Serpin family A member 1 (SERPINA1) encodes a protease inhibitor participating in many human diseases, but its value in immunoregulation and prognosis of human cancers remains unclear. In this study, through comprehensive analysis of data from The Cancer Genome Atlas (TCGA) datasets, we found that SERPINA1 was dysregulated in many cancers compared with normal tissues. SERPINA1 expression was significantly associated with prognosis, immune subtype, molecular subtype, immune checkpoint (ICP) genes, tumor mutational burden (TMB), microsatellite instability (MSI), and the estimation of stromal and immune cells in malignant tumor tissues using expression data (ESTIMATE) score. There was a strong connection between SERPINA1 expression and tumor-infiltrating lymphocytes, and SERPINA1 showed significant relation to gene markers of immune cells in digestive tumors. Fluorescence-based multiplex immunohistochemistry confirmed that SERPINA1 protein expression was related to clinicopathologic features and immune infiltrates in hepatic cancer. This study suggests that SERPINA can potentially serve as a novel biomarker for cancer prognosis and immunotherapy. Immune-based therapies have revolutionized the treatment of advanced cancer. The application of immune checkpoint inhibitor therapy targeting the PD-1/PD-L1 or CTLA-4 pathway is a breakthrough for many types of cancer . Tumor cSerpin family A member 1 (SERPINA1) is located on chromosome 14 (14q32.1), encoding the protein Alpha1-antitrypsin (A1AT) that is a highly conserved protein and the dominant protease inhibitor, the inhibition capacity of which accounts for more than 90% of all plasma proteases [SERPINA1 is expressed mainly in hepatocytes but is also synthesized in mononuclear phagocytes, neutrophils, and intestinal epithelial cells [SERPINA1 plays a crucial role in the maintenance of cell homeostasis through irreversibly inhibiting a variety of serine endopeptidases. Previous literature has paid much attention to the pathological process and treatment of SERPINA1 deficiency in the liver and lung. Recently, a growing body of research has confirmed that SERPINA1 shows a tremendous influence on multiple tumors, such as lung cancer [SERPINA1 in prognosis and TME regulation in cancers is rare.al cells . SERPINAg cancer , gastricg cancer , and breg cancer . HoweverSERPINA1 in prognosis and immunological regulation in tumors was analyzed through multiple bioinformatics approaches. The differential expressions and prognostic values of SERPINA1 in different cancers were explored comprehensively. We also analyzed the potential connection between SERPINA1 level and immune and molecular subtypes, biomarkers of therapeutic efficacy, and tumor-infiltrating lymphocytes. Furthermore, the role of SERPINA1A in liver hepatocellular carcinoma (LIHC) was detected using fluorescence-based multiplex immunohistochemistry (mIHC) to confirm the results we acquired. The study was designed to elucidate the potential of SERPINA1 in tumor prognosis and immunotherapy, thus providing novel insight into the antitumor strategy.In this study, the role of SERPINA1 mRNA expression was significantly higher in BLCA , HNSC (head and neck squamous cell carcinoma), KIRC , KIRP , STAD (stomach adenocarcinoma), THCA (thyroid carcinoma), and UCEC  than in paired normal tissues. Meanwhile, SERPINA1 expression was significantly lower in CHOL (cholangiocarcinoma), LIHC, LUAD (lung adenocarcinoma), and LUSC (lung squamous cell carcinoma) , DLBC (lymphoid neoplasm diffuse large B-cell lymphoma), and THYM (thymoma) (The TIMER database confirmed that rcinoma) A. The GEthymoma) B.SERPINA1 to differentiate tumors from normal tissue via the ROC curve , CHOL (AUC = 0.952), ESAD , GBM , HNSC (AUC = 0.757), KICH , KIRC (AUC = 0.773), LIHC (AUC = 0.723), LUAD (AUC = 0.737), LUADLUSC , LUSC (AUC = 0.984), OSCC , STAD (AUC = 0.703), THCA (AUC = 0.873), and UCEC (AUC = 0.715).In addition, we detected the diagnostic value of OC curve . SERPINASERPINA1 was identified using different databases. In the Xiantao database, higher SERPINA1 expression was connected to better overall survival (OS) in BRCA (breast invasive carcinoma), COAD (colon adenocarcinoma), DLBC, KIRC, KIRP, osteosarcoma, SARC (sarcoma), and SKCM (skin cutaneous melanoma) (SERPINA1 expression was associated with poorer OS in GBMLGG (glioma), GBM, HNSC, KICH, LGG (brain lower-grade glioma), LIHC, and LUSC  , THCA, and UCEC. In comparison, higher SERPINA1 expression was associated with poorer OS in ESCA , TGCT (testicular germ cell tumors), and THYM  A–H. Howeand LUSC I–O. Furtal (DSS) . In the and THYM . These rSERPINA1 on immune and molecular subtypes in human tumors through the TISIDB web portal. SERPINA1 expression was confirmed to be associated with immune subtypes in ACC, BLCA, BRCA, KICH, KIRC, LGG, LUAD, LUSC, OV (ovarian serous cystadenocarcinoma), PCPG (pheochromocytoma and paraganglioma), PRAD (prostate adenocarcinoma), SARC, SKCM, THCA, UCEC, and UVM   . Differeand UCEC . The resSERPINA1 in immunotherapy, we analyzed the relationship between SERPINA1 expression and ICP genes, which significantly affect immune cell infiltration, through the SangerBox database [SERPINA1 expression is positively related to ICP genes in most cancer types, especially GBMLGG, OV, KIPAN (Pan-kidney cohort), and PRAD, in which more than 90% of 60 ICP genes were connected to SERPINA1 expression , we explored the connections between SERPINA1 expression and TMB and MSI, which have been confirmed to influence immunotherapy efficacy significantly [SERPINA1 expression was positively associated with TMB in COAD, COADREAD, ESCA, GBMLGG, KIPAN, KIRC, LGG, SARC, and THYM and negatively connected to TMB in BRCA, LIHC, LUAD, PRAD, and STAD , NB (neuroblastoma), OV, PCPG, PRAD, SARC, SKMC, THCA, UCEC, UVM, and WT (high-risk Wilms tumor)  C. These SERPINA1 expressions. We further explored the correlations between SERPINA1 expression and immune cells in the TME through the Timer 2.0 website. The result demonstrated that SERPINA1 expression presented strong connections to B cells in 25 cancer types, CD4+ T cells in 28 cancer types, CD8+ T cells in 25 cancer types, neutrophils in 32 cancer types, macrophages in 31 cancer types, and dendritic cells (DCs) in 32 cancer types  and LIHC (n = 371) were examples to illustrate the potential immune value of SERPINA1. As shown in SERPINA1 had a solid connection to all enrolled markers of CD8+ T cells, general T Cells, B cells, M2 macrophages, neutrophils, and Treg cells in ESCA. SERPINA1 in LIHC also showed a good relationship with most gene markers in neutrophils and natural killer (NK) cells. These results further suggested that SERPINA1 might be vital in regulating immune cell infiltration.Finally, we analyzed the relationship between  cancers . For exaSERPINA1 was explored in digestive cancers except for READ, which was absent on the cBioPortal website. The result proved that the incidence of SERPINA1 gene alteration was 1.6% , CD4 (p = 0.005), CD3 + CD4+ (p = 0.002), CD3 + CD8+ (p = 0.041), CD68 (p = 0.018), and LAG3 (p = 0.047) (We detected SERPINA1 protein expression in 86 LIHC tissues through fluorescence-based mIHC to confirm the results above. SERPINA1 protein was detected in the cytoplasm and mesenchyme A. SERPIN= 0.047) B, which SERPINA1 has the potential to influence the TME and might be a promising biomarker for immunotherapy, but the role of SERPINA1 in TME regulation and cancer prognosis is unclear.An inflammatory microenvironment is a prerequisite and promoter for virtually all cancers. A growing body of research shows that inflammation greatly influences the TME composition, especially on the plasticity of cancer cells and stromal cells . TumorigSERPINA1 in tumor and normal tissues through multiple databases. Previous studies have demonstrated that SERPINA1 was overexpressed in pancreatic and breast cancer tissues but downregulated in lung cancer [SERPINA1 was upregulated in most cancer tissues except CHOL, LIHC, LUAD, LUSC, ACC, DLBC, and THYM. These results suggested that SERPINA1 may play a different role in different cancers.In this study, we first explored the differential expressions of g cancer ,22,23. OSERPINA1 expression in tissues [SERPINA1 expression in cancer tissues and the prognosis was analyzed. Our results, obtained from the Xiantao web tool and Kaplan–Meier plotter database, demonstrated that SERPINA1 expression in tissues had a significant prognostic value for many cancers. Next, we detected the relationship between SERPINA1 expression and immune subtypes and molecular subtypes of human cancers via the TIMER database and the R package. The results confirmed that SERPINA1 expressions in different immune subtypes and molecular subtypes of most cancers were significantly different, indicating that SERPINA1 may affect the immune microenvironment and cancer prognosis. Moreover, we confirmed that SERPINA1 expression was significantly associated with clinical features in seven digestive cancers, similar to a paper proving that different SERPINA1 levels exist in patients with varying characteristics in colorectal cancer [Previous research confirmed serum SERPINA1 as a potential biomarker for cancer diagnosis and prognosis ,24,25. H tissues . Then, tl cancer .SERPINA1 expression was significantly connected to ICP genes in most cancer types, especially GBMLGG, OV, KIPAN, and PRAD, which suggested that patients with these cancers may respond well to ICB treatment. Patients with higher TMB can generate a more significant neoantigen load and induce a more robust neoantigen-specific T cell response, suggesting that patients benefit more from ICB treatment [SERPINA1 expression was associated with TMB and MSI in some cancers, positively or negatively, indicating that SERPINA1 plays a different role in the prediction of immunotherapy effect in various human cancers. Above results suggested that SERPINA1 might be important in cancer immunotherapy.ICPs are inhibitory receptors expressed on T cells or other immune cells, and tumors can upregulate ICPs to achieve immune escape . Thereforeatment . MSI is reatment . Our resSERPINA1 was positively connected to immune scores, stromal scores, and ESTIMATE scores in many cancers. Tumor-infiltrating lymphocytes (TILs) have an outsized influence on cancer prognosis and immune therapy [SERPINA1 strongly connects to TILs, which means a high probability of regulating the TME. For example, SERPINA1 expression was closely related to CD4+ T cells, CD8+ T cells, neutrophils, and macrophages in most cancer types. Particularly in ESCA, SERPINA1 was connected to PDCD1 and CTLA4, which are classical targets of immunotherapy [SERPINA1 demonstrated significant association with CD8+ T cells, general T cells, B cells, M2 macrophages, neutrophils, and Treg cells in ESCA. These results suggested that SERPINA1 might affect immunotherapy efficiency through regulating TIL compositions in the TME.The ESTIMATE score is utilized to speculate the proportion of stromal and immune cells in the TME . Through therapy ,32. Thisotherapy ,34. FurtFinally, we explored the SERPINA1 protein (A1AT) in LIHC tissues via fluorescence-based mIHC to preliminarily prove the results acquired. Similar to the findings regarding the SERPINA1 gene, SERPINA1 protein (A1AT) in LIHC tissues was associated with clinical features. In addition, the positive relationship between SERPINA1 level and some biomarkers of immune cells suggested the SERPINA1 protein (A1AT) might affect the infiltrations of Th cells, CTLs, and macrophages, as well as the efficacy of immunotherapy targeting LAG3.SERPINA1 in pan-cancer, and cross-certified the result through different databases and R package, the study has some limitations. First, systematic bias may be caused by the difference in RNA-seq data from different databases. Second, the function of the SERPINA1 protein should be verified in vitro and in vivo. Third, we preliminarily deduced that SERPINA1 had a close connection to the immune microenvironment, but there was no direct evidence to prove the influence of SERPINA1 on immunotherapy efficiency and prognosis. In the future, more investigations are needed to certify the prognostic and immunological value of SERPINA1 and explore the underlying mechanism.Although we performed a systematic analysis on the role of http://timer.cistrome.org/ (accessed on 15 March 2022)) and the GEPIA database (http://gepia.cancer-pku.cn/ (accessed on15 March 2022)) were utilized to compare the differential expressions of SERPINA1 in various cancer tissues and normal tissues [The TIMER 2.0 tool ) and Kaplan–Meier plotter database (https://kmplot.com/ (accessed on 15 March 2022)) [The relationships between h 2022)) ,38.http://cis.hku.hk/TISIDB/ (accessed on 16 March 2022)) was used to detect the associations between SERPINA1 expression and immune or molecular subtypes of different cancers [The TISIDB web portal ) was utilized comprehensively [To explore the relationships between ensively .SERPINA1 level and six immune cells were explored in pan-cancers through the Timer 2.0 website. Then, we analyzed the connections between SERPINA1 expression and the absolute abundances of 8 immune cells and two stromal cells in seven digestive system tumors using the R package MCPcounter. The relationships between SERPINA1 expression and marker genes of immune cells were explored via the TIMER database.The associations between https://docs.cbioportal.org/ (accessed on 10 April 2022)) was used to confirm the genomic alterations of SERPINA1 in digestive system tumors.The cBioPortal for Cancer Genomics ). Data without clinical information were discarded, and the R package STATS was utilized to analyze the associations between SERPINA1 expression and clinicopathologic features in each digestive cancer.The RNAseq data of seven digestive cancers were downloaded from the TCGA database  was designed on the blank wax block, and the Quick Ray Master UATM-272A  was used to drill holes into the receptor module. Tissue columns with a diameter of 1.5 mm were taken out from donor tissue blocks and then inserted into corresponding holes in receptor blocks. The receptor blocks were cut into slices with a thickness of 3 μm and placed on polylysine-coated glass slides. The slices were dewaxed and hydrated, boiled in 0.01 mM citrate buffer (pH 6.0) to retrieve antigens, and incubated with 5% goat serum to block the nonspecific site. The nuclei were stained with DAPI . Then, the sections were incubated with anti-alpha 1 Antitrypsin antibody  at 4 °C overnight. Opal polymer HRP Ms + Rb  was added to the slices and incubated at dark for 3 h. Fluoroshield with DAPI  was used to stain nuclei and seal the sections.p < 0.05 was considered statistically significant.The experimental data of mIHC were statistically analyzed using IBM SPSS Statistics v26 . The correlation between SERPINA1 protein expression and clinicopathologic features was investigated using the Pearson Chi-square test. SERPINA1 shows potential value for the diagnosis and prognosis of many human cancers and may be involved in the immune regulation of the TME. Further study about SERPINA1 is necessary for cancer immunotherapy and prognosis.Above all,


EGFR) was identified. Tumor cells were isolated and treated with arsenic trioxide (ATO) or/and gefitinib. Additionally, six patients were intrapleurally administrated with ATO. Results showed that 25 samples bore EGFR wild type (WT) and 11 harbored EGFR mutations, including 6 with L858R, 3 with ΔE746-A750, and 2 with T790M. ATO diminished the number of tumor cells from patients with WT and mutant EGFR, down-regulated the expression or phosphorylation of EGFR, pmTOR, PI3K, PTEN, and p4E-BP1, and up-regulated the expression of LC3. Immunofluorescent experiments showed that ATO enhanced LC3 and P62. By contrast, gefitinib was only effective in those harboring EGFR sensitizing mutations. Notably, in patients with intrapleural ATO injection, the pleural effusion underwent a bloody to pale yellow color change, the volume of the pleural effusion was reduced, and the number of the tumor cells was significantly reduced. In conclusion, arsenic is effective against NSCLC with various EGFR genotypes in vitro and in vivo, and potentially circumvents gefitinib resistance.To clarify whether arsenic could exert inhibitory effects on tumor cells in pleural effusions of patients with non-small cell lung cancer (NSCLC), 36 NSCLC pleural effusion samples were collected from Changzheng Hospital and Ruijin Hospital, from 2019 to 2022. The genotype of epidermal growth factor receptor ( EGFR) mutation, anaplastic lymphoma kinase (ALK) fusions, ROS proto-oncogene 1 (ROS1) fusions, B-Raf proto-oncogene (BRAF) V600E, neurotrophic tropomyosin-receptor kinase (NTRK) fusions, rearranged during transfection (RET) fusions, and mesenchymal epithelial transition factor (MET) exon 14 skipping alterations, as well as programmed cell death protein-ligand 1 (PD-L1) immunohistochemistry (IHC), given that there are highly effective targeted therapies that have been approved for these targets  years old. All cases were confirmed by cytology and/or pathology, and the basic information of the gender, age, smoking status, type of pathology, tumor TNM stage, appearance of pleural effusions, and EGFR genotype was collected and is shown in Fetal bovine serum (FBS), antibiotic (antimycolic 100×), and 0.25% trypsin-EDTA were purchased from GIBCO . DMEM medium and phosphate buffer (PBS) were purchased from Basalmedia Technologies Co., Ltd. . Wright’s staining solution was purchased from Shanghai Beyotime Biotechnology Co., Ltd. , while the blood/body fluid DNA extraction kit was purchased from TIANGEN . Polymerase chain reaction (PCR) amplification primers and sequencing primers were synthesized by BioSune Biotechnology Co., Ltd. . The immunological reagents included the antibodies (Abs) against phosphorylated mammalian target of rapamycin (pmTOR), phosphatidylinositol 3 kinase (PI3K), gene of phosphate and tension homology deleted on chromosome ten (PTEN), and phosphorylated eukaryotic initiation factor 4E binding protein 1 (p4E-BP1) which were purchased from Cell signaling technology , and the Abs against EGFR, LC3, and P62 which were purchased from Abcam , Santa Cruz Biotechnology , and Medical and Biological Laboratories , respectively. Additionally, β-actin Ab was purchased from Sigma-Aldrich , while horseradish peroxidase (HRP)-labeled sheep anti-rabbit Ab and HRP-labeled art anti-mouse Ab were purchased from Abcam . Lymphocyte isolation solution was purchased from Shanghai Sangon Biotech , the protein quantification kit (Bicinchoninic acid (BCA) Assay) from Pierce , Polyvinylidene Fluoride (PVDF) membrane from GE-Healthcare , and HRP-labeled chromogenic substrate from Millipore .ATO was purchased from Beijing Shuang Lu (SL) Pharmaceutical Co., Ltd.  and was prepared as a 2 mmol/L stock solution in PBS at −20 °C for in vitro experiments. Gefitinib was purchased from Sigma-Aldrich  and was dissolved in dimethylsulfoxide (DMSO) to produce a stock solution of 10 mmol/L at −20 °C.2 mixture cell incubator , cell smear centrifuge (CYTOPRO) , and Western blot (WB) developer LAS-4000 .The instruments used in this study included an inverted fluorescence microscope , a laser confocal microscope SP8 MP , 5% COAfter B-ultrasound examination and localization, the patient was placed under local anesthesia with 8–14 F (French gauge system) deep venous catheter drainage, and the external end was connected with a closed drainage bag. The initial drainage volume was less than 800 mL, followed by slow drainage of 1000–1500 mL. The pleural effusion was drained as much as possible before intrapleural administration at 200 mL/d (no negative pressure suction). To treat NSCLC patients, 40 mL of saline with 10 mg of ATO was injected into the pleural cavity, and the tube was clamped after injection. The patients were asked to rotate the body position so that the drug could widely and evenly distribute within the pleura. After 24 h, the pleural fluid was released and then drained again. When the pleural effusion was less than 200 mL/d, the drainage tube was removed. The procedure was based on what previously performed .g for 10 min, and the cells in the pleural effusion were collected. Then, the cells were re-suspended in PBS, the mononuclear cells (including tumor cells) were separated with lymphocyte separation solution, and the red blood cells and other cells were removed. The isolated primary tumor cells were counted using a cell counting plate, and the cell density was adjusted to 2 × 105/mL. A quantity of 200 μL of cells was centrifuged by a cell smear centrifuge at 100× g for 5 min. The cells were evenly coated on the slide, and Wright’s staining solution was evenly dropped onto the cells for about 5 min. The cell staining was observed under a microscope. The staining was terminated when the results were satisfactory, and the cells were photographed.The pleural effusion was collected and stored in a 500 mL glass bottle. The pleural effusion in the glass bottle was photographed to facilitate the monitoring of the volume and color. Following standing for 2 h, the pleural effusion was centrifuged at 800× 6–1 × 107) derived from pleural effusion was extracted according to the protocol provided by the blood/body fluid DNA extraction kit. Specific primers for EGFR exon 18, 19, 20, and 21 were used for PCR reactions. Primer information is shown as follows: (i) exon 18F: 5′-TCCAGCATGGTGAGGGCTGAG-3′ and 18R: 5′-GGCTCCCCACCAGACCATG-3′; (ii) exon 19F: 5′-TGGGCAGCATGTGGCACCATC-3′ and 19R: 5′-AGGTGGGCCTGAGGTTCAG-3′; (iii) exon 20F: 5′-CCTCCTT-CTGGCCACCATGCG-3′ and 20R: 5′-CATGTGAGGATCCTGGCTCC-3′; (iv) exon 21F: 5′-CGGATGCAGAGCTTCTTCCC-3′ and 21R: 5′-AGGCAGCCTGGTCCCTGGTG-3′. PCR products were detected by agarose electrophoresis before sequencing by BioSune. Sequencing primers were the same as PCR amplification primers. Forward and reverse sequencing were performed. Sequencing results were compared with wild type (WT) EGFR sequences to determine the EGFR genotype for each sample. The following PCR reaction conditions were adopted: 94 °C for 3 min, followed by 41 cycles of amplification of 94 °C for 40 s, 60 °C for 40 s, and 72 °C for 35 s, with a final extension at 72 °C for 5 min. The amplified fragment sizes were 242, 217, 296, and 275 bp for exon 18, 19, 20, and 21, respectively.The DNA of the tumor cells (5 × 106–1 × 107) isolated from pleural effusion were resuspended in DMEM medium containing 10% of FBS with 1% antibiotics  together. According to the counts of tumor cells, the cells were seeded in 6-well plates or 60 mm cell culture dishes. The tumor cells isolated from each patient were seeded into 4 wells or 4 dishes, and then treated with ATO (2 μmol/L) alone, gefitinib (5 μmol/L) alone, or ATO combined with gefitinib. Cells were harvested at the 4th day after drug treatment. Wright’s staining, WB, and IF were performed.The tumor cells (5 × 106) were lysed using RIPA lysate  on ice for 30 min. After lysis, the cells were centrifuged at 13,400× g for 10 min, and the cell lysis supernatant was collected. The concentration of protein was quantified using a BCA quantification kit. After SDS-PAGE electrophoresis, the proteins were transferred to PVDF membrane and incubated with the indicated antibodies, and the corresponding secondary antibodies labeled with HRP. After incubation, HRP substrate was used for the development and LAS-4000 was used for photo capture.The total cells , anti-LC3 and anti-P62 antibodies were added for incubation overnight. After washing with PBS 3 times, the Alexa Fluor 488-labeled anti-mouse antibody  and the Alexa Fluor 647-labeled anti-rabbit antibody  were added for incubation, and then the cells were washed with PBS 3 times. The cells were sealed by adding a DAPI-containing sealing solution. A laser confocal microscope, SP8 MP, was used for observation and photo capture.n) is shown in each statistical result. Pairwise comparisons were performed using the student’s t test and two-tailed analysis , and one-way analysis of variance (ANOVA) was adopted with normally distributed data assuming equal variances . * p < 0.05, ** p < 0.01, *** p < 0.001.Data are presented as mean ± standard deviation (SD). Sample size  A,B, but p < 0.001, p < 0.01, p < 0.001, p < 0.01, Using an IF assay, we detected the intracellular expression and distribution of the autophagy marker proteins LC3 and P62 in tumor cells derived from NSCLC patients’ pleural effusion treated with arsenic and gefitinib. Results revealed that arsenic increased the cytosolic expression of LC3 and P62 to a certain extent in tumor cells harboring EGFR WT A,B, EGFROur previous study showed the effects and mechanisms of arsenic on NSCLC at cellular and animal levels , but theEGFR genotype identification, specific primers of EGFR exon 18, 19, 20, and 21 were used by PCR, and the PCR products were sequenced to ensure the accuracy of the results. However, since Sanger sequencing is unable to detect variants with mutation rates lower than 15% [The malignant pleural effusion (MPE) represents a common complication of lung cancer and is associated with poor prognosis . The celthan 15% , resultiThe occurrence of a malignant pleural effusion may be caused by many factors, such as pleural inflammation, tumor angiogenesis, and increased vascular permeability ,29. In tResults demonstrated that arsenic plays a therapeutic effect at the protein level, since it can degrade the oncoproteins through the ubiquitin-proteasome pathway or via the autophagy pathway ,33,34. OEGFR genotypes (EGFR mutations. Even in tumor cells harboring T790M (gefitinib-resistant mutant), arsenic was still effective  [EGFR mutations, EGFR-TKIs are preferably prescribed as they provide a superior survival benefit over chemotherapy [We think it is necessary to compare the efficacy, mechanisms, and safety of targeted therapy (TKIs), chemotherapy, immunotherapy (ICIs), arsenic, surgery, and radiation therapy in NSCLC . With thutations . Among tutations ,52. Alth (T790M) . To furt (T790M) . Osimert (T790M) , has sho (T790M)  and a su (T790M) . For advotherapy . In a photherapy .Platinum-based chemotherapy drugs are widely used in human neoplasms . NSCLC pImmunotherapy with ICIs, which unleash a patient’s own T cells to kill tumors, has become a “game changer” in the treatment of advanced NSCLC. The most clinically important advantage over chemotherapy is its long-term survival benefits ,71. ICIsSurgery is currently the standard treatment for patients with stage I NSCLC. Radiation therapy, stereotactic body radiation therapy (SBRT) in particular, is recommended for those who are not medically fit for surgery . RadiothThe therapeutic efficacy and safety of arsenic in clinical trials has been well demonstrated in the treatment of leukemia. The 5-year DFS rate of APL after treatment with ATO combined with ATRA is more than 95% . The lonGefitinib  and osimTo sum up, targeted drugs and ICIs are gaining increasing importance in the treatment of NSCLC and these novel treatments will supplement chemotherapy, radiotherapy, and surgery. For gefitinib-sensitive and -resistant NSCLC, TKIs might exert potently therapeutic effects. In contrast, in all stages of NSCLC, platinum-based adjuvant chemotherapy could play a synergistic therapeutic role. However, for NSCLC resistant to TKIs and platinum-based chemotherapy drugs, arsenic could still be effective. Therefore, utilization of arsenic to treat NSCLC is a promising and potential therapeutic strategy.However, this study has limitations, including the limited number of NSCLC samples, especially of the gefitinib-resistant NSCLC samples. In addition, other proteins of the PI3K/AKT/mTOR pathway were not assessed, and the sensitivity of Sanger sequencing in the detection of gene mutations was low. These limitations will be addressed in future studies.In conclusion, for NSCLC patients with pleural metastasis and pleural effusions, intrapleural administration with arsenic has a certain therapeutic effect and significance, making it suitable as a therapeutic strategy for these patients. The findings provide important preliminary data that can be used for future development of strategies using arsenic combined with TKIs, chemotherapy, and immunotherapy, and even with surgery and radiation therapy, for the treatment of NSCLC in clinical trials.


Inflammatory bowel disease (IBD) mainly includes Crohn’s disease and ulcerative colitis. These diseases have a progressive course of chronic relapse and remission and affect a large number of children and adults worldwide. The burden of IBD is rising worldwide, with levels and trends varying greatly in countries and regions. Like most chronic diseases, the costs associated with IBD are high, including hospitalizations, outpatient and emergency visits, surgeries, and pharmacotherapies. However, there is no radical cure for it yet, and its therapeutic targets still need further study. Currently, the pathogenesis of IBD remains unclear. It is generally assumed that the occurrence and development of IBD are related to the environmental factors, gut microbiota, immune imbalance, and genetic susceptibility. Alternative splicing contributes to a various diseases, such as spinal muscular atrophy, liver diseases, and cancers. In the past, it has been reported that alternative splicing events, splicing factors, and splicing mutations were associated with IBD, but there were no reports on the practical application for clinical diagnosis and treatment of IBD using splicing-related methods. Therefore, this article reviews research progress on alternative splicing events, splicing factors, and splicing mutations associated with IBD. These diseases have a progressive course of chronic relapse and remission and affect a large number of children and adults worldwide . IBD canCurrently, the pathogenesis of IBD remains unclear. It is generally assumed that the occurrence and development of IBD are related to the environmental factors, gut microbiota, immune imbalance, and genetic susceptibility, which can impact a weakened gut barrier on the microbiota, leading to inappropriate intestinal immune activation . The genOn the basis of the Web of Science core collection database, approximately 6–19 articles have been published in this category each year for the last decade (2MAOA), signal transducer and activator of transcription 3 (STAT3), and interferon regulatory factor 1 (IRF1)] and splicing factors can be used for screening and treatment of patients with CD (HDAC6) and lipase A (LIPA) had significant alternative splicing events in patients with UC involved in immune responses and inflammation, indicating that dysregulated alternative splicing of HDAC6 or LIPA may be a potential therapeutic target for patients with UC. In this section, we summarize studies on alternative splicing in adults and children with IBD  is IBD specific  is positively correlated with the severity of IBD, and a single-nucleotide polymorphism (SNP) in IFNG-AS1, rs7134599, is associated with the pathophysiology of IBD  by quantitative reverse transcriptase real-time PCR to measure relative expression of Th1, Th17, and Treg cytokines, finding that mRNA splicing for transcription factor XBP1 may be involved in immunopathogenesis of IBD  (including MLCK1 and MLCK2) could regulate paracellular and extracellular permeability in IBD  gene includes IGF-IEa and IGF-IEc (igf1 may be related to pathogenesis of fibrostenotic CD (CXCR3) splice variants is detected in CD3+ peripheral blood lymphocytes (PBL) of patients with CD, indicating that CXCR3 splicing may play a significant role in pathogenesis of CD  resistance, and therapeutic target for CD in adults –26, 33.  IGF-IEc . Researcnotic CD . Li et anotic CD . Anotheris of CD .Previous studies found that serum spliceosome-associated protein 130 (SAP130) levels in patients with active CD are significantly higher than in patients with remission CD and controls and changed with different disease activities being significantly correlated with disease severity . FurtherFOXP3) is involved in Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling pathway in Th17 lymphocytes, leading to CD  knockout mice may spontaneously develop mild colitis  are mainly enriched in intercellular items related to cell junction, cell adhesion, actin cytoskeleton, transmembrane receptor signaling pathways, and intracellular items related to RNA splicing, metabolism, and localization  were mainly enriched in intercellular items related to cell junction, cell adhesion, actin cytoskeleton, transmembrane receptor signaling pathways, and intracellular items related to RNA splicing, metabolism, and localization, splicing of above genes may be associated with pathogenesis of UC, serving as potential biomarkers for clinical diagnosis and therapy in children  observed in a study were specific to IBD rather than solely dependent on inflammation  that are significantly differentially expressed in patients with CD, playing a key role in the regulation of alternative splicing events in patients with CD  that are significantly differentially expressed in patients with UC, closely related to change in immune response in patients with UC  , 39. Acc and WAS Table 3. and WAS . However4.1CARD9), located on chromosome 9q34.3, is a susceptibility gene for IBD that encodes a connexin that integrates signals downstream from pattern recognition receptors and plays a key role in immune responses against microbes (CARD9 (c.1434+1G>C) may affect IBD risk in adults (CARD9 is involved in the nucleotide-binding domain (NOD)–like receptor signaling pathway, which is involved in the occurrence of IBD , located on chromosome 10p11.21, is a protein coding gene, and its splicing site (c.1675-5T>C) has also been reported to affect IBD risk , located on chromosome 19p13.11, has a 3′ splicing site mutation (c.1791+2T>G) that has been reported to be associated with pathogenesis of adults with CD , located on chromosome 2p21-p16.3, has a splicing variant (c.2006-6T>C) that has been reported to be associated with an excess risk of colorectal cancer for patients with UC (NCF4), located on chromosome 22q12.3, encodes the p40phox protein and is another susceptibility gene for IBD (NCF4 (c.32+1258T>C) has been reported to be involved in the pathogenesis of CD , located on chromosome 16q12.1, is associated with CD   and macrophages of CD (NOD2 (c.2798+158C>T) has been reported to affect CD risk (NOD2 (c.74-7T>A) may be associated with the pathogenesis of CD  have been reported to be related to IBD (including CD and UC) in adults (NOD2 is directly related to the genetic typing of IBD (IBD1), and further studies on these splicing sites will be of great help to the pathogenesis, diagnosis, and treatment of IBD1.Moreover, nucleotide-binding oligomerization domain containing 2 (#266600) . NOD2 si#266600) . NOD2 ises of CD Figure 2es of CD , 80, 81. CD risk , 54. Anois of CD , 51. In 4.2ANKZF1), located on chromosome 2q35, is associated with infantile-onset IBD (ANKZF1 (c.262-97G>A) has been reported to be involved in the pathogenesis of infantile-onset IBD , located on chromosome Xq26, is associated with VEO-IBD and EO-IBD (CD40LG (c.410-2A>T) may affect the treatment of VEO-IBD and EO-IBD (CTLA4), located on chromosome 2q33, is associated with pediatric IBD (CTLA4 (c.457+1G>A) may affect the treatment of pediatric IBD through regulatory T cells and immune modulation  plays a very important role in the splicing process and might be a diagnostic and therapeutic target for VEO-IBD  may be involved in the pathogenesis of VEO-IBD , located on chromosome 6p21, is associated with VEO-IBD and UC in children (SKIV2L (c.354+5G>A) may be associated with the pathogenesis of UC in children, another splicing variant (c.355-2A>C) may be involved in the pathogenesis of VEO-IBD (STXBP3), located on chromosome 1p13.3, is associated with VEO-IBD (STXBP3 (c.1029+1G>A) may be associated with the pathogenesis of VEO-IBD (TTC7A), located on chromosome 2p21, is also associated with VEO-IBD (TTC7A (c.844-1G>T and c.1204-2A>G) may be involved in the pathogenesis of VEO-IBD (TTC37), located on chromosome 5q15, is associated with VEO-IBD and EO-IBD (TTC37 (c.4497-1G>A) may affect the treatment of VEO-IBD and EO-IBD (WAS), located on chromosome Xp11.4-p11.21, is associated with EO-IBD and VEO-IBD (WAS (c.463+8G>A and c.360+1G>C) may be involved in the pathogenesis of EO-IBD and VEO-IBD, respectively  in adults, it is also associated with IBD1 in children  may be involved in the pathogenesis of IBD1 in children , located on chromosome 21q22.11, is associated with IBD25 (OMIM #612567) (IL10RB (c.646+2T>C) may be involved in the pathogenesis of EO-IBD (IBD25) , located on chromosome 11q23, is associated with IBD28 (OMIM #613148) (IL10RA (c.188+1G>A) may be a pluripotency marker for IBD28 (IL10RA (c.537G>A) may be associated with the pathogenesis of IBD28 by affecting RNA splicing (IL10RA (c.368-10C>G) may also be associated with the pathogenesis of VEO-IBD (IBD28)  . A splic (IBD25) . Interle#613148) . Splicinor IBD28 . A synonsplicing , 60–64.  (IBD28) . Both ILng to UC Figure 2ng to UC .CARD9, NOD2, and IL12RB1, whereas IL10RA and IL10RB are involved in the JAK-STAT signaling pathway connected with UC  have been reported to be associated with IBD in children  in adults, 20 splicing mutations in 13 genes have been reported to be associated with IBD in children. Splicing mutation sites and genes are much more frequently reported in children. Importantly, three of these genes are associated with IBD1, IBD25, and IBD28, including NOD2, IL10RB, and IL10RA). At present, research on alternative splicing in IBD is still limited. Future studies are needed to understand the role of alternative splicing in IBD and to find new pathogenesis, diagnostic, and therapeutic targets for children and adults with IBD. If biomarkers and therapeutic targets for IBD can be found in this field, then it will provide convenience for the diagnosis and treatment of IBD.Nevertheless, there are no validated splicing events that have a causative component of IBD. There are no validated splicing events that are just a consequence of IBD. No splicing events are employed routinely in the therapy of IBD. There are no validated or approved biomarkers of IBD that employ splicing events and are used by clinicians. No splicing-related events are employed routinely for the direct benefit of the patient with IBD. However, studies have shown that some alternative splicing may serve as potential biomarkers or therapeutic targets for IBD Table 1 JZ, QZ, YZ, YS, YL, MC, SZ, and ZW designed and drafted the article. MC, SZ, and ZW reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.


In addition to CT images and pathological features, many other molecular characteristics remain unknown about multiple primary lung cancer (MPLC) from intrapulmonary metastatic lung cancer.in situ (AIS) subtype and minimally invasive adenocarcinoma (MIA) subtype. The patient was diagnosed with more than 10 nodules and underwent precise surgery assisted by three-dimensional (3D) reconstruction at the left upper lung lobe. Whole-exome sequencing (WES) and multiple immunohistochemistry (mIHC) were performed to reveal the genomic profiling and tumor microenvironments of multiple nodules in this patient with MPLC. Based on 3D reconstruction location information, we found that the genomic and pathological results of adjacent lymph nodes were quite different. On the other hand, PD-L1 expression and the proportion of infiltrating lymphocytes in tumor microenvironments were all at a low status and did not vary in adjacent lymph nodes. Additionally, maximum diameter and tumor mutational burden levels were found to be significantly associated with CD8+ T cell proportion (p<0.05). Besides, CD163+ macrophages and CD4+ T cell proportion were higher in MIA nodules than in AIS nodules (p<0.05). This patient reached a recurrence-free survival of 39 months.In this study, we reported a patient with an early-stage MPLC with both adenocarcinoma Generally, in addition to CT imaging and pathological results, genomic profiling and tumor microenvironments may facilitate identifying the potential molecular mechanisms and clinical outcomes in patients with early-stage MPLC. The In this study, the patient was diagnosed with multiple primary lung cancer (MPLC) with more than 10 nodules of both AIS and MIA subtypes and subsequently underwent precise lobectomy assisted by three-dimensional (3D) reconstruction. Additionally, we compared molecular characteristics between AIS and MIA subtypes in terms of the CT images, 3D locations, genomic profiles, and tumor microenvironments in order to provide potential clinical evidence for identifying the molecular mechanisms in early lung cancer.A 55-year-old man who was a mild smoker, and had no medical or family history, had his annual physical examination in January 2019 and was found with a small nodule (about 1cm) in the upper lobe of the left lung. Three months later, he was observed with multiple small ground-glass nodules (about 1cm) in the upper lobe of the left lung. After oral moxifloxacin anti-inflammatory treatment, the CT images of the patient did not improve. On the contrary, in July 2019, his CT images revealed multiple enlarged nodules in the upper lobe of the left lung and he was admitted to our hospital for further treatment . Except for KRAS in nodule 5 and A subsegment (nodule 11), there were almost no collective mutations among all nodules. By associating with 3D locations, we found that the genomic and pathological results of adjacent lymph nodes were quite different, implying MPLC instead of intrapulmonary metastatic lung cancer (IPM). To illustrate the intratumor heterogeneity, we used opposite sections of a wax block for nodule 1 and nodule 4 to perform WES and mIHC analysis. Mutations at those genes, including AXIN2, CDK2, TSPAN5, SUZ12, MAP2K1, LATS2, FAT4, FAM47E, and BRAF, were observed at nodule 1A, while mutation at those genes, including CDK2, AXIN2, and DOC2A, were observed at nodule 1B. Mutations at those genes, including RAET1E, CACNB2, MAP3K13, PLCG1, IL6ST, NKX2-2, DOC2A, COL9A2, were observed at nodule 4A while mutation at those genes, including CACNB2 and RAET1E, were observed at nodule 4B. Overall, only a few mutations were simultaneously found at opposite sections of the wax block in either nodule 1 or nodule 4 .In this current case, whole-exome sequencing (WES)  for the publication of any potentially identifiable images or data included in this article.TW: supervision. ZL and YX: conceptualization and writing - original draft preparation. CL, LZ, and RZ: methodology and writing - review and editing. YX, ZZ, and DW: formal analysis. CS: editing. All authors contributed to the article and approved the submitted version.


Colorectal cancer (CRC) is one of the most prevalent gastrointestinal malignancies and is considered the third major cause of mortality globally. Probiotics have been shown to protect against the CRC cascade in numerous studies.  The goal of this systematic review was to gather the preclinical studies that examined the impact of probiotics on the alteration of gut microbiota profiles  and their link to colorectal carcinogenesis as well as the potential processes involved.  The search was performed using Scopus, Web of Science, and PubMed databases. Five parameters were used to develop search filters: “probiotics,” “prebiotics,” “synbiotics,” “colorectal cancer,” and “animal model.”  Of the 399 full texts that were screened, 33 original articles met the inclusion criteria. According to the current findings, probiotics/synbiotics could significantly attenuate aberrant crypt foci (ACF) formation, restore beneficial bacteria in the microbiota population, increase short-chain fatty acids (SCFAs), and change inflammatory marker expression.  The present systematic review results indicate that probiotics could modulate the gut microbial composition and immune regulation to combat/inhibit CRC in preclinical models. However, where the evidence is more limited, it is critical to transfer preclinical research into clinical data. Firmicutes and Bacteroidetes, which account for approximately 90% of the microbial system [Fusobacterium nucleatum, Escherichia coli, Bacteroides fragilis, Peptostreptococcus stomatis, Parvimonas micra, and Campylobacter jejuni are enriched in CRC [Bifidobacterium breve, Lactobacillus rhamnosus, and Akkermansia muciniphila which are poor in CRC subjects. Furthermore, the metagenome sequencing and metabolomics combination demonstrated that the CRC-associated microbiome can be a source of harmful metabolites . For example, B. fragilis, F. nucleatum, and some Prevotellaceae family have been reported to produce succinate [Colorectal cancer, a multifactorial gastrointestinal malignancy, is one of the most critical public health issues and the third leading cause of cancer mortality worldwide . In reced in CRC , in contuccinate , 12. Sevuccinate –16. In auccinate , 17.Therefore, the goal of this study is to evaluate and collect high-quality preclinical studies through a systematic assessment to determine the safety and effectiveness of pro/synbiotics in CRC animal models. Indeed, we performed this systematic review on the studies that explicitly assessed the efficacy of probiotics on gut microbial  community composition in CRC animal models and the effectiveness of probiotic supplements on proinflammatory marker alteration and SCFAs production was evaluated.The guidelines defined by PRISMA were followed for this systematic review  .A systematic search was conducted to evaluate the efficacy of probiotics in the CRC animal models with a focus on microbiota bacterial population. Original research papers were searched in three electronic databases  up to December 22, 2021, by two researchers independently. Also, a search was undertaken for grey literature in Google Scholar. Besides, reference lists of all related systematic reviews and articles were monitored to eliminate any potential flaws in online databases and search engines. Initial searches were conducted using terms chosen from our research questions. The terms were “probiotics,” “prebiotics,” “synbiotics,” “colorectal cancer,” “colon cancer,” “CRC,” and also “mice,” “rat,” and “animal model” were searched to improve the results. The search strategy of each database was matched to its particularities. The reference lists of all articles included in this study were also checked for any relevant or ignored studies. After the initial search, two investigators screened the titles and abstracts and excluded articles that did not meet the inclusion criteria. If duplication of the same study was found, its data were included just once. And also, by manually searching Iranian medical laboratory websites, additional relevant papers were discovered and included in a standardized data extraction form. A third investigator double-checked the results to ensure that all eligible articles were considered. Eventually, an alert was set up based on our research keywords in all databases. The flow diagram of the article selection process is represented in Studies included in the study met the criteria of being original studies in the animal CRC models, published in English and concerning the administration of probiotics and synbiotics in animal models with CRC. Non-English papers and nonoriginal articles  were excluded. We had limited access to the Embase database, as well as studies that did not meet the eligibility criteria, i.e., (a) studies that used only prebiotics agents; (b) in vitro assays; and (c) clinical studies; and after excluding these items, specifically, studies evaluating microbiota populations were finalized. So, 33 studies that assessed bacterial microbiome compositions by three methods, including high-throughput sequencing, the PCR-denaturing gradient gel electrophoresis (DGGE) fingerprint method, and real-time PCR, were reviewed.Two reviewers independently coded and extracted the data from the 33 selected studies. Again, this process was supervised by a third researcher. The data extracted included the following: first author (year of publication), location, animals, number of animals (age range), probiotic types , interventions, cancer agent and study duration, number of ACF, bacterial profile in gut microbiota, inflammatory markers, SCFAs, and fecal enzymes.The risk of bias (RoB) in animal studies was assessed using the SYRCLE's  tool . This inThe search strategy submitted a total of 744 papers in the following databases: Scopus, Web of Science, PubMed, and Google Scholar. The process of study selection is shown in the PRISMA flowchart . In the L. acidophilus (4 studies) showed the highest number of combinations with other bacteria [The summarized characteristics of 33 studies are presented in bacteria , 37, 46.7 to 6.4 × 1011 colony forming units (CFU) alone and/or in combination with each other. Based on L. acidophilus, L. casei, and L. rhamnosus (18.2%) were the most common probiotics used by the included studies.A total of 19 different probiotic species were administered daily at doses of 1 × 10C. butyricum  [E. fecalis (1 study) [P. pentosaceus  [In these studies, some unique probiotics such as  trials) , 23, E. 1 study) , P. pent1 trial) , and Kef1 trial)  were use1 trial) , 45–52, 1 trial) , 48. The1 trial) , 49–51. 1 trial) , 24, 31.1 trial) , 38. In 1 trial) , 52. Fin1 trial) .L. salivarius [L. rhamnosus [L. casei, inulin, and synbiotic (L. casei + inulin) significantly decreased the number of ACF by three, five, and six times, respectively, compared to the DMH-treated group (p < 0.001). Besides, Cruz et al. [VSL#3) consumption reduced the incidence of total ACF by 38.1% compared to the DMH group (p=0.001). Interestingly, de Almeida Brasiel et al. [Five studies presented the results of the development of ACF after the animals were exposed to the carcinogen, and ACF was identified and analyzed by methylene blue staining in all studies , 49, 50.livarius  and L. rhamnosus  treatmenl et al.  reportedLactobacillus casei variety rhamnosus (Lcr35) supplementation did not affect intestinal crypt proliferative activity. Besides, Zhu et al. [Lactobacillus salivarius Ren suppressed the cell proliferation of colonic mucosa in cancerous rats.In six studies , 45, 48,u et al.  evaluateLcr35 at the highest dose did not significantly reduce goblet cell damage in the cancerous group [In four studies , 38, 49,The integrity of the gut mucosa and the intestinal epithelial barrier was examined by mucin-2(MUC2), adherence junction protein-1 (ZO-1), and tight junction (occludin) proteins in 4 studies , 29, 51.Hematoxylin and Eosin (H & E) staining results from 8 studies , 46, 52 Altogether, the major effects of various probiotics on the development of histopathological parameters in CRC models were collected in this study including (1) Probiotic application alone or with prebiotics limited the development and incidence of ACF as preneoplastic lesions. (2) Probiotics suppressed the proliferation of intestinal tumor cells and upregulated their apoptosis. (3) Probiotics alone or in combination with prebiotics could help to recover and prevent goblet cell loss caused by CRC. (4) Probiotic and synbiotic administration ameliorated intestinal gut barrier integrity by enhancing some related proteins in CRC cancerous models. (5) Probiotic alone or with prebiotics moderated inflammatory cell infiltration in CRC animals.In Bacteroidetes (10 studies), Firmicutes (9 studies), Proteobacteria (8 studies), Verrucomicrobia (5 studies), Actinobacteria (4 studies), and Deferribacteres (3 studies), respectively. The abundance of phylum Bacteroidetes decreased in 7 studies: 3 studies by synbiotics [Bacteroidetes increased in 3 studies [Firmicutes were assessed in 9 studies (out of the 33 studies) [Firmicutes declined significantly in treated CRC animals  [B. bifidum [Actinobacteria in 12% of studies (4 out of the 33 studies) [Deferribacteres in 9% of studies (3 out of 33 studies) [In 24% of studies (8 out of the 33 studies) that detected um level , 50, 51, studies , 47) and studies , 50) trestudies) , 47, 49.studies) , 47, Whi bifidum  and synbstudies) , 46, 51 studies) , 26, 33 VSL#3 + PBY) intervention in CRC animals. In another study by Cruz et al. [VSL#3 + Yacon as synbiotic agent. Surprisingly, C. butyricum had different effects on the abundance of the two families mentioned above. Liu et al. [C. butyricum-treated cancerous animals. On the other hand, in Chen et al.'s study [C. butyricum in CRC groups. Furthermore, Lactobacillaceae had significant expansion in 3 studies [P. pentosaceus [VSL#3 + Yacon [LGG [L. casei [LGG [L. helveticus [L. helveticus [VSL# [P. pentosaceus in CRC models [At the family level, Lachnospiraceae and Ruminococcaceae (in 5 studies) , 50, 51 u et al.  showed t's study , Ruminoc studies , 40, 50 con [LGG  in cancecon [LGG , 25, 33,sei [LGG  and a reus [VSL#  in CRC ius [VSL# , 25 and us [VSL# , 50 wereLactobacillus was the most predominant genus reported among included studies and improved notably by probiotic and synbiotic agents in CRC animal groups, and then Akkermansia as one of the main genera was discovered in 7 studies (7 out of 33 studies) [Akkermansia genus declined in L. fermentum [LGG [Prevotella as butyric acid-producing bacteria at the genus level had a remarkable increase in 3 studies in probiotics-treated CRC-models [Prevotella in mice with colitis and tumors were lowered by L. helveticus NS8 and Kefir as probiotic agents. And also, Turicibacteria was shown in 5 studies [Desulfovibrio which was reported in 5 trials [Helicobacter genus (in 4 out of 33 studies) had a contraction trend in probiotic and synbiotic-treated groups [Roseburia was reported in 4 studies with an increased trend by probiotics and synbiotics in CRC models [Lactobacillus, Akkermansia, Prevotella, Turicibacteria, and Roseburia.At the genus level, in 13 out of 33 studies –47, 50, studies) , 48, 49.ermentum  and LGG tum [LGG  in groupC-models , 30, 45.C-models  and de AC-models  reported studies , 42, 49. studies , 42, 49, studies , the abu5 trials , 28–30, d groups , 29, 50.Bacteroides spp. were identified in 5 studies [Bacteroides acidifaciens increased and Bacteroides uniformis decreased by L. helveticus NS8 in groups in which AOM/DSS was inoculated into animals as a carcinogen. In addition, Lactobacillus spp. increased significantly in 5 studies [Bifidobacterium had an increment trend in Lactobacillus alone or mixed with other probiotics in cancerous models [Different  studies , 33, 37. studies , Bactero studies , 45, 52 s models , 37.Lactobacillus alone or in mixtures with Bifidobacterium [P < 0.001) in the C. butyricum-treated cancerous mice were significantly higher than in cancerous groups. Moreover, two studies by Cruz et al. [Based on acterium , 42, 44,acterium  reportedz et al. , 51 reveC. butyricum treatment. The levels of the fecal secondary BAs DCA and LCA were markedly increased in the HFD-treated mice compared with the control . Surprisingly, the DCA and LCA levels in the C. butyricum group decreased significantly .Besides, only in one study , the levβ-glucuronidase, azoreductase, and β-glucosidase have been implicated in converting procarcinogens into carcinogens; thus, the activity of these enzymes was assessed to deduce the modulating potential of probiotics in the colonic environment. Only three studies [β-glucosidase and β-glucuronidase were not significantly affected by LS treatment. Moreover, CRC animals receiving the synbiotic displayed a significant reduction in β-glucuronidase enzyme activity when compared to the control group. In addition, in both studies, SCFAs production was increased notably by probiotics and synbiotics.Fecal enzymes, i.e.,  studies , 41, 50  studies , 50. Als studies  revealedα levels in cancer models that received probiotics, and in 57% of the included studies (8 out of 14 studies) [α declined significantly in probiotic-treated groups. Besides, among the 20 preclinical trials, 13 trials (65% of studies) [ɣ) was investigated in 9 out of 20 studies (45% of studies) [β levels was detected in 6 out of 20 studies (30% of included studies) [α [The effects of probiotics on inflammatory marker expression were assessed in 20 studies , 50, 52 studies) , 50, 52,studies) , 50, 52 studies) , 43, 52,studies) , 48, 50,studies) , 48, 50.studies) , 48, 50 studies) , 42, 45 studies) , 31, 42 studies) , 40, 43,studies) , 40, 43,dies) [α , IL-13 [dies) [α , IL-18 [dies) [α , IL-21 [dies) [α , was repκB, Cox-2, Bcl-2, and β-catenin expressions were elevated in cancerous groups but depleted in probiotic and synbiotic-treated CRC groups. In two studies, da Silva Daurte et al. [The effects of probiotics/synbiotics on the regulation of apoptotic markers were measured in 11 studies , 45, 49 e et al.  and Gamae et al.  found the et al. , 23, 38,The SYRCLE's tool  was usedCRC is the third most prevalent cancer in both men and women, and most of its environmental etiological factors, such as changes in dietary habits and lifestyles, can be regulated therapeutically to minimize the risk of disease development. Regular intake of probiotics would present a more effective anticarcinogenic effect in the early stages of CRC . In the β-glucuronidase, nitroreductase, and β-glucosidase enzymes, resulting in lower ACF counts [γ as an antiproliferative and antiangiogenic protein by some probiotics and its noticeable apoptotic activity, which may diminish ACF [ACF is recognized as a CRC precancerous lesion, and significant numbers of ACF are detected in full-blown CRC models. Increased ACF numbers are associated with a higher risk of CRC . Based oF counts , 57. (c)F counts , 59. (d)F counts . (e) Hignish ACF . It is wLactobacillus and Bifidobacterium were the most widely investigated probiotic bacteria, followed by C. butyricum (2 studies) and P. pentosaceus (1 study). These reviewed studies have reported that probiotic intervention (single/mix) or along with prebiotics (synbiotic) markedly improved the abundance of microbiome-friendly bacteria such as Lactobacillus, Bifidobacterium, Akkermansia, Romboutsia, and Roseburia in the preclinical CRC context. Besides, based on ten studies [Turicibacter has been linked directly to the production of butyric acid, which acts as an immunomodulator with anti-inflammatory activity in Cruz et al. and da Silva Duarte et al.'s studies [Roseburia as a butyrate and short-chain fatty acid-producing bacteria, along with high butyric acid levels in 3 studies [Clostridium XI and Clostridium XVII (as important butyrate producers) [Prevotella and Alloprevotella from the Prevotellaceae family with higher butyric acid levels [Ruminococcaceae and Eubacterium, which are well known to produce SCFAs, were elevated together with total SCFAs in a study by Chen et al. [Lactobacillus, Bifdobacterium, Akkermansia, and Faecalibaculum that is produced simultaneously by L. coryniformis MXJ32 with high levels of SCFAs [_NK4A136_group, Faecalibaculum, Roseburia, and lactobacilli in 2 studies [Turicibacter, surprisingly, showed a negative correlation with butyrate in Wang et al.'s study [Akkermensia genus, as a type of Gram-negative bacteria and a promising probiotic candidate, declined in two studies [In this systematic review, we have focused on the association between probiotic/synbiotic supplementation and the regulation of gut microbiota bacterial profiles in CRC animal models.  studies , 50, 51  studies , 50. (2) studies , 28, 50.oducers)  after producers) , 50. (4)oducers) , Wang etd levels , 34. (6)n et al. . (7) Enrof SCFAs . Besides studies . Indeed, studies , regulat studies , preserv studies . Butyrat studies . However studies , 51 in r studies , 39 in rβ-glucuronidase, β-glucosidase, and azoreductase bacterial enzymes are associated with the conversion of procarcinogens to potential carcinogens in the colon by the release of cytotoxic and genotoxic metabolites [Bifidobacterium and Lactobacillus displayed minimal β-glucuronidase activity for modifying the microbiota [β-glucuronidase activity decreased in response to probiotic and synbiotic supplementation. While Zhu et al. [β-glucosidase and β-glucuronidase showed no alterations in L. salivarius-treated cancerous animals, still azoreductase activity was reduced in this group, indicating a possible protective effect against carcinogenesis by decreasing levels of carcinogen activation and DNA mutation.Furthermore, abolites , 64. Modcrobiota . In addicrobiota , 50 in ou et al.  identifiAkkermansia and Turicibacter while decreasing the expression of phosphorylated GSK3b and β-catenin [Protective effects of pro/synbiotic against colon cancer through increasing the phosphorylated JNK-1 expression as well as boosting beneficial bacteria in the colon such as -catenin .Probiotics might be able to suppress inflammation and improve mucositis in the intestine by inhibiting NF-B activity , and TNF- and IL-6 proinflammatory effects could be reduced by taking probiotics .+ macrophages (limiting macrophages recruitment) and then a significant reduction in proinflammatory markers such as IL-1α, IL-1β, and IL-6 by probiotics [Reducing pathogenic bacteria and infiltration of CD68obiotics .A marked increase in IL-2 and IL-4 by probiotic and synbiotic in CRC models which results in the regulation of immune cells and antitumor defense , 66.γ protein secretion by LGG (probiotic) in CRC animals is consistent with the considerable apoptotic activity, immune-regulatory function, antiproliferative, and anticancer strategies of IFN-γ [The increased IFN-of IFN-γ .Probiotics induce apoptosis in CRC tumors by significantly decreasing Bcl-2 levels while increasing Bax, caspase-3, and p53 expression. In addition, probiotics block Cox-2, which activates the downstream target proapoptotic protein p53, which links to tumor suppression , 67, 68.Bacteroides that releases metalloprotease and fragilysin toxins which help CRC promotion through boosting IL-17a production and inducing E-cadherin cleavage [Inhibitory effect of probiotics on cleavage , 69.Lactobacillus reuteri in a model of CRC by a histidine decarboxylase (HDC), which downregulated IL-22 expression levels that were enhanced in tumor tissues [Beneficial effects of a strain of  tissues , 70.κB in the CRC models by probiotics and inhibiting inflammation by preventing the release of certain reinflammatory cytokines  and CXCR2 ligand chemokines  [Attenuating the over-activation of TLR4/NF-nd CCL7) .Lactobacillus by downregulating proinflammatory markers such as IL-6, IL-17 F, and IL-22 [Great probiotic potential and anti-inflammatory effect of nd IL-22 , 71, 72.Based on 33 reviewed papers, it was demonstrated that many immune pathways inhibit carcinogens by probiotics in animal CRC models that these potential and diverse pathways briefly include the following:VSL#3 probiotic was the lack of an inhibitory effect on tumorigenesis and the tendency to enhance tumor invasion in AOM/Il10−/− mice. They have previously shown that inflammation affected the composition of the intestinal microbiota in Il10−/− mice, leading to an increase in Proteobacteria, which influenced CRC formation. In the current study [VSL#3 administration after the initiation of inflammation and dysbiosis can boost tumorigenesis and primarily induce the elimination of beneficial bacteria such as Clostridium.It is worth mentioning that none of the concluded studies reported a reverse or negative effect of probiotics against CRC in animal models except the Arthur et al.'s study . In thisnt study , they deβ, IL-6, and TNF-α). A study by Gao et al. found Roy's Lactobacillus can suppress the incidence of inflammation-related CRC in mice by secreting histamine, and this suppresses tumor growth by producing histamine [ß-catenin pathway. (vii) By modulating the activity of fecal bacterial enzymes such as β-glucuronidase, β-glucosidase, and nitroreductase, probiotics can significantly change the metabolism structure of detrimental bacteria in the gut microbiota. (viii) Probiotics increase mucin production and tight junction protein expression to improve gut-barrier function.Together, these 33 studies have revealed that the anti-CRC effects of probiotics arise through alteration of the composition of the microbiota by various mechanisms, including (i) probiotics promote microbiota hemostasis by competing with putrefactive and harmful bacteria, reducing their abundance while increasing the number of LAB bacteria. (ii) In spite of their strong adhesion to the intestinal epithelium, probiotics are noninvasive and inhibit pathogen adhesion to the intestine , 73. (iiistamine . (vi) PrLactobacillus alone or in a mixture with other probiotics with strong positive effects on modulation of gut microbiota composition by enriching beneficial bacteria, especially Lactobacillus, Akkermansia, Prevotella, Turicibacteria, Roseburia, and other markers. (2) the dose of which probiotics administration is the most effective? The analysis of probiotic dosage revealed significant differences among the 33 studies. A total of 19 different probiotic species were administered daily orally at doses of 1 × 107 (in mice) to 6.4 × 1011 (in rats) CFU in two best-recommended averages ≥109 and ≥108 CFU, respectively, displaying the best achievement such as improving the abundance of gut microbiota-friendly bacteria. (3) How many weeks was the best time to study? The duration of studies ranged between 4 and 24 weeks in three categories, as follows: (1) study period from 4–10 weeks, (2) 11–20 weeks, and (3) study period more than 20 weeks. Despite the broad study period in 33 studies, the positive effects of probiotic treatment on increasing beneficial bacteria and reducing cancer damage were demonstrated in all study periods, including the lowest and highest study periods. (4) cancer modeling agent with which optimal dosing is best? Since the optimal method and gold standard for tumor induction in CRC animal models have not yet been established, two major classes of cancer agents were discussed in these 33 studies, respectively: (1) AOM with a concentration of 8–12 mg/kg in a period between five days and six weeks, (2) 30 mg/kg and 40 mg/kg DMH used in rats in the induction period (2–10 weeks), as well as 10 mg/kg and 20 mg/kg in mice DMH (6–20 weeks). Since not all probiotics strains exhibit anti-CRC activities, screening the potent strain for the development of a probiotic-based therapeutic agent to control or prevent the incidence of CRC is crucial, and as regards, 85% of these 33 original studies used Lactobacillus species alone or mixed with other probiotics. With superior effects on CRC attenuation and successful modulation of the bacterial population in the microbiota, the Lactobacillus genus can be suggested for comprehensive clinical studies in the treatment of colon cancer and promoting the reproduction of beneficial bacteria in the gut microbiota.Taken together, the remarkable strength of this systematic review is that we have presented all the latest preclinical papers that investigated the potential beneficial effects of probiotics in addressing colorectal cancer animal models by focusing on microbiome bacterial populations. In addition, histopathological changes, signaling pathways, inflammatory markers alteration, short-chain fatty acids, and fecal enzyme release were gathered systematically in these studies. Nevertheless, there are some limitations in our study because of the different methodological designs and protocols of the included studies such as the use of diverse ranges of probiotic strains, prebiotics products, and cancer induction agents, as well as the administration of lots of dosages with different duration of treatment and different pathways for the preparation of probiotic supplements in these studies, which prevented us from drawing absolute conclusions and pooling the included studies in a meta-analysis. Despite this heterogeneity, we tried to collect some homogeneous data based on 33 studies; these include (1) the bacteria which are the most beneficial? Based on Since the precise mechanisms of probiotics for ameliorating human CRC as a multifactorial cancer, and circumstances of rapid shifts of the bacterial microbiota compositions from health to disease are still poorly understood, standard protocols must be applied in preclinical settings in order to ensure reproducibility and generalization of probiotics function results to clinical studies and develop treatment pathways based on a balance in fecal microbial structure. On the other hand, since animal metabolisms differ markedly from those of humans, animal models may not always reflect what occurs in humans. It is suggested to conduct additional studies, particularly long-term, randomized double-blind, placebo-controlled clinical trials to clarify and confirm preclinical findings in dealing with probiotics prior to advising their routine use as an adjunctive therapy for CRC prevention and treatment. Finally, the combination of CRC cell line studies, animal models, and clinical trials will help researchers develop a comprehensive picture of probiotic therapeutic pathways for guiding health care policies in the global fight against CRC.


All our cases of this variant also harbored other potentially actionable mutations in other genes, which could also be amenable to novel targeted therapy. Patients with this variant may benefit from additional molecular screening to identify potential therapeutic targets that could improve the clinical outcome of such patients. Urothelial carcinoma of the large, nested variant is a specific histological morphology subtype of urothelial carcinoma. Although it is a rare variant, it requires specific attention due to its bland histology and the fact that it may potentially be missed in routine biopsies. In this study, we identify Fibroblast Growth Factor Receptor-3 (FGFR-3) activation and missense mutation in 38 cases, including 6 cases diagnosed with LNVUC and 32 with metastatic invasive urothelial carcinoma (UC). Initially, six formalin-fixed paraffin-embedded (FFPE) tissue samples of the LNVUC were subjected to whole-exome sequencing (WES), and then we performed targeted sequencing on 32 cases of metastatic invasive UC of various morphological subtypes, which were interrogated for the FGFR3. Our results revealed 3/6 (50%) LNVUC cases evaluated by WES in our study showed an activating mutation in FGFR-3, 33% showed an activating mutation in PIK3CA, and 17% showed activating mutation in GNAS or MRE11. Additionally, 33% of cases showed a truncating mutation in CDKN1B. All LNVUC in our study that harbored the FGFR-3 mutation showed additional activating or truncating mutations in other genes. Overall, 6/32 (18.75%) cases of random metastatic invasive UC showed missense mutations of the FGFR-3 gene. The LNVUC variant showed the higher incidence of FGFR-3 mutations compared to other types of mutations. Additionally, all LNVUC cases show additional activating or truncating mutations in other genes, thus being amenable to novel targeted therapy. The large, nested variant of urothelial carcinoma (LNVUC) is characterized by bland histomorphology mimicking that of benign von Brunn nests. In the current study, we aimed to investigate the  Urothelial carcinoma is the most common pathological  type of bladder cancer . ApproxiThe nested variant of urothelial carcinoma is a very rare subtype that is characterized by bland nests of neoplastic urothelial cells that are often seen invading muscularis propria . The larFibroblast growth factor receptor-3 (FGFR-3) is one of the four highly conserved fibroblast growth factor receptor tyrosine kinases (RTKs) and has been shown to have a role in tumor growth and survival regulations [FGFR-3 has been implicated in multiple malignant neoplasms including multiple myeloma, urothelial and cervical carcinoma [FGFR-3 as therapeutic and prognostic marker in urothelial carcinoma [FGFR mutations is highlighted with the development of the pan-FGFR inhibitor, Erdafitinib, and Pembrolizumab, which was approved by the Food and Drug Administration [FGFR-3 mutations [ulations ,10,11. Farcinoma ,13,14. Darcinoma . The clistration ,17. Thisutations .FGFR-3 mutation to be among the most common mutated oncogene in urothelial carcinoma overall [FGFR-3 mutations have been reported in about 75% of noninvasive urothelial carcinoma and about 15–20% of high-grade tumors [Studies have shown  overall . FGFR-3 e tumors ,22,23, bThis study is designed to characterize the molecular backgrounds of the large, nested variant of urothelial carcinoma, in the hope of identifying potential targetable mutations linking this specific histomorphology with specific genetic mutations.FGFR-3 target seq.In the current study, we recruited 38 metastatic UC samples of which 6 cases were diagnosed with LNVUC at Alberta Precision labs/University of Calgary Cumming School of Medicine between 2015 and 2019. Of these 6 cases, 2 belonged to the same patient (cases 2 and 3), who developed a primary LNVUC in the lower urothelial system (bladder) which was treated with partial cystectomy, and subsequently, after this diagnosis, he developed another primary LNVUC in the upper urothelial system (ureter) which was treated with bilateral nephroureterectomy. These cases were re-reviewed by an experienced genitourinary pathologist to confirm the diagnosis of this variant and select the most suitable areas for sequencing. The remaining 32 cases of metastatic invasive urothelial carcinoma, including 3 cases of the LNVUC, were also analyzed for All cases were classified based on the 2016 edition of the World Health Organization Classification of Tumors of the Urinary System. LNVUC cases demographic data, stage at the time of diagnosis, treatments and outcomes up to date were documented for each case , and furPathologically reviewed samples confirmed the diagnosis and determined tumor content, and marked tumor areas on hematoxylin and eosin (H&E) slides used to accurately obtain tissue samples from formalin-fixed paraffin-embedded (FFPE) samples. Briefly, the study pathologists cored or scrolled the selected tumor areas. Then tumor DNA was extracted using the QIAamp DNA FFPE Tissue kit . Germline DNA was extracted from normal kidney tissue adjacent to the tumor. Qubit used to quantify the DNA using Qubit DNA HS assay . Whole-exome sequencing (WES) was carried out by The Centre for Applied Genomics (TCAG), Toronto, ON, Canada. Briefly, 750 ng of DNA was used for WES exome library preparation and sequencing using SureSelect XT Human All Exon V5 Kit . Genomic DNA was fragmented to 200-bp on average using a Covaris LE220 instrument. Prior to ligation, sheared DNA was end-repaired, and the 3′ ends were adenylated on adapters with overhang-T. Then, the genomic library was amplified by PCR using 10 cycles and hybridized with biotinylated probes that target exonic regions; the enriched exome libraries were amplified by an additional 8 cycles of PCR. The exomic libraries were validated using DNA High-Sensitivity chips on a Bioanalyzer 2100 (Agilent Technologies) for size and by qPCR using the Kapa Library Quantification Illumina/ABI Prism Kit protocol (KAPA Biosystems) for quantities. Exome libraries were pooled and sequenced with the TruSeq SBS sequencing chemistry using a V4 high throughput flowcell on a HiSeq 2500 platform , as per Illumina’s recommended protocol.Around 6–8 gigabases of raw paired end data of 126-bases were generated per exome library. Reads were aligned to the hg19 build human reference genome using BWA (version 0.5.9). PCR duplicates were marked using picard-tools-1.108, and local re-alignment and base recalibration were performed using GATK 1.1-28. Variants  were called using GATK UnifiedGenotyper 1.1-28. An Annovar-based pipeline was used for adding gene-based, feature-based and frequency-based annotations for variant filtering and prioritization .FGFR Fusion proteins: Driver genes partner genes FGFR1: ADAM32, BAG4, BCR, CNTRL, CPSF6, CUX1, ERVK3-1, FGFR1OP, FGFR1OP2, FN1, LRRFIP1, MYO18A, NTM, PLAG1, RANBP2, SQSTM1, TACC1, TPR, TRIM24, WHSC1L1, ZMYM2, FGFR2ZNF703 : AFF3, AHCYL1, BICC1, CASP7, CCAR2, CCDC6, CD44, CIT, COL14A1, CREB5, CTNNB1, FAM76A, KCTD1, MGEA5, NOL4, OFD1, PARK2, PDHX, PPHLN1, SHTN1, SLC45A3, SNX19, TACC3, TXLNA, FGFR3USP10 : AES, BAIAP2L1, ELAVL3, ETV6, FBXO28, JAKMIP1, TACC3.Targeted seq testing was performed at Cancer Genetics Clinic, Jewish General Hospital, using an NGS panel which analyzes both DNA and RNA extracted from FFPE material and detects sequence changes involving the following FGFR-3. Two cases showed an activating mutation in PIK3CA (33%), and one showed activating mutations in GNAS (17%), and another case showed mutations in MRE11 (17%). Whole-exome sequencing results  for the CDKN2A, ARID1B, ARID1A and KDM6A was seen only in one case each (17%). It was interesting to note that cases that showed activating mutations in FGFR-3 also showed additional activating and truncating mutations in other genes including PIK3CA. Detailed genetic mutations detected in the six cases studied are presented in A truncating mutation in CDKN1B was seen in two cases (33%), and truncating mutations in FGFR-3 mutations showed additional activating or truncating mutations. One case showed simultaneous mutations in FGFR-3, PIK3CA, CDKN1B, ARID1B and PPP2R1A. Another case showed mutations in FGFR-3 as well as MRE11 and KDM66, and an additional case had simultaneous mutations in FGFR-3, CDKN1B and CDKN2A. Of the cases that did not harbor any FGFR-3 mutations, one case showed simultaneous mutations in PIK3CA, ARID1A and GNAS  were positive for FGFR-3 missense mutations . Three out of six cases exhibited dedifferentiated histology (poorly differentiated or squamous/sarcomatoid differentiation), five of six were from metastatic sites, and one was from the upper urothelial tract. However, no significant association was noted to specific histopathological morphology or metastatic site.As exhibited in FGFR-3 mutation is among the most common mutated oncogene in urothelial carcinoma, and it is even more common in the LNVUC. In our small study, we report 50% of cases as having a positive activating mutation in FGFR-3.Our results indicates that the Additionally, we observed that many cases of LNVUC harbor simultaneous multiple activating mutations. In our series, four out of six (66%) LNVUC cases showed multiple activating mutations in oncogenes and truncating mutations in tumor suppressors, simultaneously. Of note, one patient who had two separate primaries of bladder and renal pelvis LNVUC demonstrated different mutational landscape between the two tumors, thus supporting different mutational landscape even in same-patient tumors, based on location.FGFR-3 sequencing of 32 cases of metastatic invasive urothelial carcinoma, 16% of cases showed FGFR-3 missense mutations. None of the cases that did not harbor FGFR-3 mutations by whole-exome sequencing showed FGFR-3 mutations, raising the possibility that point mutations in FGFR-3 gene are likely more frequent in LNVUC. Based on the targeted FGFR-3 analysis and comparison table analysis of two studies, including a study by Pietzak et al. [To illustrate our results in relation to published reports using genomic sequencing data, we added a review table for k et al.  and the FGFR-3 mutation, whereas compared to data provided by TCGA, the incident of FGFR-3 mutations in our variant was significantly higher compared to unselected variants of urothelial carcinoma. The incidence of CDKN1B, GNAS, MRE11 and PPP2R1A mutations was also significantly higher in our cases compared to both studies. As demonstrated in our data, in non-muscle-invasive urothelial carcinoma, our results showed a slight but non-significantly higher incidence of FGFR-3 mutations, ranging from 11% to 52% suggesting that the rate of FGFR-3 mutations may vary significantly depending on the methods used, site of assessment and variants of urothelial carcinoma included in the studies [Similarly, other studies characterizing non-muscle-invasive and muscle-invasive high-grade urothelial carcinoma reported similar incidence of  studies ,27,28,29FGFR-3 mutation, whereas only 13% of the mixed tumor variant harbored this mutation. Their finding raises the possibility of different neoplastic pathways for mixed and pure LNVUC in their study.As in our study, Weyerer et al.  focused It is also important to note that LNVUC and advanced UC shows a response to pembrolizumab especially with the recurrent LNVUC; however, our study does not focus on therapeutic strategies but the incidence associated with the presence of FGFR3 mutations ,18.FGFR-3 mutations in urothelial carcinoma, and they support that the LNVUC variant may exhibit an even higher incidence of FGFR-3 mutations especially in more pure histological type. Additionally, the rate of FGFR-3 mutations varies depending on the samples analyzed, whether it is resection or TURBT as well as histological grade, patient’s demographics, and underlying risk factors such as history of smoking. Finally, the studies document that FGFR-3 mutations usually occur in association with other activating or truncating mutations, especially in the PI3CKA pathway, and that LUCNV may harbor simultaneous activating and truncating mutations making them amenable for targeted therapy. Overall, all of these studies document a high incidence of FGFR-3 in the diagnosis and treatment of the large, nested variant of urothelial carcinoma, possibly implicating other targetable pathways compared to random unselected variants of urothelial carcinoma. We do acknowledge the limitations of our study, including small sample size and the fact that most cases were those of TURB. To better evaluate the role of these mutations in this rare variant of urothelial carcinoma, more studies, with a larger number of cases, focused on histomorphology, grade, stage as well as patient demographics and prognosis should be designed. Our study provides further evidence of the promising role for


Dear Editor,+ uptake, release, and distribution, playing important roles in plant growth and development2. Among these channels, the Stelar K+ outward rectifier channel (SKOR) functions as a K+ efflux channel for long-distance distribution of K+ from roots to the upper parts of the plant4. There are only two structures of voltage-gated K+ channels, KAT1 and AKT1 from Arabidopsis, reported to date7. Notably, KAT1/AKT1 and SKOR represent two different types of K+ channels that share an extensively similar structural background but display profoundly different biophysical characteristics, belonging to Kin and Kout channels, respectively2. Interestingly, SKOR contains an intracellular ankyrin repeat (ANK) domain, which is absent in all the depolarization-activated K+ channels with known structure. Here, we reported the cryo-EM structure of SKOR, and investigated the role of the ANK domain in channel structure and function modulation.In plant, the family of voltage-gated potassium channel is crucial for Kwt1 and SKORwt2) with global resolutions of 3.1 Å and 3.5 Å were obtained 164 Fig. . This inwt1 and SKORwt2 reveals subtle differences  and CBL-interacting protein kinase (CIPK) was reported to phosphorylate the ANKs of GORK, an outward potassium channel in Arabidopsis, to activate K+ transport14. However, when CIPK alone or in pairs with CBL were co-expressed with SKOR, no direct influence on SKOR current was found , which was reported to convert outwardly rectifying SKOR into an inward rectifier15, at a resolution of 3.1 Å (Supplementary Figs. wt1 and SKORmut showed that the pore domains of these two structures are almost identical (Supplementary Fig. mut is also in a closed state. Notably, our electrophysiological data demonstrated that SKORmut is a non-functional channel (Supplementary Fig. Besides the wild-type SKOR, we also determined the structure of an SKOR mutant L271P-D312N (SKOR+ channels with an ANK domain. The tetrameric assembly of SKOR around a central pore, the TMD, C-linker, CNBD, and ANK domain, as well as their interactions, are well characterized. Unlike the canonical potassium channel, SKOR does not adopt a C4 but a C2 symmetry, which occurs in the C-linker, CNBD and ANK domain. The reduction in symmetry suggests a unique regulatory mechanism for SKOR activation. Together, this study paves the way for further investigations of the functional properties and physiological roles of SKOR and other depolarization-activated K+ channels with an ANK domain.In summary, the cryo-EM structures of SKOR provide valuable insights into the architecture of depolarization-activated KSupplementary Information