Both systemic SHP2 inhibition and tumor cell–autonomous SHP2 depletion reduced tumor-infiltrated CD4+ T cells and M2-polarized tumor-associated macrophages. Analysis of TCGA datasets revealed that phosphorylation of SHP2 is important for immune-cell infiltration, T-cell activation and antigen presentation. To investigate this mechanistically, we conducted in vitro T-cell killing assays, which demonstrated that pretreatment of tumor cells with FGF2 and PDGF reduced the cytotoxicity of CD8+ T cells in a SHP2-dependent manner. Both growth factor receptor signaling and three-dimensional culture conditions transcriptionally induced PD-L1 via SHP2. Finally, SHP2 inhibition reduced MAPK signaling and enhanced STAT1 signaling, preventing growth factor–mediated suppression of MHC class I. Overall, our findings support the conclusion that tumor cell–autonomous SHP2 is a key signaling node utilized by MBC cells to engage immune-suppressive mechanisms in response to diverse signaling inputs from TME.SH2 containing protein tyrosine phosphatase-2 (SHP2) is recognized as a druggable oncogenic phosphatase that is expressed in both tumor cells and immune cells. How tumor cell–autonomous SHP2 contributes to an immunosuppressive tumor microenvironment (TME) and therapeutic failure of immune checkpoint blockades in metastatic breast cancer (MBC) is not fully understood. Herein, we utilized systemic SHP2 inhibition and inducible genetic depletion of SHP2 to investigate immune reprogramming during SHP2 targeting. Pharmacologic inhibition of SHP2 sensitized MBC cells growing in the lung to α-programmed death ligand 1 (α-PD-L1) antibody treatment via relieving T-cell exhaustion induced by checkpoint blockade. Tumor cell–specific depletion of SHP2 similarly reduced pulmonary metastasis and also relieved exhaustion markers on CD8Findings present inhibition of SHP2 as a therapeutic option to limit breast cancer metastasis by promoting antitumor immunity. Metastatic breast cancer (MBC) is the most advanced stage of breast cancer (stage IV) with lower 5-year survival rates and higher treatment costs than localized disease . Cases o+ cytotoxic T cells and M2-polarized tumor-associated macrophages (The tumor microenvironment (TME) is composed of numerous immune cell populations such as CD8rophages . These crophages . This imin vivo (SH2 containing protein tyrosine phosphatase-2 (SHP2) is a promising candidate for a multifunctional therapeutic target as it is a druggable oncogenic phosphatase expressed in both tumor cells and immune cells . In tumoin vivo . Before in vivo . Hence, in vivo . Systemiin vivo .Herein we sought to address the hypothesis that tumor cell–autonomous SHP2 contributes to an immune suppressive TME through its regulation of receptor tyrosine kinase (RTK) and extracellular matrix (ECM) signaling. Using a doxycycline-inducible approach, we demonstrate that MBC-cell specific depletion of SHP2 reduces pulmonary metastasis. Mechanistically, inhibition of SHP2 in MBC cells biases upstream signaling toward STAT1 signaling, leading to enhanced expression of MHC class I. Overall, our studies further expand the notion of SHP2 inhibition as a promising strategy to combine with ICB to treat MBC.Mycoplasma contamination by PCR.The growth conditions of the cell lines in this study are described in in vivo studies were performed in 4-to 6-week old, female BALB/cJ mice purchased from Jackson Laboratories. For the combination study in D2.A1 model, 1  ×  106 cells were injected via the lateral tail vein. The SHP099 was administered via oral gavage, and the α-PD-L1 antibodies were administered via intraperitoneal injection at the indicated concentrations and frequencies. The mice were sacrificed at the end of study, and the tumor-bearing lungs were fixed by 10% formaldehyde overnight. Paraffin tissue sectioning and hematoxylin and eosin (H&E) staining were executed by AML Laboratories, Inc. In the 4T1 spontaneous metastasis model, the 4T1 cells bearing doxycycline-inducible depletion of SHP2 were constructed, sorted and verified as previously described . No randomization or blinding was done.All escribed . Then, 5Tumor bearing lungs were harvested, imaged, weighed, and dissociated with Mouse Tumor Dissociation Kit (Miltenyi Biotec) and GentleMACS Dissociator (Miltenyi Biotec) immediately after sacrificing the mice. The spleens were harvested, weighted, and mechanically disrupted by grinding. The cell suspension was filtered through 70-μm sterile cell strainers and treated with ACK buffer to lyse red blood cells. The single-cell suspension was incubated with TruStain FcX (BioLegend) at 1:50 and Zombie violet (BioLegend) at 1:100. The single-cell suspension from pulmonary tumors was separated into two tubes and subsequently stained with panels of lymphoid antibodies and panels of myeloid antibodies at 1:200 per antibody, respectively. The single-cell suspension from the spleens was subsequently stained with panels of lymphoid antibodies only. Considering the influence of GFP induction with doxycycline induction, the antibody panels were different for the two models. The antibodies for the D2.A1 model and 4T1 model were listed in http://firebrowse.org/) hosted by Broad Institute by selecting the cohort as “Breast Invasive Carcinoma (BRCA)” on the left panel, and clicking “Reverse Phase Protein Array”, “mRNA” and “Clinical” bars on the right panel. The primary files “RPPA_AnnotateWithGene (MD5)” for RPPA data, “mRNA_Preprocess_Median (MD5)” for mRNA data and “Merge_Clinical (MD5)” for clinical outcomes were downloaded as txt file, and stored locally as raw files named “RPPA_raw.csv”, “mRNA_raw.csv” and “Clinical_raw.csv”.Reverse-phase protein array (RPPA) dataset, mRNA dataset, and clinical outcomes dataset of patients with breast cancer in TCGA were achieved from Firebrowse (https://bioinformatics.mdanderson.org/estimate/disease.html) by selecting the “Disease Type” as “Breast Cancer” and the “Platform Type” as “RNA-Seq-v2”. The immune scores and stroma scores here were calculated by ESTIMATE  at backend of the tool , M1 macrophages and M2 macrophages were estimated with R package Immundeconv .These four locally stored files were the inputs of the downstream analyses described in the undeconv . The CD88 T cell . The gen8 T cell . The oth5 4T07 cells were engrafted into the mammary fat pads of BALB/cJ mice via an intraductal injection (+ cells were isolated from the splenocytes using EasySep Mouse CD8+ T Cell Isolation Kit (STEMCELL Technologies Inc.) following the manufacturer's instructions. The D2.A1 cells were pretreated with growth factors and inhibitors listed in + cells. The ratio of tumor cells to T cells was 1:10. The coculture system was stained with Incucyte Cytotox Dye for Counting Dead Cells (Essen BioScience), and monitored using the Incucyte S3 (Essen BioScience).To induce antitumor immunity, 1  ×  10njection . The enlThe tumor cells were treated with growth factors and inhibitors listed in Immunoblotting was performed as previously described . BrieflyThe process was performed as previously described . Brieflyt test was used for comparing differences between two groups of measurements in analyses of immune composition and in vitro assays. A Mann–Whitney U test was used for comparing differences between two patient groups with differential CD4+ T-cell, Tregs, M1 macrophages, and M2 macrophages’ infiltration; while a Student t test was used for the comparison in analyses of other clinical datasets. Group measurements of in vivo assays were compared with a Mann–Whitney nonparametric test. Error bars show the SEM. No exclusion criteria were used in these studies. All statistical tests were appropriate in which the groups are assumed with similar variance.A Student http://firebrowse.org/ and ESTIMATE online tool at https://bioinformatics.mdanderson.org/estimate/disease.html.The data analyzed in this study were obtained from Firebrowse at + lymphocytes into pulmonary tumors (We previously reported that depletion of fibroblast growth factor receptor 1 (FGFR1) increases infiltration of CD8y tumors . SHP2 isy tumors . As detey tumors . We did y tumors . These d+ cells within the CD45+ splenic population significantly decreased upon combination of SHP099 and α-PD-L1  was significantly reduced with SHP099 and the combination therapy as compared with the control (+ in F4/80+CD11b+CD45+ cells) was significantly reduced by α-PD-L1 antibody, which was rescued by SHP099; while the percentage of M2-polarized macrophages (CD206+ in F4/80+CD11b+CD45+ cells) was significantly reduced by SHP099 and combination therapy  . The perf SHP099 . The per control . The per therapy . Hence,  therapy . These d therapy .ex vivo bioluminescent imaging of the lungs upon necropsy  and PD-1 (CD247), in patients with higher phosphorylation levels of SHP2  is the key to improved therapeutic outcomes.Our current study did not elucidate a combinatorial effect in terms of tumor growth between SHP099 and α-PD-L1, which might require further dosage optimization and timing, but we did observe the combination group achieved faster regression in pulmonary tumor burden, which could be a benefit from combination therapy . This ad+ T cells in pulmonary tumors, but no increase in CD8+ infiltration was observed. In contrast, combination of SHP099 and α-PD-L1 led to a dramatic increase in splenic CD8+ T cells. Overall, these data suggest that increasing cytotoxic T-cell infiltration into metastatic tumors remains a challenge for immune therapy that is not overcome by SHP2 targeting.Besides T-cell exhaustion, the amount of T-cell infiltration and composition of TAMs within tumors are critical factors for the response to ICB . We obse+ and induction of CD8+ T cells were observed in both pulmonary tumors and spleens, and the T-cell exhaustion markers were also reduced, matching the effects of systemic SHP2 inhibition. The enhancement of T-cell cytotoxicity by SHP2 inhibition in MBC cells was also confirmed with in vitro T-cell cytotoxicity assays. The M1/M2 ratio of TAMs was also modulated in similar fashion as compared with systemic inhibitors, but total TAM populations were not significantly changed. These findings are consistent with the notion that SHP2 function in tumor cells can influence the lymphoid, but not the myeloid components of the metastatic TME (As SHP2 is expressed in both tumor cells and immune cells, we sought to investigate how tumor cell–autonomous SHP2 contributes to immune escape by MBC. Indeed, previous reports suggest SHP2 in T cells is dispensable for their function and that the tumor-facilitating role of SHP2 lies in myeloid cells and tumor -associated endothelial cells . Using tatic TME . Overall+ T-cell cytotoxicity downstream of multiple growth factors via regulation of PD-L1 and MHC class I. Our studies also identify the ability of 3D culture environment with ECM signaling to promote PD-L1 via SHP2. Studies to determining the mechanistic details behind this event are ongoing, but our studies herein strongly suggest that the ability of SHP2 to balance STAT1 and MAPK signaling always for its regulation of PD-L1 expression at several levels (The clinical significance of phosphorylation of SHP2 has been supported by multiple studies, including those herein, where we demonstrate that phosphorylation of SHP2 at Y542 is a promising marker to predict immune profiles in patients with MBC . These fl levels .In summary, we show that tumor cell–autonomous SHP2 is a key signaling node by which MBC cells induce immune suppression from a variety of signaling inputs within the TME . We estaSupplementary Information SI1This is the code information for publicly available data analysis.Click here for additional data file.Supplementary Tables S1-S7These are supplementary tables to provide detailed information about the materials used in the study including cell, culture conditions, growth factors, inhibitors, and antibodies.Click here for additional data file.Supplementary Figure S1Combination of SHP099 and α-PD-L1 delays D2.A1 pulmonary growth in vivoClick here for additional data file.Supplementary Figure S2The gating ancestry for the populations in the study of D2.A1 modelClick here for additional data file.Supplementary Figure S3Corresponding representative dot plots for the quantification in figure 2A and additional T cell composition analysis in the study of D2.A1 model.Click here for additional data file.Supplementary Figure S4Corresponding representative dot plots for the quantification in figure 2B-C and additional T cell exhaustion marker analysis in the study of D2.A1 model.Click here for additional data file.Supplementary Figure S5Corresponding representative dot plots for the quantification in figure 2D-F and additional myeloid composition analysis in the study of D2.A1 model.Click here for additional data file.Supplementary Figure S6Depletion of SHP2 in tumor cells delays 4T1 pulmonary metastasis in vivoClick here for additional data file.Supplementary Figure S7The gating ancestry for the populations in the study of 4T1 modelClick here for additional data file.Supplementary Figure S8T cell composition analysis in mice bearing SHP2 manipulated 4T1 metastases and representative dot plots for data shown in figure 4A.Click here for additional data file.Supplementary Figure S9Representative dot plots for data shown in figure 4B-C and additional exhaustion marker analysis of CD4+ T cells in mice bearing SHP2 manipulated 4T1 metastases.Click here for additional data file.Supplementary Figure S10Representative dot plots for data shown in figure 4D-F and additional exhaustion marker analysis of CD8+ T cells in mice bearing SHP2 manipulated 4T1 metastases.Click here for additional data file.Supplementary Figure S11Representative dot plots for data shown in figure 4G-H and additional myeloid composition analysis in mice bearing 4T1 metastases.Click here for additional data file.Supplementary Figure S12Analyses of Tregs and macrophage scores in clinical datasets with respect to SHP2 phosphorylation.Click here for additional data file.Supplementary Figure S13Inhibition or depletion of SHP2 and PD-L1 blockade rescue T cell cytotoxicity.Click here for additional data file.Supplementary Figure S14PD-L1 expression is induced by growth factor stimulation in the D2.A1 cells.Click here for additional data file.Supplementary Figure S15Growth factors and 3D culture environment induce PD-L1 in MBC cells.Click here for additional data file.Supplementary Figure S16SHP2 regulates MHC class I expression via the balance between MAPK and STAT1 signaling in human MBC cells.Click here for additional data file.


Antimicrobial susceptibility testing and PFGE typing were used to identify tigecycline resistance and assess clonal relationships among isolates. Plasmids present and genome sequences were analyzed by conjugation, S1 pulsed-field gel electrophoresis (PFGE), plasmid stability testing and whole-genome sequencing.tet(X4)-carrying E. coli and these strains exhibited a high degree of multidrug resistance. Based on the geographical location distribution of the sampled areas, number of samples in each area and isolation rate of tigecycline-resistant strains and tet(X4)-carrying isolates, 72 tet(X4)-positive isolates were selected for further investigation. Tigecycline resistance was shown to be mobile in 72 isolates and three types of tet(X4)-carrying plasmids were identified, they were IncHI1 (n = 67), IncX1 (n = 3) and pO111-like/IncFIA(HI1) (n = 2). The pO111-like/IncFIA(HI1) is a novel plasmid capable of transferring tet(X4). The transfer efficiency of IncHI1 plasmids was extremely high in most cases and IncHI1 plasmids were stable when transferred into common recipient strains. The genetic structures flanked by IS1, IS26 and ISCR2 containing tet(X4) were complex and varied in different plasmids.A total of 204 tigecycline-resistant E. coli were isolated from 662 samples. Of these, we identified 165 E. coli is a major threat to public health. This data suggests careful use of tetracycline on farms is important to limit spread of resistance to tigecycline. Multiple mobile elements carrying tet(X4) are in circulation with IncHI1 plasmids the dominant vector in this setting.The widespread dissemination of tigecycline-resistant  The global transmission of multidrug resistant bacteria is a major threat to public health . Tigecyctet(X) was first reported in anaerobes, but plays an important role in many bacteria, including E. coli  in recent years presents a threat for clinical treatment of infections (tet(X) into different variants has been observed with extended spectrum of activity of some conferring resistance to tigecycline (tet(X), tet(X3)/tet(X4)/tet(X5) can mediate high-level resistance to tigecycline which is a clinical problem (tet(X4) has recently been detected from food-producing animals and is the most prevalent tet(X) subtype in China (tet(X4) can be mediated by diverse vectors, including IncX1, IncX4, IncX1/N, IncX1/FIA/B/Y, IncX1/FIA/B/HI1A/B, IncFIA/B/HI1A/B, IncFII, Col, IncQ, and IncFIB/FIA(HI1)/X1 plasmids (tet(X4) genes can be spread by many vectors, but it is unclear which is of most importance. In this study, we investigated mechanisms of transmission of tet(X4) obtained from a single large chicken farm. Combining this data with the literature, we provide firm evidence for the growing problem of tigecycline resistance in the farm environment and characterize the dominant vectors responsible for transmission.The wide transmission of plasmid-mediated fections , mutatioecycline . Among t problem . Of thesin China . The emeplasmids . This suE. coli were isolated using MacConkey agar containing 4 mg/L tigecycline. The presence of tet(X4) was identified by PCR using primers tet(X4)-F (ATCGTTATGGGTAATGGAC) and tet(X4)-R (ACCTGGTAAGAAGAAGTGG) (495 bp). In addition, a tet(X2/3/5) universal PCR test  was used to identify the presence of other tet(X) variants. The following strain nomenclature was used in this study: area of the farm/serial number of samplings . Seventy-two tet(X4)-carrying strains were selected for subsequent experiments from all tet(X4)-carrying E. coli strains in this study to explore the transmission mechanisms of tet(X4). These 72 strains were chosen based on the geographical location distribution of the sampled areas, number of samples in each area and isolation rate of tigecycline-resistant strains and tet(X4)-carrying isolates to ensure the reliability of the results.A total of 662 samples were recovered from 10 areas across a large-scale chicken farm in Guangdong Province, China in August 2020, 526 were collected from feces, 47 were collected from water, 81 were collected from soil and 8 samples were collected from flies . TigecycE. coli ATCC25922 was used as a quality control strain.Antimicrobial susceptibility testing was performed by agar dilution method according to the CLSI guidelines . The folXbaI restriction enzyme to analyze the diversity of tet(X4)-carrying E. coli isolates to assess the relationship between different isolates. Bacterial DNA samples were electrophoresed with pulse times from 2.2 s to 54.2 s over 19 h and PFGE was performed at 14°C in 0.5 × TBE. Isolates with a profile possessed >80% similarity were considered to belong to the same PFGE group .The 72 tested isolates were subjected to PFGE analysis using GE group . SalmoneE. coli strains, filter mating was performed with E. coli C600 as a recipient. The recipient and donor strains were combined 1:3/1:1/3:1 on a filter disk placed on LB agar the mating mixture was incubated at 37°C overnight. Transconjugants were selected on MacConkey agar containing tigecycline (4 mg/L) and streptomycin  following incubation (tet(X4) target PCR of the transconjugants/donors/recipients were subsequently performed to confirm whether the plasmid was successfully transferred to the recipient (tet(X4)-carrying plasmids. Two additional replicon primers  were included.To investigate the transfer ability of plasmids isolated from 72 tested cubation . Enterobecipient . Non-conecipient  to deterE. coli strains as recipients to generate transconjugants containing tet(X4)-carrying IncHI1 plasmids. These strains were reference strains C600 and MG1655 and a duck isolate BYZ3 (which contains no plasmids). IncHI1 plasmid-carrying transconjugants were grown overnight at 37°C in LB broth without any antibiotics. The following day the strains were passaged by 1,000-fold dilution into fresh medium and were allowed to grow for another 24 h. The same procedure was repeated, and the strains were cultured for 7 days. After day 7, cultures were serially diluted (10-fold) in saline  and plated on LB agar plates. Single colonies (n = 100) were picked, and the loss of plasmids was confirmed by the tet(X4) and IncHI1 replicon target PCR. Three parallel samples were analyzed for each strain.The stability of plasmids was studied as previously described . We seletet(X4)-carrying plasmid and isolation source of sample, six strains to represent the diversity in the panel were selected for whole genome sequencing. Three of them carrying a transferable IncHI1-tet(X4) plasmid , the other three strains carrying untransferable tet(X4)-carrying plasmids .Based on results of PFGE, type of E. coli strains was extracted by Hipure Bacterial DNA kit . The genomes were sequenced using both long-read Nanopore and short-read Illumina Miseq sequencing platforms. The clean reads were then used to create hybrid assemblies using the CLC Genomics Workbench. Genes and insertion element confirmation and annotation were done with RAST,Genomic DNA of the 6 E. coli strains were isolated and identified from a large chicken farm. The tigecycline resistant isolates contained 165 tet(X4)-carrying E. coli, including 110 strains isolated from feces samples, and 55 strains isolated from soil samples (E. coli strains not harboring tet(X4), 22 isolates carried tet(X2/3/5) according to PCR with these universal primers. Of the 165 tet(X4)-carrying E. coli isolates, 72 were selected to represent each region of the chicken farm  for subsequent experiments  were resistant to minocycline. The resistance rates to enrofloxacin, ciprofloxacin, cefotaxime and amikacin were 38.89% (28/72), 13.89% (10/72), 22.22% (16/72), and 4.17% (3/72), respectively . All strE. coli isolates were divided into 39 different PFGE clusters with 80% genetic similarity. Based on the PFGE results, it was concluded that horizontal transmission of tet(X4) in the large chicken farm of this study is common (The s common .tet(X4)-carrying plasmids that ranged in size from 45 to 250 kb. IncHI1 plasmids were by far the most prevalent type (n = 67) as assessed by PCR replicon typing demonstrating these plasmids are the dominant vectors of tet(X4) in this setting. The remaining five plasmids were IncX1 (n = 3) and pO111-like/IncFIA(HI1)  (n = 2)  .−7 and 3.4 × 10−1. The transfer efficiency of four IncHI1 plasmids was found to be extremely high (above 1.0 × 10−1)  . The sta × 10−1) .E. coli strains carrying tet(X4) plasmids  were fully sequenced and annotated. The tet(X4)-carrying plasmid types were IncHI1 (n = 3), pO111-like/IncFIA(HI1) (n = 2) and IncX1 (n = 1), respectively. See Six tet(X4) were present in these related plasmids by comparing the complete sequence of p19A20–1 and 13 reference sequences (1R-abh-tet(X4)-ISCR2-orf2-ISCR2-orf3-△Tn2-IS26-orf4-IS15DI-△Tn2-IS26-orf5-IS1R) containing multiple resistance genes (tet(X4), floR, blaTEM-1, aadA22, et al.) in one region flanked by IS1R was observed -ISCR2 (The three IncHI1 plasmids varied in size (from 190 to 244 kb) but there was more than 80% similarly in common regions. One noticeable difference is the BREX  structure differentiating p19A20–1 (190.657 kb) and the other two  . We founequences . One 25,observed , TypeI. 4)-ISCR2 , TypeII.tet(X4) were also identified in pO111-like/IncFIA(HI1) plasmids. Four distinct genetic structures were identified by comparing the complete sequence of p13Q15 and other 9 reference plasmids. A core structure contained IS1R-rep (HIA)-ISSen9-hypothetical protein-yadA-hypothetical protein-IS1R-abh-tet(X4)-ISCR2-△virD2-expZ-cat-marR-ISCR2-△virD2-floR-lysR-hypothetical protein-△tnpA-IS26 . Based on this structure, different variants  were identified as inserted downstream of tnpA-IS26 in the other three structures (tet(X4) organization was identified in p13QT4. The only major difference in this variant is the insertion of ISCR2 downstream of the lysr-hp compared with p13Q15 and results in a truncated form of the Type VI structure with a loss of △hypothetical protein-△tnpA-IS26 (Further genetic environments (named here III to VII) of npA-IS26 , TypeVIItet(X4) confirmed in this study was found in IncX1 plasmids. Compared with reference plasmid pCD33-6-1, the remaining four plasmids  carried the tet(X4) bearing genetic structure and the structure was an IS26-flanked (in the same orientation) transposition unit, IS26-orf1-ISCR2-hypothetical protein-abh-tet(X4)-ISCR2-orf2-TnAs2-IS26  (tet(X4)-abh in p13QT11 showed significant inconsistencies compared to the other seven upstream structures. And the virD2-expZ-cat-marR structure, downstream of tet(X4)-ISCR2 were found to have complete deletion in p13QT11 compared with genetic environments of IncHI1 and pO111-like/IncFIA(HI1) plasmids found in this study (Type VII).The 8th genetic environment of  537 bp) . The upsE. coli carrying tet(X4) have been reported in pigs, cows, ducks, sheep, migratory birds, and humans to date with the detection rate being less than 5% (tet(X4)-positive E. coli in this study is much higher than that reported in previous studies. It is interesting that the positive rate of tet(X4)-harboring E. coli isolated from soil was also much higher when compared with fecal samples. Therefore, soil may be an important repository of tet(X4) resistant genes and the flow of strains between animals and the farm environment may provide a route for tet(X4) resistant gene transmission. Animal feces and the farm environment has been considered an important reservoir of antibiotic resistance genes. Animal feces can be used as fertilizer in agricultural production, which could aid transmission of tet(X4). The tet(X4) resistant gene would then be able to access a wide range of hosts, including strains presebt in agricultural soil and edible produce (tet(X4)-positive E. coli in this study also carried other antibiotic resistance genes, consistent with the high rate of MDR in this study and this observation is consistent with several other studies (Isolates of  than 5% . The iso produce . Tetracy produce . The tet studies .E. coli strains harboring tet(X4) in this study was detected by PFGE suggesting that the wide distribution of tet(X4) is mediated by horizontal genetic transfer rather than expansion of a single predominant clone. The transmission of tet(X4) in farming systems has become increasingly serious in recent years, including many food-producing animals (tet(X4). We found that IncHI1 plasmids were by far the most common isolated from tigecycline resistant strains carrying tet(X4) in this study and have played the most important role in the transmission of tet(X4) in this setting. This is similar to recent evidence showing transferable tet(X4)-carrying IncHI1 plasmids were isolated from pork and duck farms (tet(X4)-bearing IncHI1 plasmids were highly transferable and demonstrated stability in host strains in this study. The stability and the high transfer efficiency of IncHI1 plasmids observed could contribute to both high rates of tet(X4) transmission but also stable existence in the environment and gives further evidence for the potential of IncHI1 plasmids as a major vector mediating transmission of resistant genes in farms. We also discovered two additional families of plasmids, IncX1 and pO111-like/IncFIA(HI1) carrying tet(X4). To the best of our knowledge, pO111-like/IncFIA(HI1) is a novel family of plasmid carrying tet(X4). While we found the dominant vector mediating widespread of tet(X4) in this study to be IncHI1 plasmids we also believe that the gradual diversification of vector types might facilitate their interaction with a broad range of hosts  into different types of plasmids and facilitates the chances of tet(X4) to spread among different bacterial species and expand its host range (tet(X4)-genetic environment has previously been largely reported as ISCR2-tet(X4)-abh, we expanded the region in this study and documented more complex tet(X4)-carrying structures containing multiple resistant genes and insertion elements. We found that IS1R/A, ISSen9, IS26 insertion elements also played a key role when extending the analysis for the genetic background of tet(X4). These insertion elements carry other resistant genes such as floR, qnrS1 et al. and concomitant with tet(X4), which causes multi-resistant genes co-transfer. This can lead to multidrug-resistant bacterial infections occurring in more hosts. This greater complexity of insertion elements reflects the increased genetic diversification of tet(X4) evolution. It will also bring about more possibilities and opportunities for spread of tet(X4).Insertion elements lead to the gradual formation of complicated genetic structures and promote the transfer of resistant genes. The ISrganisms . The spest range . The conE. coli isolated from different areas across one large chicken farm exhibit a high prevalence of tigecycline resistance. tet(X4)-production is a primary mechanism of resistance to tigecycline and IncHI1 plasmids are common vectors mediating transfer of tet(X4) in a representative large farm which is a matter of concern. The genetic structure of tet(X4) appears to be evolving and becoming more complicated. Our results suggest a mechanistic basis for the high detection rate of tet(X4) in recent years and it emphasizes the potential risk of transmission of tet(X4)-positive strains in farming systems.In conclusion, we found that There was a close relation between human health and food-producing animals with antibiotic resistance and it constitute a growing threat to sustainable agriculture. Increased surveillance of the multidrug-resistant bacteria in agriculture is urgently needed, such as control for antibiotics use as feed additives. In addition, we should also increase monitoring of plasmid mediated transfer of resistant genes in farms and improve antibiotic regimens in poultry farms as soon as possible to limit the spread of resistant bacteria.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: NCBI- p13Q15 (ON934549), p13QT4, (ON934550), p13QT11 (ON934551), p13QT31-1 (ON934552), p19A20-1 (ON934554), p22Q34-1, (ON934556).YZ, JZ and H-XJ conceived and designed the experiments. YZ, JZ, PC, YL, M-TC, and X-LX performed the experiments. YZ and JZ analyzed the data. R-YS uploaded the sequencing data to the NCBI. YZ, MW, and H-XJ wrote and revised the manuscript. All authors approved the final draft and contributed to the article and approved the submitted version.This work was funded by the National Natural Science Foundation of China (Grant No. 31972734) and the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (Grant No. 2019BT02N054).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The analysis of “big data” identified superoxide dismutase-1 (SOD1) as a novel target of CHI3L1 and interacted with CHI3L1. The depletion of CHI3L1 increased SOD1 expression, resulting in ER stress. Furthermore, the depletion of SOD1 reduced the expression of ER chaperones and ER-mediated apoptotic marker proteins, as well as apoptotic cell death induced by the depletion of CHI3L1 in in vivo and in vitro models. These results suggest that the depletion of CHI3L1 increases ER stress-mediated apoptotic cell death through SOD1 expression, and subsequently inhibits lung metastasis.Chitinase-3-like protein 1 (CHI3L1), which is secreted by immune and inflammatory cells, is associated with several inflammatory diseases. However, the basic cellular pathophysiological functions of CHI3L1 are not well characterized. To investigate the novel pathophysiological function of CHI3L1, we performed LC-MS/MS analysis of cells transfected with Myc-vector and Myc-CHI3L1. We analyzed the changes in the protein distribution in Myc-CHI3L1 transfected-cells, and identified 451 differentially expressed proteins (DEPs) compared with Myc-vector-transfected-cells. The biological function of the 451 DEPs was analyzed and it was found that the proteins with endoplasmic reticulum (ER)-associated function were much more highly expressed in CHI3L1-overexpressing cells. We then compared and analyzed the effect of CHI3L1 on the ER chaperon levels in normal lung cells and cancer cells. We identified that CHI3L1 is localized in the ER. In normal cells, the depletion of CHI3L1 did not induce ER stress. However, the depletion of CHI3L1 induces ER stress and eventually activates the unfolded protein response, especially the activation of Protein kinase R-like endoplasmic reticulum kinase (PERK), which regulates protein synthesis in cancer cells. CHI3L1 may not affect ER stress owing to the lack of misfolded proteins in normal cells, but instead activate ER stress as a defense mechanism only in cancer cells. Under ER stress conditions induced by the application of thapsigargin, the depletion of CHI3L1 induces ER stress through the upregulation of PERK and PERK downstream factors (eIF2α and ATF4) in both normal and cancer cells. However, these signaling activations occur more often in cancer cells than in normal cells. The expression of Grp78 and PERK in the tissues of patients with lung cancer was higher compared with healthy tissues. It is well known that ER stress-mediated PERK-eIF2α-ATF4 signaling activation causes apoptotic cell death. ER stress-mediated apoptosis induced by the depletion of CHI3L1 occurs in cancer cells, but rarely occurs in normal cells. Consistent with results from the  The endoplasmic reticulum (ER) is an important cellular organelle responsible for the synthesis of proteins, protein modification through glycosylation or disulfide bond formation, protein folding into a normal structure, calcium storage, and signaling transduction The activation of UPR by ER stress is mediated by IRE-1 (inositol-requiring 1), PERK (Protein kinase R-like endoplasmic reticulum kinase), and ATF6 (activating transcription factor) Chitinase-3-llke protein 1  was first discovered in mouse breast cancer cells and was named breast regression protein 39 (BRP-39). CHI3L1 binds to chitin; however, unlike chitotriosidase (CHIT1) and acid mammalian chitinase (AMCase), it has an intrinsic function independent of chitin. In humans, CHI3L1 plays an important role in maintaining homeostasis in many organs Mus musculus skin melanoma cells were obtained from the American Type Culture Collection . U-2 OS cells were cultured in McCoy's 5a medium supplemented with 10% fetal bovine serum (FBS), 100 μg/mL penicillin, and 100 μg/mL streptomycin. A549, MRC-5, and B16F10 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 μg/mL penicillin, and 100 μg/mL streptomycin. A172 and Hep3B cells were cultured in DMEM medium supplemented with 10% FBS, 100 μg/mL penicillin, and 100 μg/mL streptomycin. Cell cultures were maintained in an incubator with a humidified atmosphere of 5% CO2 at 37 °C. Reduced serum medium and X-tream GENE HP DNA transfection reagent were used for transfecting plasmid and siRNA oligonucleotides following the manufacturer's instructions.A549 lung carcinoma, MRC-5 normal lung cell, U-2 OS bone osteosarcoma, A172 glioblastoma, Hep3B hepatocellular carcinoma, and B16-F10 Animal experiments were performed as described previously All protocols involving mice in this study were reviewed and approved by the Chungbuk National University Institutional Animal Care and Use Committee (IACUC) and complied with the Korean National Institute of Health Guide for the Care and Use of Laboratory Animals (CBNUA-1073-17-01).g for 20 min at 4 °C. Cells were harvested and lysed using lysis buffer and centrifuged. The proteins were loaded to SDS-PAGE. The specific primary antibodies and HRP-conjugated secondary antibodies were incubated and visualized by using a FUSION Solo S chemiluminescence detection system .The tissues were homogenized with lysis buffer  and centrifuged at 13,000 X For immunoprecipitation, the cell lysates were incubated with specific primary antibodies for 4 h at 4 °C. Then protein A/G PLUS-Agrose (Santa cruz) were added to antibody containing cell lysates and incubated for 2 h at 4 °C. Immunoprecipitated proteins were loaded to SDS-PAGE.Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Cells then were permeabilized with 100% cold-Methanol for 5 min and blocked by 4% BSA in PBS with 0.1% Triton X-100 (PBST) for 1 h. Primary antibodies were incubated for overnight at 4 °C and Alexa Fluor 488  or Texas Red  conjugated secondary antibodies were incubated for 1h at room temperature. Fixed cells were incubated with 1 μg/mL of DAPI  for 5 min at room temperature and then covered with Fluoromount-G Mounting Medium . Cells were visualized using Ziess AxioObserver  fluorescence microscope system. Digital images were analyzed using the ZEN 2.1 (Carl Zeiss) software.The tumor and lung tissue sections were blocked with 3% normal goat serum diluted in PBS, for 30 min; the sections were then incubated with antibodies for CHI3L1, Grp78, PERK, CHOP, caspase 12, and SOD1, at the appropriate dilution in blocking serum, for overnight at 4 ºC. The slides were washed in PBS, followed by the avidin-biotin-peroxidase complex . The slides were washed, and the peroxidase reaction was developed with diaminobenzidine and peroxide , mounted in Aqua-Mount, and evaluated under a light microscope (Olympus).A549 cells were transfected with either control siRNA or CHI3L1-siRNA for 1 day with thapsigargin for 18 h. Then cells were fixed with 4% paraformaldehyde in DPBS for 30 min at room temperature, permiabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 10 min at room temperature. The cleavage DNA fragments were stained for 1 h in the dark at 37 °C using the TUNEL reaction mixture. Cells were observed on an Axio Observer Z1 inverted fluorescence microscope.For trans-well assay, A549 cells were seeded on upper chamber inserts . After incubation for 18 h, the cells were fixed with 4% formaldehyde for 5 min, permeated with 100% methanol for 15 min, and stained with 0.1% crystal violet for 20 min. In the upper chamber, non-migrated cells were removed with a cotton swab. The migrated cells were visualized using a light microscope (Olympus) and analyzed using ImageJ software (NIH).Cell pellets were stored at -80 °C until cell lysis was performed. Lysis of cell pellets was done at room temperature. Biological replicates (one cell pellet from one cell line) were processed simultaneously to minimize the effect of error. Pellets were resuspended in 100 μL 8 M Urea  were added. Protein concentration was estimated with the bicinchoninic acid assay . Proteins were reduced with 10 mM dithiothreitol (Sigma) and alkylated 25 mM iodoacetamide (Sigma). Samples were diluted in 50 mM AmBic and trypsinized  overnight at 37 °C at a trypsin/protein ratio of 1:50, w/w). The resulting peptide mixture was lyophilized overnight and digested peptides were cleaned by flowing through an Oasis HLB 1cc (10 mg) solid phase extraction (SPE) catridges . Samples were dried using a Speed-Vac  and stored at -80 °C until time for analysis. The peptides digestion protocols were performed modifying the method described previously 6, and a maximum injection time of 80 ms. Fragmentation was performed with a normalized collision energy of 25.LC-MS/MS was performed as described previously Human tissue samples from lung cancer patients and normal controls were obtained from Chungbuk National University Hospital Biobank, Keimyung University Dongsan Hospital Biobank and the Biobank of Ajou University Hospital, members of Korea Biobank Network. All studies using human samples were conducted in accordance with the Declaration of Helsinki and were approved by the Ethics Committee of Chungbuk National University Medical Center (IRB No. CBNU-201910-BR-941-01).t-test. Multiple comparisons were using one-way analysis of variance followed by Tukey's tests. Differences between groups were considered significant at P-values of <0.05.Statistical analyses were performed using the GraphPad Prism 5 software. All error bars reported are the standard deviation (SD) unless otherwise indicated. Pairwise comparisons were performed using Student's To evaluate the global changes in protein abundance in CHI3L1-expressing cells, we performed nano-high-performance liquid chromatography (nano-LC). In total, we identified 1146 proteins and 1120 proteins in CHI3L1-expressing cells and 1053 proteins in control vector-expressing cells. There were 1027 proteins differentially expressed in CHI3L1-expressing cell and vector-expressing cell samples  treatment, an ER stress inhibitor. The western blotting results showed that 4-PBA treatment had no obvious effect on ER chaperone protein levels in cells transfected with control siRNA. However, ER chaperone protein levels were lower in CHI3L1 depletion in the presence of 4-PBA, indicating that the degree of ER stress is reduced . Compared with DMSO-treated control cells, thapsigargin treatment did not alter cell proliferation (75.34±4.07%). The proliferation of cancer cells transfected with CHI3L1 siRNA and treated with thapsigargin was significantly decreased (49.88±7.80%)  Figure A. In sevin vivo metastasis in CHI3L1 WT and KO mice, A549 cells were injected into tail veins and control siRNA and SOD1 siRNA were intravenously injected into CHI3L1 WT and KO mice a three times per week for 7 weeks. The CHI3L1 KO group had significantly fewer metastatic nodes in lung tissues compared with the CHI3L1 WT group  and PERK signaling proteins compared with the depletion of CHI3L1 in lung cancer cells and metastatic lung tumor tissues. In addition, ER stress-mediated apoptotic marker protein levels also much greatly decreased in the dual depletion of CHI3L1 and SOD1 in in vitro and in vivo. Here, we showed that the depletion of CHI3L1 induces ER stress-mediated lung cancer cell death through SOD1 upregulation.However, the exact mechanism of how CHI3L1 causes ER stress and thus induces apoptosis and inhibits tumorigenesis is unknown. From the GENEMANIA database, we found that the SOD1 protein forms an interaction network between CHI3L1 and ER stress. In addition, we confirmed that CHI3L1 physically interacts with SOD1. Several papers reported that SOD1 accumulation activates the ER stress-mediated UPR pathway The present study provides important insights into the role of CHI3L1 in the regulation of ER stress associated UPR function and apoptosis, as well as the SOD1 dependent regulation of tumor growth.Supplementary figures and table.Click here for additional data file.


P3221 space group) for fixing[Rh2(OAc)4] and developing a heterogeneous catalystto perform reactions in an aqueous medium. The structure of the [Rh2(OAc)4]/RNase A adduct was investigated by X-raycrystallography: the metal complex structure remains unperturbed uponprotein binding. Using a number of crystal structures, metal complexaccumulation over time, within the RNase A crystals, and structuresat variable temperatures were evaluated. We also report the large-scalepreparation of microcrystals (∼10–20 μm) of the[Rh2(OAc)4]/RNase A adduct and cross-linkingreaction with glutaraldehyde. The catalytic olefin cyclopropanationreaction and self-coupling of diazo compounds by these cross-linked[Rh2(OAc)4]/RNase A crystals were demonstrated.The results of this work reveal that these systems can be used asheterogeneous catalysts to promote reactions in aqueous solution.Overall, our findings demonstrate that the dirhodium paddlewheel complexescan be fixed in porous biomolecule crystals, like those of RNase A,to prepare biohybrid materials for catalytic applications.Due to their uniquecoordination structure, dirhodium paddlewheelcomplexes are of interest in several research fields, like medicinalchemistry, catalysis, etc. Previously, these complexes were conjugatedto proteins and peptides for developing artificial metalloenzymesas homogeneous catalysts. Fixation of dirhodium complexes into proteincrystals is interesting to develop heterogeneous catalysts. Poroussolvent channels present in protein crystals can benefit the activityby increasing the probability of substrate collisions at the catalyticRh binding sites. Toward this goal, the present work describes theuse of bovine pancreatic ribonuclease (RNase A) crystals with a poresize of 4 nm ( Porous RNase A crystals were stabilizedby cross-linkingand functionalized with dirhodium tetraacetate. The metal/proteinadduct was extensively characterized by X-ray crystallography. Cross-linkedcrystals of the metal/protein adduct were used as heterogeneous catalystsfor reactions in aqueous solutions. The study of the interaction of proteins with organicmolecules, metal ions, or complexes has provided insightful informationon the binding mechanism of these ligands to proteins.6Protein crystals are useful materialsfor structural analysis.7 This drawback can be overcome using the covalent cross-linkingstrategy, which allows the formation of cross-linked protein crystals(CLPCs). CLPCs are stable and insoluble in both organic and aqueoussolvents and display good mechanical and thermal stability.9 CLPCs are usually prepared using glutaraldehyde (GA) as the cross-linkingagent because both monomeric and oligomeric GA species can interactwith proteins,12 making the cross-linking process nonspecific andthus useful for linking crystals of different proteins, regardlessthe nature of the protein or crystal packing.7 CLPCs have been used for several applications ranging from biosensingto drug delivery.17 CLPCs are of great interest for the preparation of heterogeneouscatalysts based on artificial metalloenzymes.18 In fact, when a protein crystal is stabilized by cross-linking,metal complexes can diffuse through solvent channels of the crystallattice and interact with protein molecules, forming a stable metal/proteinadduct crystal.20 This system can be used as aneffective heterogeneous catalyst because it allows one to screen betterreaction conditions for catalysis. Indeed, it can tolerate the presenceof high concentrations of organic compounds in aqueous solvents andthermal and pH variations.9 Moreover, CLPCs overcomethe limitation of both metal complexes and proteins alone. In fact,in CLPCs, the metal first, second, and third coordination spherescan be modulated by protein engineering, providing a catalyst withunprecedented performances,18 while theprotein can retain its folding within the crystal lattice, even atextreme pH or in the presence of a high percentage of organic solvents.Hence, these systems can, in principle, improve the performances notonly of the metal complexes but also of artificial metalloenzymes.Some of us have functionalized cross-linked hen egg white lysozyme(HEWL) (CL_HEWL) crystals with [Ru(benzene)Cl2]2 and have tested the reactivity of cross-linked crystals of thismetal/protein adduct toward transfer hydrogenation of acetophenonederivatives.21 CLPCs catalyzed the transferhydrogenation reaction better than the metal/protein adduct in solution.21 CL_HEWL crystals have also been used for growingplatinum nanoparticles (from H2PtCl6), usefulas hydrogen evolution promoters.22 CLPC-basedcatalysts can be recycled several times before inactivation.22 Cross-linked NikA (a nickel binding protein)crystals have been functionalized with Fe-EDTA-like compounds (EDTA= ethylenediaminotetraacetate) and employed as oxidation catalystsfor C–C double bond, using O2 as an oxidant.23However, the weak interactions between symmetry mates andthe largepores that are formed within protein crystals make these systems veryfragile and unstable when they are removed from their mother liquor,limiting their applications as biohybrid materials.Despite the tremendous advantages thatarise from the metal functionalizationof CLPCs, those mentioned above are the only known examples of theuse of these systems as catalysts.2(μ-O2CR)4L2] are versatile catalysts for several reactions,25 like decomposition of diazo compounds,26 carbene insertion into aliphatic and aromatic C–H bonds,28 nitrene transfer reaction,29 olefinecyclopropanation,30 aromatic cycloaddition,31 hydrosilylation of alkynes,33 oxidation of alcohols,34 and photochemicaland thermal hydrogen evolution.36 Bioconjugation of thesecomplexes with peptides and proteins has provided artificial metalloenzymesuseful for stereoselective C–H insertion reaction and hydrosilylation,38intermolecular cyclopropanation,41 and selective(E)-alkene coupling.42 Some of us have previously studied the interaction of these complexeswith proteins by X-ray crystallography,47 revealing that they degrade within HEWL crystals,47 while they react with bovinepancreatic ribonuclease (RNase A) in the solid state, forming a metal/proteinadduct where the dirhodium core remains intact.45 Crystals of the [Rh2(OAc)4]/RNaseA adduct (OAc = acetate) belonged to the C2 spacegroup, with two protein molecules in the asymmetric unit. In bothprotein molecules, two dirhodium binding sites were observed, in theproximity of His105 and His119 side chains. In both cases, the dimetallicpaddlewheel scaffold is preserved and Rh atoms are linked to the Natom of the His imidazole rings at the axial position. The interactionof [Rh2(OAc)4] with RNase A does not suppressthe reactivity of the dimetallic compound toward small ligands, likeimidazole, in the solid state.48 Hence,[Rh2(OAc)4]/RNase A adduct crystals seem tobe ideal candidates for preparing heterogeneous catalysts based onCLPCs. However, crystals of RNase A used for structural determinationof the [Rh2(OAc)4]/RNase A adduct grow in 2weeks and need one additional week for the adduct formation by soaking.45 For this reason, RNase A crystals obtained inother experimental conditions and belonging to another space group(P3221) were prepared here. The protocolfor preparing good-quality cross-linked crystals of the [Rh2(OAc)4]/RNase A adduct in this space group was optimized,and cross-linked [Rh2(OAc)4]/RNase A adductcrystals were prepared, characterized by X-ray crystallography, andtested for the first time as heterogeneous catalysts toward olefincyclopropanation and self-coupling of the diazo compound reactions(Dirhodium tetracarboxylates[Rheactions1. This iP3221 to prepare the [Rh2(OAc)4]/RNase A adduct because they have pores of 40.2Å . This pore size isadvantageous for the diffusion of small molecules into the crystals.Hence, to prepare more effective systems to be used as heterogeneouscatalysts, RNase A crystals were grown under different experimentalconditions. An efficient system was obtained by growing RNase A crystalsin 2.5 M NaCl, 3.3 M sodium formate, and 0.1 M sodium acetate bufferat pH 4.7–5.5,49 using the hanging-dropvapor diffusion technique. Under these conditions, RNase A crystallizeswith one protein molecule in the asymmetric unit in 1 day. This crystalform shows wider pores than those found in crystals obtained in the C2 space group .We chose RNase A crystals from spacegroup 2(OAc)4] were formed viasoaking1 using RNase A crystals grown atpH 5.2. Soaking was performed using a saturated solution of [Rh2(OAc)4] dissolved in the reservoir. Crystals turnedinto light pink after 1 h of soaking with the metal compound. To characterizethe metal binding process within the protein crystals, X-ray diffractiondata were collected on RNase A crystals exposed to [Rh2(OAc)4] for different soaking times . Hence,three different structures of the [Rh2(OAc)4]/RNase A adduct in the P3221 space groupwere solved (Table S1), as observedfor the structure of the metal/protein adduct derived from crystalsin the C2 space group.45It was verified that these crystals can be used to form metal/proteinadducts upon reaction with metal compounds in the solid state. Crystalsof the adduct with /RNase A adduct in the C2 space group .In all of the structures, two binding sites for the metal compoundwere observed, close to His105 and His119 side chains, as observedin previous reports.2(OAc)4] (occupancy = 0.70–0.78) is linked to theNε atom of the imidazole ring and to a Cl– ion 4]/RNase A adduct as catalysts,they should be stabilizedby cross-linking. To prepare cross-linked crystals of the [Rh2(OAc)4]/RNase A adduct (CL_[Rh2(OAc)4]/RNase A crystals), cross-linked RNase A (CL_RNase A) crystalswere prepared and then treated with [Rh2(OAc)4]. Using this procedure, RNase A crystals resist both soaking andcross-linking treatment. A screening of the GA percentage and cross-linkingtime was carried out to obtain CL_RNase A crystals with enhanced mechanicalproperties. The gentle cross-linking technique50 was used: GA was added to the reservoir solution, so thatit can reach the crystals by vapor diffusion.50 CL_RNase A crystals were obtained by exposing protein crystals for2 h to the reservoir enriched with 0.5% GA. After the cross-linkingtreatment, few cracks were observed on the lattice surface, but crystalsstill diffract X-rays and were insoluble in aqueous solutions differentfrom their mother liquor . Itis worth noting that when RNase A crystals are exposed to GA vaporsfor more than 2 h, they lose their diffraction power.To use crystalsof the [Rh2(OAc)4]/RNase A crystals were then prepared. Theywere obtained by soaking CL_RNase A crystals in a saturated solutionof [Rh2(OAc)4] dissolved in the reservoir overnight.A soaking time higher than 1 h was used to prepare CL_[Rh2(OAc)4]/RNase A crystals because it is expected that cross-linkingcould, at least in part, slow down the diffusion of ligands withincrystal channels.CL_[Rh2(OAc)4]/RNase A crystals were collected at two differenttemperatures,−180 and 0 °C, to evaluate if the temperature could havean effect on the structure of the adduct, when crystals were treatedwith GA. The two structures solved using data collected at −180and 0 °C refine at 1.5 and 1.6 Å resolution, respectively.Data collection and refinement statistics are reported in α rmsd analysis revealed that cross-linking does notalter the overall protein structure (Table S1).The X-ray diffraction data on CL_[Rh2(OAc)4] is linked to the protein via axial binding tothe His105 side chain, with high occupancy (0.75–0.80). Theremaining axial positions are occupied by a Cl– ion4]/RNase A adduct formed in the absence andin the presence of GA indicates that the binding of [Rh2(OAc)4] to the protein close to the His105 side chainis not affected by the cross-linking, while a decrease of the occupancyis observed for the dirhodium center bound to the His119 side chain,upon treatment of the crystals with GA.A comparison of the results obtained from crystals of the [Rh2(OAc)4] using a soaking technique andthat the structure of the adduct is unaffected by the data collectiontemperature.Overall, these resultsreveal that CL_RNase A crystals can be metalatedby [Rh2(OAc)4] .Catalysis requires CLPCs in bulk scale thatcannot be obtained by a hanging-drop vapor diffusion technique. Hence,RNase A crystals were grown via the batch technique in 2.5 M NaCl,3.3 M sodium formate, and 0.1 M sodium acetate buffer at pH 5.2. Thesmaller size of these crystals compared to those obtained using thehanging-drop vapor diffusion method is useful for catalysis becausesmall crystals allow better substrate diffusion toward the catalyticcenter. These crystals were treated with GA and functionalized with[Rh2(OAc)4]/RNase A crystals asheterogeneous catalysts toward self-coupling of diazo compounds andolefin cyclopropanation reactions was assayed (2(OAc)4]/RNase A crystals.Theability of CL_[Rh assayed 1. The re2(OAc)4]/RNase A crystals . Also, itshydrolysis derivative was found at tR =9.34 min (2(OAc)4]/RNase A crystals canbe used as heterogeneous catalysts .Analysis of the reaction products by gas chromatography(GC)–massspectrometry (MS) indicated that both reactions were promoted by CL_[Rh9.34 min 2.Althou2(OAc)4]/RNase A crystals. The GC–MSelution profile reported in tR = 13.38 and 14.08 min; Figure S5). The chromatogram of cis-ethyl phenylcyclopropane-1-carboxylateshowed a peak at tR = 13.38 min, perfectlymatching the results derived from the reaction catalyzed by CLCPs.Hence, the peak at tR = 14.08 min (trans-ethyl phenylcyclopropane-1-carboxylate.The ratio between these two peaks (cis/trans = 1:2.4) indicates aslight preference of the CL_[Rh2(OAc)4]/RNaseA crystals in promoting formation of the trans isomer rather thanthe cis isomer.Then, a catalytic styrene cyclopropanation reactionwas promotedusing CL_-functionalizedcross-linked RNase A crystals considering the 4 nm porous channelspresent in the P3221 space group. Cross-linkingtreatment was done for stabilization of the crystals and for potentialapplication as heterogeneous catalysts. The structure of the adductand the dirhodium accumulation mechanism in these crystals over timewere investigated via X-ray crystallography. Structural analysis demonstratesthat 1 h of soaking is sufficient to metalate the protein and thatthe crystal packing does not influence the binding of the metal compoundto RNase A: two binding sites were identified for the metal compound,close to the His105 and His119 side chains, in agreement with thatobserved using RNase A crystals in a different crystal form.45 These sites are on the pore surface, thus ina position that is potentially useful for catalysis. Cross-linkingtreatment and structure determination at −180 and 0 °Crevealed no significant change, thus suggesting perfect stabilizationof Rh in the RNase A scaffold. Thus, CL_[Rh2(OAc)4]/RNase A crystals were used as heterogeneous catalysts toward self-couplingof the diazo compound and olefin cyclopropanation reactions. Althoughside reactions were observed, GC–MS analysis of the reactionproducts revealed that both reactions are promoted by CLPCs functionalizedwith Rh and that CL_[Rh2(OAc)4]/RNase A crystalscan be used as heterogeneous catalysts in an aqueous medium. To thebest of our knowledge, metalated RNase A crystals were used for thefirst time as heterogeneous catalysts. Due to the presence of a 4nm porous channel, the current results reveal that the metalationof CLPCs can expand the functionality of a protein beyond its naturalone. This could give rise to a new generation of heterogeneous catalyststhat take advantage of the synergistic action of the metal complexes,protein scaffold, and crystal lattice.Insummary, we have prepared  was added to the solution. A solution of ethyl diazoacetate was added dropwise to the reaction blend at roomtemperature over 5 h. After 4 h, 1 mg of [Rh2(OAc)4] was added to the solution, and the reaction mixture wasleft at room temperature over magnetic stirring for 24 h. Then, themixture was concentrated under reduced pressure and purified by columnchromatography (Merck Kiesgel 60) of the crude residue over silicagel (ethyl acetate/hexane 9:1). cis-Ethyl phenylcyclopropane-1-carboxylatewas obtained  as a pale-yellow oil. 1H NMR spectra were coherent with those reported elsewhere .51A total of 43 mmol (4.5 g) of styrene was dissolvedin 5.0 mL ofCHP3221 spacegroup were prepared at 20 °C using thehanging-drop vapor diffusion technique. A RNase A solution (50 mg/mL)was equilibrated with a reservoir consisting of 2.5 M NaCl, 3.3 Msodium formate, and 0.1 M sodium acetate buffer at pH 5.2. Crystalswere obtained in 1 day. Soaking was performed by transferring proteincrystals into a solution consisting of the reservoir saturated with[Rh2(OAc)4] for 1, 2, or 6 h. Crystals turnedinto light pink upon exposure to the metal complex.RNase A crystals in the Figure S2). Crystals were transferred to a solution containing the originalreservoir to remove the excess of GA and then soaked with [Rh2(OAc)4] overnight.RNase A crystals (prepared as described above)were washed and equilibrated versus a reservoir consisting of 2.5M NaCl, 3.3 M sodium formate, and 0.1 M sodium acetate buffer at pH5.2 enriched with 0.5% GA. After 2 h, crystals turned into pale yellowand CL_RNase A crystals were formed (2(OAc)4] for 6 h. Upon metalation,crystals turned pink . The excessof [Rh2(OAc)4] on the surface of the crystalswas washed out using the reservoir solution.A total of 100 μLof RNase A (300 mg/mL) was mixed with 400 μL of a solution consistingof 2.5 M NaCl, 3.3 M sodium formate, and 0.1 M sodium acetate bufferat pH 5.2 . Upon 24 h, manycrystals were obtained. Cross-linking of these crystals was performedby adding 0.1% GA to the solution containing the crystals. After 90min, yellow CL_RNase A crystals obtained upon this treatment werewashed with the reservoir solution and soaked with the same solutionsaturated with [Rh2(OAc)4]/RNase A adduct were flash-frozenusing the crystallization buffer containing 30% (v/v) glycerol asa cryoprotectant. No cryoprotectant was used for CLPCs. Diffractiondata on crystals of the adducts were collected at −180 °C.Data collection for CPLCs was performed at both −180 and 0°C. Data were processed and scaled using Aimless.52 The structures were solved in the P3221 space group with a single molecule in theasymmetric unit by a molecular replacement method using Phaser.53 Metal-free RNase A under the accessioncode 5OGH wasused as starting model.54 Restrained refinementand model building were carried out using Refmac5 and Coot, respectively.56 Anomalous difference electron density maps were used to identifythe Rh positions in the electron density maps. The PDB validationserver  was used for structure validation. The structures were depositedat PDB with accession codes 8OQC, 8OQD, 8OQE, 8OQF, and 8OQG.X-ray diffraction data werecollected using a Rigaku XtaLAB Synergy-DWdiffractometer equipped with a HyPix-6000HE detector at Tokyo Instituteof Technology, Yokohama, Japan. Before data collection, crystals ofthe [Rh2(OAc)4]/RNase A crystals prepared through the batchtechnique were suspended in 500 μL of a solution consistingof 1.25 M NaCl, 1.75 M sodium formate, and 0.05 M sodium acetate atpH 7.0.CL_/RNase A crystals, 1.25 M NaCl, 1.75 M sodiumformate, and 0.50 M sodium acetate at pH 7.0 . The reaction wasstirred at 4 °C for 17 h. The reaction was quenched using CHCl3 and extracted three times with the same solvent. The resultingmixture was analyzed by GC–MS. The Rh concentration (0.9 mM)in the reaction mixture was determined by ICP-MS.A totalof 15 μL of ethyl diazoacetate (0.50 M stock solutionin acetonitrile) was dissolved into a solution consisting of CL_[RhThe GC–MS data were acquiredwith a Shimadzu GC-MS QP2010 spectrometer equipped with a ShimadzuSH-Rxi-5Sil column . The temperature was increased from 35 °C up to 320°C using a gradient of 10 °C/min. The final temperaturewas maintained for 2 min before cooling. The substrate conversionof the two reactions was calculated as the ratio between the peakareas of the reaction products and reactants. The ratio between thecis and trans isomers of ethyl phenylcyclopropane-1-carboxylate wasevaluated by calculating the ratio between their peak areas.2(OAc)4]/RNase Acrystals were dissolved in 1 mL of HNO3 (70%v/v). The solution was diluted up to 5 mL. A total of 77 μLof this solution was diluted to a final volume of 10 mL. The metalconcentration in the solution was determined by ICP-MS . Standard solutions were prepared using StandardSolution G purchased from Kanto Chemical Co. (Rhconcentration = 10 mg/L).CL_[Rh


Colorectal cancer (CRC) is one of the most prevalent malignant neoplasms worldwide, responsible for over 900,000 deaths yearly. As the immunotherapies targeting the PD-1/PD-L1 axis in CRC are effective only in microsatellite unstable tumours, which are 15% of all CRC cases, new targets of immune evasion are still needed. B7H3 has been reported to mediate immune escape and promote tumour progression in numerous malignancies, but it has yet to be fully elucidated in CRC. The study investigates whether B7H3 expression is related to MSI/MSS status, tumour infiltrating lymphocytes and cytokine composition in CRC. We found that B7H3 expression is upregulated in CRC tumours and independent of MSI/MSS status. B7H3 correlated positively with cytokines supporting tumour growth and was associated with M2-macrophage polarization. Additionally, TCGA analysis showed that high B7H3 expression in CRC tumours is related to decreased survival in CRC patients. Our findings provide a novel insight into B7H3’s role in CRC immunity.The study aimed to assess the expression of B7H3 concerning clinicopathological and histological parameters, including MSI/MSS status, CD-8 cells, tumour-infiltrating lymphocytes (TILs), budding, TNM scale and grading. Moreover, we analyzed the B7H3-related pathways using available online datasets and the immunological context of B7H3 expression, through the 48-cytokine screening panel of cancer tissues homogenates, immunogenic features and immune composition. The study included 158 patients diagnosed with CRC. To assess B7H3 levels, we performed an immunohistochemistry method (IHC) and enzyme-linked immunosorbent assay (ELISA). To elucidate the immune composition of colorectal cancer, we performed the Bio-Plex Pro Human 48-cytokine panel. To study biological characteristics of B7H3, we used online databases. Expression of B7H3 was upregulated in CRC tumour tissues in comparison to adjacent noncancerous margin tissues. The concentrations of B7H3 in tumours were positively associated with T parameter of patients and negatively with tumour-infiltrating lymphocytes score. Additionally, Principal Component Analysis showed that B7H3 expression in tumours correlated positively with cytokines associated with M2-macrophages and protumour growth factors. The expression of B7H3 in tumours was independent of MSI/MSS status. These findings will improve our understanding of B7H3 role in colorectal cancer immunity. Our study suggests that B7-H3 is a promising potential target for cancer therapy. Further studies must clarify the mechanisms of B7H3 overexpression and its therapeutic importance in colorectal cancer. Immunotherapy is a recently discovered treatment that stimulates or inhibits the immune system from intensifying or decreasing an immune response and has rapidly become a significant treatment for multiple types of solid cancers .In colorectal cancer, immune checkpoint therapy is approved for treating tumours with a mismatch repair deficiency (MMR-D) and microsatellite instability-high (MSI-H) phenotype . MSI resPresent immune checkpoint inhibitors (ICIs) are ineffective in tumours that are microsatellite-stable (MSS) or have low levels of microsatellite instability (MSI-L) . ResearcThe B7 family molecules have received growing attention in recent years and are essential for regulating T-cell responses. The members of the B7 family function as immune regulatory ligands that exhibit interaction with the CD28 receptors family ,6. B7H3 Here, we aimed to assess the expression of B7H3 regarding clinicopathological parameters, including MSI/MSS status, CD8+ T-cells, histopathological features: budding, tumour-infiltrating lymphocytes (TILs), TNM scale, and grading. Moreover, we examined the B7H3-related pathways using available online datasets. Our research also aimed to investigate the immunological context of B7H3 expression through the 48-cytokine screening panel, immunogenic elements, and immune landscape.The samples used in the study were obtained from 158 patients who had undergone surgery due to CRC. Patients were operated in the 1st Specialistic Hospital in Bytom, Poland . The obtained specimens included colorectal tumour tissues and adjacent “tumour-free” surgical margins, confirmed in histological examination. The qualification for the study was led according to the inclusion and exclusion criteria described in our previous studies ,18. The To weigh and homogenize examined tumour and surgical margin tissue, we have followed our previous research papers’ homogenization protocol ,19. We cB7H3 expression was assessed in 77 randomly selected cases using B7H3 immunostaining. The tissue specimens from formalin-fixed paraffin-embedded tissue blocks were deparaffinized and rehydrated. Subsequently, antigen retrieval was conducted by incubating slides in EnVision Flex Target Retrieval Solution High pH  at 95 °C for 20 min. The prepared samples were treated with Peroxidase-Blocked Reagent (Dako) and then exposed to the B7H3 Polyclonal Antibody  with a 40 min incubation time and a dilution of 1:300. EnVision FLEX HRP (Dako) was applied afterwards. Later, 3,3′-diaminobenzidine was used to stain the antigen-antibody complexes. Next, the tissue sections underwent counterstaining with hematoxylin, and dehydration and were covered with coverslips for subsequent analysis. Two independent pathologists conducted a histological evaluation using an Olympus BX51 microscope. Tumours were classified as B7H3 positive if staining was detected in 1% or more cancer cells. Furthermore, the percentage of tumour cells exhibiting positive B7H3 staining was utilized for additional statistical analyses.To determine the MSI/MSS status, immunohistochemical (IHC) staining for MSH2, MSH6, PMS2, and MLH1 was conducted following the procedure described in our previous publications ,21. A reThe nuclear staining of invasive tumour cells for MSH2, MSH6, PMS2, and MLH1 was evaluated in an internal positive control  to assess the tumours. The tumours showing nuclear staining for at least 1% of invasive tumour cells were classified as having positive marker staining. The interpretation of immuno-histochemistry testing followed the criteria established by Olave and Graham  and descFor immunohistochemical (IHC) analysis, tissue sections measuring 4 µm in thickness were utilized. These sections underwent deparaffinization using xylene, rehydration in a series of graded alcohol, and thorough rinsing with deionized water. Subsequently, antigen retrieval was achieved by incubating the slides in EnVision Flex Target Retrieval Solution High pH  at 95 °C for 20 min. Prepared samples were then incubated with a peroxidase-blocked reagent (Dako), followed by treatment with the CD8+/144B mouse monoclonal antibody diluent. The incubation time was set for 40 min, with a dilution ratio 1:100. Subsequently, the samples were exposed to EnVision FLEX HRP (Dako). The antigen-antibody complexes were then subjected to staining with 3,3′-diaminobenzidine, which led to their visualization. Lastly, the tissue sections were counterstained with hematoxylin, dehydrated, and covered with coverslips for subsequent analysis, as described in the previous publications .The extent of lymphatic infiltration associated with the tumour was assessed in a semi-quantitative manner on the same H&E-stained slides using a five-grade scale, as per the guidelines established by Salgado et al. in breast cancer , which wThe levels of chemokines/cytokines/growth factors were assessed in 77 tissue homogenates using the Bio-Plex Pro Human cytokines screening panel 48 cytokines assay  following the manufacturer’s procedure. Briefly, 50 µL aliquot of tissue homogenate was diluted 1:2 with sample diluent, incubated with anti-body-coupled beads, biotinylated secondary antibodies, and subsequently with streptavidinphycoerythrin. Standard curves for each examined molecule were performed using corresponding cytokine standard solutions. The beads were measured using the Bio-Plex 200 System. The intra-assay %CV varied up to 15%, and the inter-assay %CV varied up to 25%, depending on the studied protein. We transformed the obtained levels to the corresponding total protein concentrations. This method was applied in our previous research .p.adj. < 0.05). For elucidating the association between B7H3 expression, immune cell infiltration, Mantis score, and patient survival, the web-based tool CAMOIP was utilized to analyze TCGA-COAD data (http://220.189.241.246:13838/#shiny-tab-188home (accessed on 1 March 2023)).Functional annotation analysis based on mRNA expression profiles was conducted on the CRC online dataset obtained from the FieldEffectCrc Package, specifically among cohort A consisting of 311 CRC samples . The DESt-test  and paired samples Wilcoxon test  were applied. Independent variables were compared using the Student’s t-test and Mann–Whitney U test. Correlations between the examined variables were assessed using Pearson’s or Spearman’s coefficients, depending on the distribution of the variables. The relationship between the levels of the studied proteins and the TNM scale was determined using Tau-Kendall’s tau rank correlation coefficient. Principal component analysis was carried out to decrease the number of correlated variables and elucidate the connections between B7H3 expression and cytokine concentrations obtained from the used screening panel. The principal components exhibiting the greatest explained variance were employed as fresh variables for subsequent analyses. Significance at a statistical level was defined as p-values ≤ 0.05. The statistical analysis was executed using STATISTICA 13 software  and R Studio .The distribution of the analyzed data were evaluated utilizing the Shapiro–Wilk test. For a better fit to the Gaussian distribution, a logarithmic transformation was applied to the levels of the analyzed molecules. Mean ± SD was used to present variables with a normal distribution, while median with interquartile range was used for variables with a non-normal distribution. To compare tumour and margin levels, the paired Student’s In this research, to investigate the expression of B7H3 in CRC patients, the B7H3 concentrations were determined in tumour tissue and normal colon mucosa adjacent to CRC. In the 154 pairs of tumour tissues and adjacent tumour-free surgical margins, the B7H3 levels were assessed with an ELISA test. The concentrations of B7H3 were significantly higher in the tumour tissue compared to the margin tissue . Furthermore, the B7H3 levels in tumour tissue homogenates correlated positively with the T parameter and were negatively associated with the TILs score .p = 0.52). We did not observe any association between B7H3 IHC expression in tumour cells and clinicopathological features of the patients   and a grIn PCA for protumour cytokines , factor CAMOIP allows us to perform survival analysis according to the low or high expression level of each gene in the TCGA cohort. To investigate the difference in overall survival between individuals with high and low B7H3 expression in the TCGA COAD cohort, we performed a survival analysis in CAMOIP. The patients with high B7H3 expression showed significantly decreased overall survival time compared to the low expression group .To elucidate the link between B7H3 expression and immune infiltration landscape in CRC tumours, we used CAMOIP- a tool for analyzing the expression and mutation data from TCGA which provides comprehensive analysis on potential immunotherapy targets. Immune Infiltration cells analysis was estimated using CIBERSORT algorithm. The percentages of CD4+ memory resting T-cells, CD4+ memory activated T-cells were lower in CRC tumours with upregulated B7H3 expression, while conversely an increased fraction of regulatory T-cells (Tregs), monocytes, macrophages M0, eosinophils and neutrophils was associated with higher B7H3 expression . The immp. adjusted < 0.05) in the enrichment of the Molecular Signature Database Collection for Hallmark gene sets are provided in To determine pathways activated in tumours with upregulated B7H3 expression, gene set enrichment analysis (GSEA) was performed to analyze the difference in gene expression between low and high B7H3 expression datasets. Significantly upregulated and downregulated pathways (Immune Checkpoint Therapy (ICT) is a new approach to cancer treatment aimed at strengthening anti-tumour response mediated by T-cells and is characterized by high efficiency. B7 family and its most known member—PD-L1—are known for regulating immune response mediated by T-cells and exhibiting significant antitumour effect, which has been widely described in several malignancies. Due to the limited possibilities of therapies targeting the PD-1/PD-L1 axis in CRC, new members of the B7 family, including B7H3, are widely investigated. B7H3, a recently discovered protein belonging to the B7 family that acts as a T-cell inhibitor promoting tumour cell invasiveness rather than a T-cell stimulator as previously classified, has gained increasing attention in the anti-cancer battlefield . NumerouOn the mRNA level, B7H3 is widely expressed in most normal tissues, but the presence of B7H3 protein is strictly limited, suggesting a significant role in post-transcriptional regulation of the B7H3 gene . On the We explored the relationship between B7H3 expression and the clinicopathological parameters of the patients. The results showed a positive correlation between B7H3 tumour concentration and the T parameter, thus suggesting that B7H3 contributed to CRC progression. Similarly, Ingebrigtsen et al. reported that high B7H3 expression was associated with the advanced TNM stage in CRC . FurtherAs we suspected, B7H3 levels in the tumour were negatively correlated with tumour infiltrating lymphocytes (TILs) score. In CRC, tumour-infiltrating lymphocytes (TILs) consist of different populations of cells  and are regarded as positive prognosis factors . B7H3 waTumour-associated macrophages (TAMs) that promote tumour invasion and metastasis elicit phenotypes of M2-polarized macrophages . MacrophExploring the cytokine network in the CRC tumour microenvironment may augment the current understanding of interrelationships between cytokines, chemokines, growth factors, and immune checkpoints and their common influence on TME modulation and tumour progression. As immune checkpoints blockade may reshape TME and restore antitumour response, investigating associations between B7H3 expression and functional groups of TME molecules could be extremely valuable for predicting the effects of B7H3 targeted therapy. Analysis of cytokines showed that B7H3 is related to protumour cytokines including IL-1β, IL-1Rα, IL-4, Il-5, IL-8, IL-9, IL-10, IL-13, IL-17A and TNFβ. Factor 1 obtained from PCA containing these molecules was positively related to B7H3 expression. IL-1β secreted by TAMs and neutrophils was found to promote cancer cell proliferation and recruit myeloid-derived suppressor cells (MDSCs) supporting tumour growth . IL-4 exWe also used CAMOIP, a web-based tool analyzing the TCGA-COAD dataset, to explore the immune composition of CRC according to high and low B7H3 expression. We found that upregulation of B7H3 affects only a few populations of immune cells, including CD4+ memory resting T-cells, CD4+ memory activated T-cells, regulatory T-cells, monocytes, macrophages M0, eosinophils and neutrophils. However, the immune score for Th1 cells, stromal fraction, macrophages regulation, lymphocytes infiltration, IFN gamma response and TGF beta was higher in a group with high B7H3 expression, indicating B7H3 contribution to immunological processes associated with tumourigenesis. Additionally, high B7H3 expression was associated with decreased survival in the TCGA-COAD dataset. This finding is consistent with clinical studies in which B7H3 was demonstrated to predict shorter overall survival and disease-free survival in CRC cohorts . Analyzing the FieldEffectCrc dataset, we explored functions, processes and upregulated pathways associated with high B7H3 expression using GSEA and GO enrichment analysis.Enrichment analysis showed that B7H3 is related to upregulated pathways involved in oxidative phosphorylation, fatty acid metabolism and downregulation of KRAS signalling. Recently, Picarda et al. demonstrated that B7H3 may play a pivotal role in regulating adipose tissue metabolism. B7H3 expression was upregulated in adipose tissue, with the highest levels in adipocyte progenitor cells and lower levels with the initiation of adipocyte differentiation. Additionally, the knockout of the B7H3 gene in progenitor cells led to impairment in aerobic metabolism and accumulation of fatty acids . MutatioFurthermore, high B7H3 expression is associated with downregulating pathways related to protooncogene MYC and cell-cycle control systems, such as G2/M Checkpoint and E2F target. Downregulation of the G2/M Checkpoint and E2F target indicates that one of the effects of B7H3 could be impaired G2/M phase cell cycle arrest. However, literature data about the association of B7H3 and the MYC gene are limited and concern only malignant gliomas in which a knockout of the B7H3 gene inhibits MYC expression . Functional analyses of B7H3 revealed its involvement in immunological-associated processes. Significantly enriched GO terms were related to various immunological functions, including innate and adaptive immunity, such as phagocytosis, complement activation, antigen binding, activation of B-cells and their receptors, and regulation of immunoglobulin complex.Our findings improved our understanding of B7H3′s role in cancer immunity. In addition, the studies on colorectal tumour tissues allowed us to estimate clinical, pathological, and immunological parameters. We assessed B7H3 concentrations by ELISA, and IHC, determined MSI/MSS status, and estimated the immune landscape by cytokines screening panel and tumour-infiltrating CD8+ T-cells. The analysis based on mRNA expression profiles has significantly expanded the scope of research by reporting many significant molecular roles of B7H3. CAMOIP, a tool for comprehensive analysis of potential immunotherapy targets, allowed us to perform survival analysis according to low or high B7H3 expression in the TCGA cohort and determined the link between B7H3 expression, immune cell infiltration, Mantis score, and patients’ survival in CRC tumours. We studied the pathways activated in tumours with upregulated B7H3 expression with gene set enrichment analysis. Our future research direction focuses on further studies of B7H3, as a potential target for immunotherapy which requires extending research to cellular, animal, and clinical models. The other cancer therapy trends require studying the diagnostic and therapeutic potential of B7-H3 thoroughly, its cellular and molecular mechanisms, B7H3-mediated metastases, tumour-associated vasculature, and recurrence. Clinical data with a long-term follow-up are essential to understand the relevance of B7H3 expression.There are some potential limitations in this research. The first one concerns using cytokines screening panels and in silico analyses to explore the immune landscape of CRC tumours without indirect assessment of selected immune cell populations through immunostaining or flow cytometry. Furthermore, the study has an observational character, and the main findings should be further evaluated using cell lines and animal tumour models.Our study showed that B7H3 is overexpressed in CRC tumours and is independent of MSI/MSS status. High concentrations of B7H3 were positively associated with the patients’ T parameter suggesting its role in promoting tumour growth. Additionally, B7H3 may support tumour progression by shaping immunosuppressive tumour microenvironment mediated by M2-like macrophages and anti-inflammatory cytokines. The findings of this study suggest that B7-H3 is a promising potential target both in MSI and MSS CRC tumours.


To understand the phenotypic and genotypic spectrum of genetic forms of rickets in 10 families.Detailed clinical, radiographic, and biochemical evaluation of 10 families with phenotypes suggestive of a genetic cause of rickets was performed. Molecular testing using exome sequencing aided in the diagnosis of six different forms of known genetic causes.CYP27B1:c.1319_1325dup, p.(Phe443Profs*24), VDR:c.1171C>T, p.(Arg391Cys), PHEX: c.1586_1586+1del, PHEX: c.1482+5G>C, PHEX: c.58C>T, p.(Arg20*)) and six novel variants , CYP27B1: c.1376G>A, p.(Arg459His), CYP2R1: c.595C>T, p.(Arg199*), CYP2R1:c.1330G>C, p.(Gly444Arg),SLC34A3:c.1336-11_1336-1del, SLC2A2: c.589G>C, p.) in the genes known to cause monogenic rickets were identified.Eleven disease-causing variants including five previously reported variants (The authors hereby report a case series of individuals from India with a molecular diagnosis of rickets and provide the literature review which would help in enhancing the clinical and molecular profile for rapid and differential diagnosis of rickets.The online version contains supplementary material available at 10.1007/s12098-022-04393-9. Rickets is a disorder of growing bone caused by a deficiency of calcium/phosphorous/vitamin D or defects in their metabolism. Defective mineralization and widening of the cartilaginous growth plates are the characteristic features of rickets . Since tCYP3A4 has been reported . Sub. SubSLC3SLC2A2 (solute carrier family 2 member 2) or GLUT2 (glucose transporter 2 protein) known to mediate bidirectional glucose transport [SLC2A2) observed in the present study (in P10) is a novel alternate variant for the first-ever reported variant in SLC2A2 in the homozygous state which causes a different missense change at the same amino acid codon [SLC2A2) has been in discussion since its first description as it was also reported in heterozygous state in African American women with diabetes mellitus (DM). Genotype–phenotype correlation by a recent study highlights that biallelic/nonfunctional variants show a complete spectrum of FBS including hepatonephromegaly owing to glycogen accumulation, renal tubular dysfunction, and hypophosphatemic rickets whereas subjects with single-heterozygote variants are susceptible to NIDDM owing to impaired sugar transport in the kidney [Hypophosphatemia is a common feature of many other diseases which are not considered to be a classical form of HR. Fanconi–Bickel syndrome, FBS MIM # 227810) an autosomal recessive disease is one such entity that mimics hypophosphatemic rickets as well as osteogenesis imperfecta (OI) [810 an auransport . Affecteransport . The varid codon . The repe kidney . ClinicaDiagnosis of rickets requires a multifaceted approach which begins with the collection of detailed family history, clinical evaluation, radiographic and biochemical investigations, and use of next-generation sequencing (NGS) to receive a definitive molecular diagnosis. Often, the clinical profile of monogenic rickets is similar to classical nutritional deficiency rickets, which leads to a delay in diagnosis and failure of treatment. However, in the present era, a low index of suspicion of genetic rickets and early deployment of genetic tests are necessary to achieve a rapid and accurate diagnosis. Recently, Marik et al. reported a cohort of 63 individuals affected with rickets and proposed a gene panel for the diagnosis of hypophosphatemic rickets . MoleculCYP27B1, CYP2R1, SLC34A3, and SLC2A2), the present study provides an update to the mutation spectrum.In this study, the authors have presented profiles of six different genetic forms of rickets including vitamin-D-dependent  rickets, hypophosphatemic rickets (XLDHR and HHRH), and a disease entity with rickets as one of the clinical features, Fanconi–Bickel syndrome (FBS). With the addition of six novel variants in four known genes (Supplementary file1 (DOCX 272 KB)Supplementary file2 (DOCX 24 KB)Supplementary file3 (DOCX 13 KB)Supplementary file4 (DOCX 16 KB)Below is the link to the electronic supplementary material.