13.43%), atezolizumab (14.45%), nivolumab (15.72%), pembrolizumab (16.58%), tremelimumab (22.04%), and ipilimumab (28.27%). CONCLUSIONS: This meta-analysis confirmed that anti-PD-1 and anti-PD-L1 inhibitors had a lower incidence of irAEs compared with anti-CTLA-4 inhibitors.

A novel photostable near-infrared-to-near-infrared fluorescent nanoparticle for in vivo imaging.

Water-soluble K5 HoLi2 F10 (KHLF) nanoprobes with the excitation and emission both in the near-infrared (NIR) region were developed and first demonstrated for in vivo imaging of living mice. The PEG400 coating endows the nanoprobes with good water solubility and biocompatibility. Doping with Ho3+ ions is capable of emitting NIR fluorescence with two peaks centered, respectively, at 887 and 1,180 nm once excited by a 808 nm laser; meanwhile, it also possess good photothermal conversion performance. The KHLF matrix with specifically structure of large ion-distance and low photon energy imparts the nanoprobes low quenching effect and excellent photostability (fluorescence decrease <5% upon 120 min illumination of 808 nm continuous laser with a power density of 1 W/cm2 ). The nanoparticles (NPs) were tested for in vitro bioimaging with living mice. The results show the NPs have low biotoxicity, rapid metabolism, normal biodistribution, together with the photothermal imaging performance and a high-contrast fluorescence images (signal-to-background ratio of 14:1). The superior performances of these nanoprobes in vivo imaging of mice proclaim the great potential of this type of probe for high-contrast imaging and photothermal treatment in practical applications.

Application of rare earth-doped nanoparticles in biological imaging and tumor treatment.

Rare earth-doped nanoparticles have been widely used in disease diagnosis, drug delivery, tumor therapy, and bioimaging. Among various bioimaging methods, the fluorescence imaging technology based on the rare earth-doped nanoparticles can visually display the cell activity and lesion evolution in living animals, which is a powerful tool in biological technology and has being widely applied in medical and biological fields. Especially in the band of near infrared (700-1700 nm), the emissions show the characteristics of deep penetration due to low absorption, low photon scattering, and low autofluorescence interference. Furthermore, the rare earth-doped nanoparticles can be endowed with the water solubility, biocompatibility, drug-loading ability, and the targeting ability for different tumors by surface functionalization. This confirms its potential in the cancer diagnosis and treatment. In this review, we summarized the recent progress in the application of rare earth-doped nanoparticles in the field of bioimaging and tumor treatment. The luminescent mechanism, properties, and structure design were also discussed.

Relationship between the efficacy of immunotherapy and characteristics of specific tumor mutation genes in non-small cell lung cancer patients.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have greatly improved the prognosis and overall management of non-small cell lung cancer (NSCLC) patients, but in the long term less than 20% of patients benefit from treatment with ICIs. Therefore, it is necessary to guide the choice of immunotherapy population through biomarkers in order to maximize the benefit for NSCLC patients. This article mainly explores the relationship between the efficacy of immunotherapy and specific tumor mutation gene characteristics in an NSCLC population. METHODS: This was a prospective analysis of patients with advanced NSCLC who visited the Department of Respiratory Medicine of Peking Union Medical College Hospital from March 2018 to June 2019 and were instructed to use PD-1 inhibitors. The follow-up deadline was 31 December 2019. The tumor pathological tissues were tested for tumor mutation genes, and the patients were evaluated for efficacy according to RECIST 1.1. The patients were divided into the durable benefit group (DCB) and the nonsustainable benefit group (NDB). DCB/NDB was used as the outcome variable. Various statistics methods were used to explore the independent predictors of long-term benefits associated with immunotherapy and to draw a progression-free survival curve for the relevant predictors. RESULTS: A total of 44 patients were examined for tumor mutation genes in pathological tissues; 20 in the DCB group and 24 in the NDB group. Specific gene mutations occurred in TP53 38.64%, KRAS 31.82%, EGFR 20.45%, BRCA 20.45%, ERBB (excluding EGFR) 18.18%, PTEN 15.91%, CDK4/6 13.64%, POLE 11.36%, MET 11.36%, PIK3CA 9.10%, FGFR 9.10%, BRAF 9.10%, JAK 9.10%, ALK 6.82%, POLD1 4.55%, BLM 4.55%. Chi-square test results showed that there were statistically significant differences between DCB and NDB groups with eight mutations such as KRAS. Logistic regression showed that the KRAS mutation was statistically significant (P < 0.001). Two accuracy indicators, Random Forest Classification of Mean Decrease Gini and Mean Decrease Accuracy, evaluated the importance of the impact of different gene mutations on the outcome. Under two different measures, the variables were all KRAS mutations. It is suggested that the mutation of the KRAS gene is an independent predictor of the long-term benefit of immunotherapy. CONCLUSIONS: The mutation of KRAS gene in tumor tissues is an independent predictor of the long-term benefit of immunotherapy, and the predictive ability is better.

Relationship between intestinal flora structure and metabolite analysis and immunotherapy efficacy in Chinese NSCLC patients.

BACKGROUND: Many immune checkpoint inhibitors (ICIs) have been approved in China to treat non-small cell lung cancer (NSCLC). However, in the long term, less than 20% of patients benefit from ICIs. To maximize the benefit for NSCLC patients, it is necessary to guide the choice of immunotherapy through biomarkers. Recent studies have shown that gut microbiota can affect tumor response to immunotherapy and might be a potential predictive biomarker. This study analyzed the relationship between intestinal flora structure and metabolomic characteristics in NSCLC and the efficacy of ICIs. METHODS: Prospective analysis of samples from 63 patients with advanced NSCLC who attended the Department of Respiratory Medicine of the Peking Union Medical College Hospital from March 2018 to June 2019, and were prescribed programmed cell death 1 (PD-1) inhibitors, was carried out. The follow-up deadline was 31 December 2019. Stool samples were collected from all patients before the start of immunotherapy. DNA was extracted from all samples and libraries were constructed. This was followed by sequencing using the Illumina sequencing platform, and results were studied using a biological information data analysis process. We divided the data into two groups based on progression-free survival (PFS) ≥ six months and PFS < six months. RESULTS: The median PFS was 7.0 months, not reaching the median overall survival (OS). We obtained 373.5 G of original sequencing data. The phyla Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria accounted for most of the bacterial communities in the stool samples studied. Compared with the PFS < six-month group, the patients in the PFS ≥ six-month group had significantly higher ß-diversity in the intestinal microbiome at the baseline level. There were also differences in composition between the two groups. Samples in the PFS ≥ six-month group were rich in Parabacteroides and Methanobrevibacter, while those in the PFS < six-month group were rich in Veillonella, Selenomonadales, and Negativicutes. The KO, COG, and CAZy databases were used to study functional group protein families, yielding 390 (KO), 264 (COG), and 859 (CAZy) functional group abundances, with significant differences between the two groups. Bacterial metabolites analysis suggested significant differences in the metabolic potential of methanol and methane between the two groups. CONCLUSIONS: We found a close correlation between intestinal microbiome ß-diversity and anti-PD-1 immunotherapy response in Chinese patients with advanced NSCLC. The intestinal flora composition, functional group protein family, and KEGG metabolism also differed between the two groups. Differences in pathways and flora metabolites were also noted.