Tumors harboring nuclear β-catenin were associated with a type II TIME , whereas tumors with membranous β-catenin expression were predominantly type IV . Combined, but not individual, high stromal CD8+ TILs and membranous β-catenin expression was independently associated with better disease-specific survival . Taken together, a combination of high stromal CD8+ T-cell infiltration and membranous β-catenin in the tumor emerges as an independent predictor of better survival in OSCC patients.The aim of this study was to investigate the prognostic relevance of β-catenin expression in oral squamous cell carcinoma (OSCC) and to explore relationships with the tumor immune microenvironment. Expression of β-catenin and PD-L1, as well as lymphocyte and macrophage densities, were evaluated by immunohistochemistry in 125 OSCC patient specimens. Membranous β-catenin expression was detected in 102 (81.6%) and nuclear β-catenin in 2 (1.6%) tumors. There was an association between β-catenin expression, tumoral, and stromal CD8 When Fzrulation .+ T-cell activation, and promoted tumor growth. Conversely, β-catenin depletion reduced PD-L1 expression levels in tumor cells, enhanced CD8+ T-cell infiltration, and inhibited tumor growth [The Wnt/β-catenin pathway is aberrantly activated in numerous tumor types , includiIn this study, we investigated the significance of the β-catenin expression pattern in a large cohort of OSCC patients using tissue microarray immunohistochemistry, its association with the type of tumor immune microenvironment (TIME), and the potential prognostic relevance in OSCC.A cohort of 125 OSCC patients who received surgical treatment at the Hospital Universitario Central de Asturias (HUCA) between 1996 and 2007 was retrospectively selected. This study was conducted following the ethical criteria of the Declaration of Helsinki and approved by the HUCA Ethics Committee and also by the Regional CEIC from Principado de Asturias . Written informed consent was obtained from all patients. We retrieved clinical and histopathologic information from the patients’ files, and pathology reports, which are summarized in Formalin-fixed paraffin-embedded (FFPE) tissue samples were sourced from the Principado de Asturias BioBank (PT20/0161) and processed following standard operating procedures. Clinicopathologic data were collected from clinical records. The inclusion criteria for all the participants enrolled were: (i) primary OSCC , (ii) treatment-naïve patients from whom we had formalin-fixed paraffin-embedded (FFPE) primary biopsy tissue samples, and (iii) with a minimum follow-up of at least three years for alive patients. In addition, the exclusion criteria were: (i) OSCC with immediate postoperative death, (ii) recurrent disease, (iii) neoadjuvant chemo- or radiotherapy, and (iv) missing survival data. All patients underwent surgery of the primary tumor with curative intention as well as neck dissection. None of the patients received any treatment before surgery, but complementary radiotherapy and/or chemotherapy were administered in 75 (60%) and 14 (11%) cases, respectively. During the follow-up period (6 to 230 months), 19 (15%) patients suffered from a second primary tumor in the oral cavity, and 51 (42%) patients died of OSCC. The clinical endpoint of this study was disease-specific survival (DSS), calculated as the period of time between the initial treatment and the death caused by the tumor or the presence of a non-treatable recurrence.Samples and data from donors included in this study were provided by the Principado de Asturias BioBank (PT20/0161), integrated in the Spanish National Biobanks and Biomodels Network financed with European funds and they were processed following standard operating procedures with the appropriate approval of the Ethical and Scientific Committees.Tissue microarrays (TMAs) were constructed by collecting 1 mm tissue cores from the most morphologically representative areas of formalin-fixed, paraffin-embedded (FFPE) tissue blocks. Three individual cores were taken per tumor block. Then, 3 μm tissue sections dried on Flex IHC microscope slides  were heated with high-pH Envision Flex Target Retrieval solution (Dako) and stained on an automatic staining workstation  using monoclonal antibodies against β-catenin , CD8 , FoxP3 , PD-L1 antibody , CD20 , CD4 , CD68 , and CD163 . The antibody-antigen complexes were visualized using the Dako EnVision Flex + Visualization System (Dako) and diaminobenzidine chromogen as a substrate. Each TMA also contained three cores of morphologically normal oral mucosa from non-oncological patients undergoing oral surgery, used as internal controls. No staining was observed when the primary antibody was omitted. Negative control was included by replacing the primary antibody with serum and positive controls using appropriate positive control tissue slides. +, CD68+, CD163+, CD4+, CD8+, and FOXP3+ cells in both the tumor nests and the tumor stroma was counted in each 1 mm2 area from three independent HPFs at 400x, as we previously reported [+ lymphocyte densities into two groups, above (high density) and below (low density) the median number of positive stained cells for the total patient population. Additionally, TIL intensity in the tumor nests and the surrounding stroma was subdivided into three groups: negative, mild-moderate, or intense. PD-L1 expression in more than 10% of the tumor cells was previously found to significantly associate with poorer survival [The IHC results were independently evaluated by two observers (VB-L and FJS-S), blinded to clinical information. The number of CD20reported ,22. The survival , and thesurvival .Statistical analyses were carried out using SPSS software version 27 . Continuous variables were expressed as the mean ± standard deviation (SD), and absolute and relative frequencies were calculated for categorical variables. Associations between the numbers of immune cells infiltrating the tumors and β-catenin expression were assessed by using the Kruskall–Wallis test. Fisher’s exact test was used to evaluate the relationship between β-catenin expression, the type of TIME, and TIL intensity. Potential associations of β-catenin and the type of TIME with the different clinicopathological variables were assessed using the Chi-square or Fisher’s exact test. p-values less than 0.05 were considered statistically significant. Disease-specific survival (DSS) was estimated using the Kaplan–Meier method, and the log-rank test was used for comparisons between survival rates. Hazard ratios (HR) with their 95% confidence intervals (CI) were calculated using univariable and multivariable Cox regression models. All tests were two-sided, and + TILs in the tumor nests was 47.88 ± 57.16 cells per mm2 (range: 0.00 to 288.33), and the mean stromal CD8+ TILs 178.45 ± 203.21 (range: 0.33 to 1202.67) cells per mm2. β-catenin immunostaining was evaluated in 125 OSCC samples. Membrane β-catenin expression was detected in 102 (81.6%) tumors, nuclear β-catenin was only found in 2 cases (1.6%), and no expression of β-catenin neither in the membrane nor the nucleus was observed in 21 cases (16.8%). Representative images of positive β-catenin staining (membranous and nuclear) are shown in + T cells in both stroma and tumor nests, and β-catenin immunoexpression . Similarly, the mean number of tumoral CD8+ T cells was higher in tumors with negative β-catenin expression, and also the lowest CD8+ TIL density was seen in the subset of tumors with positive nuclear β-catenin . Nevertheless, since only two cases exhibited nuclear β-catenin, these figures should be cautiously taken into account. Moreover, there was a tendency of association between PD-L1 positivity and membranous β-catenin expression, although this relationship did not reach statistical significance .There was an association between the infiltration of CD8pression . The meap = 0.012). Tumors harboring nuclear β-catenin were associated with type II TIME , whereas membranous β-catenin was predominantly associated with type IV   . + (p < 0.0001), CD163+ (p = 0.006), and tumoral CD8+ (p = 0.02)  .p = 0.06).Absence/loss of β-catenin expression or nuclear β-catenin expression were more frequently observed in larger tumor sizes (T3–T4) (21% vs. 17% in T1-T2), tumors with neck lymph node metastasis (pN+) (25% vs. 15% in pN0 cases), and recurrent tumors (22% vs. 16% in non-recurrent tumors); however, these differences did not reach statistical significance. Regarding histological degree of differentiation, absence or nuclear β-catenin expression was more frequently detected in moderately and poorly differentiated tumors, (22% vs. 16% in well-differentiated tumors), almost reaching significance (+ TILs were divided into two categories (high vs. low density) according to their respective median values. Only stromal CD8+ TILs, but not tumoral CD8+ TILs, were significantly associated with DSS   and positive membranous β-catenin showed the highest DSS (p = 0.012)  A as prev= 0.045) B. Howeve= 0.012) C.+ TIL infiltration combined with tumoral β-catenin expression  showed that the parameters independently associated with worse DSS were T3-T4 classification , and neck node metastasis . Conversely, high CD8+ TIL density combined with membranous β-catenin was a significant independent predictor of better DSS . Finally, the degree of differentiation was not significantly associated with better DSS . If we included in the multivariable analysis the stromal CD8+ TIL infiltration instead of this variable combined with tumoral β-catenin expression, T and N parameters retained their significant association with a worse DSS; however, CD8+ TILs density did not reach a significant independent association with survival .Multivariable Cox regression analysis, including T classification (T1-T2 vs. T3-T4), N classification (N0 vs. N+), differentiation grade (well-moderately vs. poorly differentiated), and stromal CD8β-catenin is an 88 kDa multifunctional and evolutionary-conserved protein, a member of the armadillo family of proteins, and involved in two independent processes, which are cell-cell adhesion and signal transduction . β-catenHerein, we did not find any significant relationship between β-catenin expression and the clinicopathological characteristics of our sample. Nevertheless, abnormal β-catenin expression (defined as absence/loss of expression or nuclear expression) was more frequent in T3-T4 tumors, with the presence of nodal metastasis as well as recurrent OSCC tumors. Similar to our results, Al-Rawi et al.  found noAmong the reasons that may explain the differences and inconsistencies from the available data are small sample sizes in most studies, methodological differences in immunohistochemical evaluation and scoring, often subjective, and different antibodies/epitopes used. + TILs in the TME, both in the tumor as well as in the surrounding stroma, and with a tendency to a low tumoral PD-L1 expression. The interaction between PD-1 and PD-L1 inhibits the activation and effector functions of CD8+ T cells and induces suppressive Treg cells, ultimately leading to cancer immune evasion [In this study, we showed that β-catenin expression in the nucleus of oral cancer cells was associated with the lowest density of CD8 evasion ,38. In t evasion . + TIL density and β-catenin expression in OSCC. Furthermore, tumors harboring nuclear β-catenin were associated with a type II TIME , as reported in other HNSCC subsites [CD274 gene to induce PD-L1 expression [Tumors are closely related to the TME, and there is a continuous interplay between tumor cells and stromal cells. It has been recently reported that most tumors are inversely related to a T cell-inflamed gene expression signature, potentially linked to Wnt/β-catenin pathway activation . Here, wsubsites . Notablysubsites ,39. We asubsites . The molsubsites . Howeverpression . + helper T cells [The Wnt/β-catenin pathway has been identified as one of the most important oncogenic signaling pathways associated with tumor immune evasion , and its T cells  and limi T cells . Additio T cells . Moreove T cells . + and CD163+ macrophages infiltrating the tumor, and also with the tumor-infiltrating CD8+ T cells. In a recent review, Li et al. [+ T-cell infiltration within the TME and production of Treg cells with subsequent inhibition of cytotoxic T-cell activity. Glycogen synthase kinase 3 (GSK3) inhibition in cancer cells promotes β-catenin activation and subsequently PD-L1 stabilization, driving in turn cytotoxic T-cell activity exhaustion. On the other hand, non-canonical Wnt signaling is initiated by Wnt5a-type ligands [Furthermore, we also found a significant relationship between the density of TILs within the tumor nests and stroma, with the density of CD68i et al.  summariz ligands . Wnt1 li ligands , whereas ligands ,48,49,50 ligands . Further+ T cells. As far as we know, this is the first study to analyze the relationship between β-catenin expression and the immune infiltrate TME in OSCC, mainly focused on CD8+ T cells in the OSCC TME and potentially increase the efficacy of immunotherapy in the clinical setting. Hence, β-catenin emerges as a valuable molecular target to improve the efficacy of immunotherapy in OSCC by increasing TIL density in the TME. Notably, a combination of stromal CD8+ T-cell infiltration and membranous β-catenin expression in the tumor was found to be an independent predictor of better DSS for OSCC patients.In conclusion, our findings support that the Wnt/β-catenin pathway could be a molecular target of paramount relevance in an effort to elicit an infiltration of CD8


Introduction: Wnt/β-catenin signaling controls cell division and lineage specification during embryonic development, and is crucial for stem cells maintenance and gut tissue regeneration in adults. Aberrant activation of Wnt/β-catenin signaling is also essential for the pathogenesis of a variety of malignancies. The RNA-binding protein IGF2BP1 is a transcriptional target of Wnt/β-catenin signaling, normally expressed during development and often reactivated in cancer cells, where it regulates the stability of oncogenic mRNA.Methods: In this study, we employed iCLIP and RNA sequencing techniques to investigate the role of IGF2BP1 in the post-transcriptional regulation of Wnt/β-catenin-induced genes at a global level within colorectal cancer (CRC) cells characterized by constitutively active Wnt/β-catenin signaling.Results and Discussion: In our study, we show that, in contrast to normal cells, CRC cells exhibit a much stronger dependency on IGF2BP1 expression for Wnt/β-catenin-regulated genes. We show that both untransformed and CRC cells have their unique subsets of Wnt/β-catenin-regulated genes that IGF2BP1 directly controls through binding to their mRNA. Our iCLIP analysis revealed a significant change in the IGF2BP1-binding sites throughout the target transcriptomes and a significant change in the enrichment of 6-mer motifs associated with IGF2BP1 binding in response to Wnt/β-catenin signaling. Our study also revealed a signature of IGF2BP1-regulated genes that are significantly associated with colon cancer-free survival in humans, as well as potential targets for CRC treatment. Overall, this study highlights the complex and context-dependent regulation of Wnt/β-catenin signaling target genes by IGF2BP1 in non-transformed and CRC cells and identifies potential targets for colon cancer treatment.  The canonical Wnt/β-catenin signaling pathway is a highly intricate and conserved process that plays a vital role in various developmental and physiological functions, such as embryonic development, stem cell maintenance, and tissue homeostasis . In embrThe Wnt signaling pathway is initiated when Wnt ligands bind to their receptors, which then recruit co-receptors and activate the Frizzled receptor. This initiates a cascade of events that results in the stabilization of β-catenin and its translocation into the nucleus. Once in the nucleus, β-catenin regulates target gene expression directly by interacting with the TCF/LEF family of transcription factors. The TCF/LEF proteins act as transcriptional activators or repressors, depending on their interaction with β-catenin . When thThe canonical Wnt pathway regulates the expression of several genes involved in cell proliferation and differentiation, such as c-Myc, Cyclin D1, and Axin2 . MoreoveIn this study we demonstrated the crucial role of IGF2BP1 as a modulator of Wnt/β-Catenin-regulated genes on transcriptome-wide level in CRC cells. Additionally, we conducted an Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) assay to identify the direct binding targets of IGF2BP1 in wild-type MEFs and DLD1-dnTCF7L2 cells. Our study categorized the Wnt-regulated genes into three groups based on their mode of regulation . The first group comprises Wnt transcriptional target genes that are not regulated by IGF2BP1 . The second group includes genes that are regulated both transcriptionally by TCF/β-catenin and post-transcriptionally by IGF2BP1, such as cMYC and MITF. The third group consists of genes that are regulated by Wnt solely post-transcriptionally by IGF2BP1  . This stTo investigate the role of IGF2BP1 in expression of Wnt-regulated transcriptome of CRC cells, we selected the DLD1 cell line, that harbors a homozygous APC mutation that results in constitutive activation of Wnt/β-catenin signaling . We geneConsidering the role of the RNA-binding protein IGF2BP1 as a modulator of mRNA stability and translation, we conducted a comprehensive transcriptome analysis using total mRNA sequencing in CRC DLD1 cells expressing either dnTCF7L2 alone or in combination with IGF2BP1 . The intSubsequently, a proliferation assay was conducted within these cell lines, showing, the deactivation of Wnt/β-catenin signaling led to a decline in proliferation. Remarkably, upon the overexpression of IGF2BP1 into the cells with β-catenin/Tcf inhibition, the proliferation rate was reinstated to that observed in the original cell line. This underscores the potential role of IGF2BP1 in upholding the Wnt-dependent tumorigenic phenotype .To identify a subpopulation of genes that are transcriptional targets of Wnt/β-catenin signaling and post-transcriptional targets of IGF2BP1, we conducted an analysis to match the list of TCF7L2 ChIP genes from Hatzis et al.'s study with the 177 genes (IGF2BP1 dependent regulation by Wnt) obtained from our transcriptome analysis . From thWe conducted an individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) assay to investigate and compare the IGF2BP1 interactome between DLD1 cells with constitutively active Wnt/β-Catenin signaling and DLD1-dnTCF7L2 cells with inhibited Wnt/β-Catenin signaling . We obseNext, we compared the list of direct IGF2BP1 RNA targets  with the list of genes that are regulated by Wnt/β-catenin and IGF2BP1 . Comparing these two lists revealed a common set of 17 genes  Supplem. These f−/− gene knockout (Igf2bp1-KO), and a recombinant Wnt-3a ligand to study the role of Igf2bp1 upon Wnt induction in vitro. We have chosen MEFs for these studies for the following reasons: i) in contrast to embryonic cells, the majority of non-transformed adult cells  do not express appreciable levels of IGF2BP1; ii) CRISPR-mediated knockout of IGF2BP1 results in chronic effects on gene expression that is not suitable for these studies. Therefore, we took advantage of our tamoxifen-inducible loxP-mediated excision of Igf2bp1 that produce efficient knockout in short-term cell cultures. MEFs isolated from UBC-CreERT2:Igf2bp1flox/flox mouse embryos were treated with tamoxifen to induce IGF2BP1 KO  with Igf2bp1F2BP1 KO . To actiF2BP1 KO . These rTo assess IGF2BP1’s role on genes activated by Wnt signals, we conducted mRNA sequencing in both wild-type (WT) and IGF2BP1 knockout (KO) MEFs. Wnt signaling was stimulated by Wnt3a treatment in both cell types. In WT MEFs, Wnt3a treatment prompted notable changes in 170 genes  < 0.05). Among these, 82 genes exhibited increased expression, while 88 genes showed decreased expression  comparedThe Gene ontology (GO) analysis of these 24.1% of genes regulated by both Wnt/β-Catenin and IGF2BP1 showed significant enrichment in cell processes involved in mitochondrial metabolism . The XTTTo investigate the Wnt-induced changes in RNA-binding characteristics of IGF2BP1 using MEFs Igf2bp1-KO system, we performed iCLIP analysis in MEFs treated with Wnt3a and MEFs treated with vehicle control . The anaWe also found that the number of binding events for IGF2BP1 considerably increased with the activation of Wnt/β-Catenin signaling. We identified 13,292 binding sites on all identified transcriptional targets of IGF2BP1 in Wnt3a-induced MEFs, with 8,834 identified as protein-coding transcripts and 4,458 as non-protein coding transcripts, compared to 2,495 binding targets in MEFs treated with the vehicle control, with 1,591 identified as protein-coding transcripts and 904 as non-protein coding transcripts . A searcTo identify Wnt-regulated genes that directly depend on interactions of their mRNAs with IGF2BP1, we analyzed the intersection of IGF2BP1 target genes obtained from Wnt3a-treated MEFs iCLIP and 24.1% of genes (whose regulation by Wnt depends on IGF2BP1) obtained from transcriptome analysis. We found eleven genes in the intersection analysis, with Ptgs2 being a known binding target for IGF2BP1  Figure .Taking into consideration that IGF2BP1 plays role in regulating Wnt/β-catenin-induced transcriptome in CRC cells, we, next, were seeking to identify a prognostic significance based on the 17 genes identified above as a direct IGF2BP1 targets in DLD cells. We analyzed the expression of these 17 gene’s mRNA levels in CRC patients using the Gene Expression Profiling Interactive Analysis-2 (GEPIA2)  online tNext, we proceeded to verify the binding of IGF2BP1 with the aforementioned five signature genes by tracking the regions of read enrichment for IGF2BP1 binding from our DLD1 iCLIP data. Our analysis revealed that four out of five genes, namely MGAT5, MET, ZNF33A, and DPY19L1, had read enrichment for IGF2BP1 at different regions compared with the iCLIP data for DLD1-dnTCF7L2  Supplem. To furtWe utilized the online GEPIA2 tool with the COAD TCGA cancer dataset to conduct survival analysis and identify the most prominent genes associated with survival, as outlined in Studies have shown that being regulated by Wnt/β-catenin signaling, IGF2BP1 also interacts with several components of the Wnt signaling pathway and controls their expression and function. Another potential mechanism of IGF2BP1 involvement in regulation of canonical Wnt signaling is its reported ability to regulate the expression of β-TrCP1 ubiquitin ligase receptor that target β-catenin for ubiquitination and proteasomal degradation . HoweverIGF2BP1 has been shown to enhance β-TrCP1, expression in colorectal cancer, establishing its role in CRC progression . HoweverIn contrast, in CRC cells, where Wnt signaling is constitutively active, we observed that only 25.6% of Wnt regulated genes were independent of IGF2BP1, as shown in The intricate regulatory mechanism of IGF2BP1 for Wnt signaling target genes sparked our interest in investigating how IGF2BP1 interacts with its targets in diverse conditions of Wnt signaling. In order to gain deeper insights into the interaction of IGF2BP1 with its targets under different Wnt signaling conditions, we carried out iCLIP experiments on non-transformed and CRC cells. Specifically, we induced Wnt signaling in one set of experiments (non-transformed) and blocked it in another set (CRC) to study the effects on IGF2BP1-target interactions. Our findings indicate that in non-transformed cells, the interactome in Wnt-induced conditions is significantly larger than the interactome in uninduced conditions. Specifically, we observed an interactome of 13,292 in the former condition, which is more than five times greater than the 2,495 interactomes identified in the latter condition. Notably, both mRNA and ncRNA were present in these interactomes, with mRNA comprising the majority of the interactomes. Moreover, we observed an increase in both mRNA and ncRNA interactomes in response to Wnt-induction . Based oOverall, in non-transformed cells, the majority of upregulated genes were activated by Wnt ligand treatment independent of IGF2BP1. However, a subset of Wnt ligand-induced genes in non-transformed cells were regulated by IGF2BP1, including 11 direct binding targets. In contrast, in CRC cells where Wnt signaling is constitutively active, a vast majority of downregulated genes upon β-catenin/Tcf7L2 blockage were found regulated by IGF2BP1, and 17 of these genes were identified as direct binding targets of IGF2BP1. Among the 177 genes affected by both Wnt signaling and IGF2BP1, only 9.6% (17 genes) were found to directly interact with IGF2BP1. This outcome could be attributed to our comparison of the CRC cell DLD1 IGF2BP1 interactome list with the known human Transcription factor list. This analysis revealed potential interactions and regulatory roles of IGF2BP1 with 139 transcription factors, which in turn might be influencing the remaining 160 genes in our transcriptome data . NotablyFurther, matching the IGF2BP1 interactome data with transcriptomic data, we have found 17 common genes, including MGAT5 and MET, which showed a higher degree of expression in colon tumors analyzed from the TCGA database. Our study uncovered a five-gene signature, including IGF2BP1, which showed a significant association with disease-free survival in human CRCs. MGAT5 was found to be a direct target of IGF2BP1 , and incOverall, our study emphasizes the importance of IGF2BP1 in regulating Wnt target genes. Activation of the Wnt/β-Catenin-IGF2BP1 pathway in cancer cells leads to increased gene expression of a subset of genes, promoting an aggressive tumor phenotype and unfavorable prognostic outcomes.Mycoplasma contamination was assessed in all cell lines using the MycoAlert PLUS mycoplasma detection kit (Lonza).DLD1 cells, a human colon cancer cell line, and its transformed counterparts were cultured and maintained as monolayers in RPMI medium  supplemented with 10% (v/v) fetal bovine serum  and 100 units/ml penicillin and streptomycin (Corning). The cells were incubated at 37°C with 5% CO2. All animal studies were conducted following approved protocols by the Institutional Animal Care and Usage Committee of Penn State College of Medicine, Hershey. Embryos from UBC-CreERT2:Igf2bp1flox/flox transgenic mice were isolated between E13.5. After removing the heads, tails, limbs, and most internal organs, the embryos were minced and trypsinized for 20 min, then seeded into 100 mm cell culture dishes coated with gelatin in 10 mL of complete MEF media (DMEM supplemented with 10% heat-inactivated FBS). When the cells reached confluency, they were split at 1:2–1:3 ratios. This process was repeated two or three times to obtain a morphologically homogeneous culture, after which the cells were either frozen or expanded for further studies. Tamoxifen treatment was used to induce Igf2bp1 knockout in MEF cells. To activate Wnt signaling in MEF cells, Wnt3a ligand was employed.http://n2t.net/addgene:42592; RRID: Addgene_42592), into the pInducer-20 vector. A doxycycline-inducible overexpression lentiviral plasmid for IGF2BP1 was created by cloning PCR-amplified human IGF2BP1 ORF into the pInducer-21 vector. The pInducer20 and pInducer21 were gifted from Stephen Elledge and Thomas Westbrook ,   . LentiviFor SDS-PAGE and immunoblotting, DLD1-dnTCF7L2-GFP, DLD1-dnTCF7L2-IGF2BP1, MEF wild-type, and MEF-Igf2bp1-KO cells were harvested, and whole-cell extract (WCE) was prepared by adding 1 mL of cell lysis buffer per 5 × 107 cells. WCE protein content was quantified using a Bio-Rad protein estimation kit. The WCE was subjected to SDS-PAGE (10%) and subsequently transferred to a membrane for immunoblotting. Blots were probed with antibodies against Igf2bp1, Myc, and β-actin obtained from Cell Signaling. Horseradish peroxidase-conjugated secondary antibodies (anti-mouse or anti-rabbit) were used to detect the respective primary antibodies. Quantitative luminescence of immunoblots was performed using ImageJ software.Quantitative real-time PCR was performed to measure mRNA levels. Total RNA was extracted using a RNeasy minikit from Qiagen, and cDNA was synthesized using an iScript cDNA synthesis kit (Bio-Rad) following the manufacturer’s instructions. Real-time PCR was conducted using a CFX-96 RT-PCR machine with Bio-Rad SYBR Green mix. The PCR conditions included an initial denaturation at 95°C for 3 min, followed by 40 amplification cycles consisting of denaturation at 95°C for 20 s, annealing at 58°C for 20 s, and elongation at 72°C for 20 s. Analysis of the results was performed using CFX software from Bio-Rad to determine the fold change in expression of specific genes. Three independent experiments were carried out, with each sample run in triplicate.To evaluate the transcriptional activity of Wnt/β-catenin in MEF-WT, MEF-Igf2bp1-KO, DLD1-dnTCF7L2-GFP, and DLD1-dnTCF7L2-IGF2BP1 cells, we employed the TOP/FOP reporter system in conjunction with the dual-luciferase kit . All cells (10 × 106/500 μL MEF medium/RPMI) were transiently transfected with 1 μg of a constitutively active vector encoding Renilla luciferase (Promega) and 10 μg of a β-catenin-responsive firefly luciferase reporter plasmid (TopFlash) or the negative control (FopFlash) using the MEF transfection reagent (Altogen Biosystems) or lipofectamine 2000 (Invitrogen). After 24 h, the cells were harvested, and both firefly and Renilla luciferase activities were measured in duplicate or triplicate according to the manufacturer’s instructions. Firefly luciferase activity was normalized against Renilla luciferase activity, and the fold increase in TOPFlash activity compared to FOPFlash was determined.in vivo. After cell lysis, RNA was partially fragmented using micrococcal nuclease, and IGF2BP1-RNA complexes were immunopurified using anti-IGF2BP1 antibody  immobilized on protein A-coated Dynabeads (Invitrogen). Following stringent washing and dephosphorylation , RNAs were ligated at their 3′ ends to a 3′ preadenylated RNA adaptor, radioactively labeled, separated by MOPS-based protein gel electrophoresis, and transferred to a nitrocellulose membrane. Protein-RNA complexes located 15–80 kDa above free protein were excised from the membrane, and RNA was recovered through proteinase K digestion under denaturing conditions (3.5 M urea). Reverse transcription was performed using oligonucleotides containing two inversely oriented adaptor regions, separated by a BamHI restriction site and a barcode region at their 5′ end. The barcode region contained a 4-nt experiment-specific barcode within a 5-nt random barcode to identify individual cDNA molecules. The cDNA molecules were size purified using denaturing PAGE, circularized using CircLigase II , annealed to an oligonucleotide complementary to the restriction site, and cleaved using BamHI (New England Biolabs Inc.). The linearized cDNAs were then PCR-amplified using AccuPrime Super Mix I (Invitrogen) with primers (IDT) complementary to the adaptor regions and subjected to high-throughput sequencing using the Illumina HiSeq platform. For a more detailed description of the iCLIP protocol, refer to the published literature  developed  for gene


TP53 gain-of-function and KRAS variants were poor prognostic factors, while the NOTCH3 sole variant was a favorable prognostic factor for left-sided metastatic colorectal cancer. The TP53 non-gain-of-function variant, BRAF V600E, and MYC amplification were poor prognostic factors for right-sided metastatic colon cancer. Prognostic gene alterations in metastatic colorectal cancer differed according to the sidedness of the primary tumor.The treatment strategies and prognoses of patients with metastatic colorectal cancer differ according to the sidedness of the primary tumor. The aim of this study was to evaluate the sidedness-dependent prognostic impact of gene alterations in metastatic colorectal cancer. Among patients diagnosed with metastatic colorectal cancer enrolled from April 2017 to March 2019, 531 patients who underwent complete gene sequencing were assessed. TP53 gain-of-function (GOF) and non-GOF variants have been reported to be differentially associated with prognosis by sidedness. We aimed to evaluate the sidedness-dependent prognostic impact of gene alterations in metastatic CRC. Patients enrolled between April 2017 and March 2019 were included in this study. Those excluded were individuals whose tumor tissues were obtained after chemotherapy and those who were enrolled in the study more than six months after starting first-line chemotherapy. Finally, we assessed 531 patients who underwent complete gene sequencing. The study revealed a significant difference in overall survival between individuals with left-sided CRC (n = 355) and right-sided colon cancer (CC) (n = 176) when considering the TP53 non-GOF variant, KRAS wild-type, NOTCH1 wild-type, NOTCH1 covariant, NOTCH3 sole variant, and MYC amplification. Multivariate analysis on each side revealed that the TP53 GOF and KRAS variants were independent poor prognostic factors for left-sided CRC , and the TP53 non-GOF variant, BRAF V600E, and MYC amplification for right-sided CC . The NOTCH3 sole variant was an independent and favorable prognostic factor for left-sided CRC (p < 0.01). The prognostic significance of gene alterations differed between left-sided CRC and right-sided CC.The treatment strategies and prognoses of patients with metastatic colorectal cancer (CRC) differ according to the sidedness of the primary tumor.  RAS, BRAF, PIK3CA, and TP53), microsatellite instability, chromosomal instability, gene methylation, and consensus molecular subtypes .The relationships among clinicopathological features, major gene alterations (>5%), and sidedness are shown in p = 0.16). Significant differences were noted when comparing OS between left-sided CRC and right-sided CC       . Six patients with truncated NOTCH3 survived for 28.5–56.8 months, and four of the six patients were alive (The median follow-up time was 25.5 months (range: 0.7–59.7 months), and 349 patients (65.7%) died. The median OS durations were 28.5 and 26.3 months in the left-sided CRC and right-sided CC, respectively  a, KRAS w = 0.04) b, NOTCH1 = 0.04) c, NOTCH1 = 0.03) c, NOTCH3 = 0.01) d, and MY = 0.01) e. The di variant a, and in variant d. There re alive d.BRAF non-V600E and NOTCH1, NOTCH2, and NOTCH3 covariants were excluded from the analysis because of their low numbers , and the NOTCH3 sole variant for better prognoses , in left-sided CRC  in right-sided CC , whereas no gene alterations were prognostic in 82 left-sided CRC patients treated with anti-EGFR antibody combined therapy  were integrated into the analyses as RAS variants combined with KRAS variants, the results of the prognostic variants did not change (When t change .TP53 GOF and KRAS variants in patients with left-sided CRC, and the TP53 non-GOF variant, BRAF V600E, and MYC amplification in patients with right-sided CC, were independent predictors of a shorter OS. Notably, the NOTCH3 sole variant in patients with left-sided CRC was the only independent predictor of a longer OS. The results are summarized in We demonstrated that the The distribution of clinicopathological features and gene alterations in left-sided CRC and right-sided CC was similar to that in previous reports, such as the Cancer Genome Atlas Project of human CRC, suggesting that our cohort represents common mCRC ,20,21.TP53 status and sidedness was distinctly dichotomized into different prognostic populations: poor survival of TP53 GOF variants in both sidedness and TP53 non-GOF variants in right-sided CC (TP53 variants, OS significantly differed between left-sided CRC and right-sided CC in the TP53 non-GOF variant, but not in the TP53 GOF variant. This was consistent with the results of the previous report, and was considered to be confirmed in our patient cohort [TP53 GOF and non-GOF variants were independent poor prognostic factors in left-sided CRC and right-sided CC, respectively. The mechanism underlying the different survival behaviors of the TP53 GOF and non-GOF variants in each side remains unclear. Because the right-sided colon arises from the midgut, and the left-sided colon and rectum arise from the hindgut, the difference in anatomical origin and gut microbiome are the likely reasons, as was recently suggested [TP53 variants is influenced by the microbial environment in mouse models. In the proximal gut, lower microbial load and diversity are associated with lower levels of gallic acid, which is a polyphenol metabolite produced by gut commensal microbes. In the distal gut, higher microbial load and diversity are associated with higher levels of gallic acid. When the TP53 GOF variant is exposed to gallic acid, it loses its tumor-suppressive effects, switches to oncogenic effects by activating the WNT signaling pathway, and enhances the proliferation and invasion of tumor cells. Therefore, the influence of the gut microbiome on TP53 variants should be further investigated.The OS of the six groups divided by sided CC a. From et cohort . The novuggested ,23,24. TTP53 GOF variants may contribute differently to tumor progression. CRC patients with the TP53 R273H variant had more progressive disease and poorer survival than those with TP53 R175H variant [Distinct  variant . HoweverKRAS variant in left-sided CRC, BRAF V600E, and MYC amplification in right-sided CC were also independent poor prognostic factors. Previous studies have shown that these gene alterations are poor prognostic factors for mCRC [RAS wild-type is more promising in left-sided CRC than right-sided CC [BRAF V600E variant is more promising in left-sided CRC than in right-sided CC [MYC amplification in right-sided CC was small, its negative impact on OS was substantial, with a median OS of approximately 12 months (MYC amplification in right-sided CC should be the focus of future research.The for mCRC ,26,27,28sided CC . Similarsided CC . Interac2 months e. MYC amNOTCH3 sole variant was an independent favorable prognostic factor in patients with left-sided CRC. Truncating NOTCH3 among NOTCH3 sole variants was only observed in left-sided CRC. Truncating NOTCH3 causes it to lose its intracellular domain, which acts as a transcription factor in the Notch3 receptor signaling pathway. This may result in a favorable prognosis in patients with left-sided CRC who harbor the NOTCH3 sole variant. We could not confirm this plausible explanation because we did not examine the protein expression of the Notch3 intracellular domain in the same samples.Notch3 activates cancer stem cells; promotes tumor growth, invasion, metastasis, angiogenesis, and epithelial–mesenchymal transition; and contributes to chemoresistance. Its overexpression is a poor prognostic factor in patients with mCRC ,32,33,34RAS wild-type patients treated with the anti-EGFR antibody.Our study had several limitations. We did not establish a validation cohort for this study. We investigated the publicly available cBioPortal database targeted sequencing of 471 unresectable colorectal adenocarcinoma samples in MSK-IMPACT™ . HoweverTP53 variant classification into GOF and non-GOF, other than KRAS, BRAF, and MYC, is a useful prognostic biomarker for each-sided mCRC. The NOTCH3 sole variant in left-sided CRC was found to be the only favorable prognostic factor. These gene alterations are potential stratification factors for clinical trials involving patients with mCRC.The prognostic significance of gene alterations differed between left-sided CRC and right-sided CC. This study suggests that


Kinetin riboside is a naturally produced cytokinin that displays strong antiproliferative activity in various human cancer cells. However, the mechanism of chemoprevention in colorectal cancer cells has not been elucidated. We used a cell-based reporter system to identify kinetin riboside as an antagonist of the Wnt/β-catenin pathway, which is aberrantly upregulated in colorectal cancer. Kinetin riboside suppressed β-catenin response transcription (CRT) by accelerating the degradation of intracellular β-catenin via a proteasomal degradation pathway. Pharmacological inhibition of glycogen synthase kinase-3β did not affect CRT downregulation. Kinetin riboside decreased the intracellular β-catenin levels in colorectal cancer cells with mutations in adenomatous polyposis coli (APC) and β-catenin. Consistently, kinetin riboside repressed expression of c-Myc and cyclin D1, β-catenin/T-cell factor (TCF)-dependent genes, and inhibited the proliferation of colorectal cancer cells. In addition, kinetin riboside stimulated apoptosis, as measured by an increase in annexin V-FITC-stained cells. These findings suggest that kinetin riboside exerts its anti-cancer activity by promoting β-catenin degradation and has significant potential as a chemopreventive agent for colorectal cancer cells. The Wnt/β-catenin pathway plays essential roles in regulating various cellular behaviors, including proliferation, survival, and differentiation -3. The iAPC gene are observed in familial adenomatous polyposis coli (FAP) and sporadic colorectal cancers [c-Myc and cyclin D1, which leads to colorectal tumorigenesis [Colorectal cancer is a significant cause of cancer-related deaths worldwide. Abnormal up-regulation of the Wnt/β-catenin pathway is a major pathological event in intestinal epithelial cells during human colorectal cancer oncogenesis . Genetic cancers . In addi cancers . These migenesis -14. TherN6-substituted purine derivatives; they promote cell division in plants and regulate developmental pathways. Natural cytokinins are classified as isoprenoid , aromatic , or furfural (kinetin and kinetin riboside), depending on their structure [Plant cytokinins are tructure , 16. Kintructure . HoweverHEK293, SW480, HCT116, and Wnt3a-secreting L cells were obtained from the American Type Culture Collection (USA) and cultured in Dulbecco’s Modified Eagle’s medium (DMEM) containing 1% penicillin/streptomycin and 10% fetal bovine serum (FBS). HEK293-firefly luciferase reporter cells were established as previously described . Wnt3a-cCytosolic fractions were prepared as previously described . ProteinTotal RNA was isolated with Trizol reagent  following the manufacturer’s protocols. cDNA synthesis, reverse transcription, and PCR were performed as described previously . Amplifi4 cells/well) were seeded into 96-well plates and treated with kinetin riboside for 48 h. Cell viabilities for each treated sample were measured in triplicate using a Celltiter-Glo assay kit (Promega) according to the manufacturer’s instructions.SW480 and HCT116 cells  and propidium iodide (PI), using an ApoScanTM annexin V-FITC apoptosis detection kit (BD Transduction Laboratories), according to the manufacturer’s instructions. Apoptosis rates were calculated using a Cellometer Vision Image Cytometer .t-test was used to compare the means between the control and experimental groups. All experiments were performed in triplicate. Statistical significance was set at p < 0.05 or p < 0.01. Results are expressed as mean ± standard deviation (SD).The student’s We used HEK293-firefly luciferase (FL) reporter cells that were stably transfected with synthetic β-catenin/Tcf-dependent FL reporter gene (TOPFlash) and human Frizzled-1 (hFz-1) expression plasmid (hereafter referred to as HEK293-FL reporter cells) to determine whether kinetin riboside, a purine derivative , suppresThe effect of kinetin riboside on intracellular levels of β-catenin protein was investigated by Western blot analysis with anti-β-catenin antibody. Consistent with the effects of kinetin riboside on CRT, incubating HEK293-FL reporter cells with different concentrations of kinetin riboside attenuated the Wnt3a-CM-mediated increase in intracellular β-catenin protein levels in a dose-dependent manner . In contMG-132, a proteasome inhibitor, was used to examine the involvement of proteasomes in the kinetin riboside-induced downregulation of β-catenin protein. Kinetin riboside treatment consistently decreased intracellular β-catenin levels in HEK293-FL reporter cells; however, the addition of MG-132 nullified kinetin riboside-mediated β-catenin downregulation . BecauseGSK-3β-mediated β-catenin phosphorylation at residues Ser33/37 is a prerequisite for proteasome-dependent β-catenin degradation via the Wnt/β-catenin signaling pathway. Therefore, we used 6-bromoindirubin-3’-oxime (BIO), an inhibitor of GSK-3β , to deteAPC gene and a Ser45 (CK1 phosphorylation site) deletion mutation in β-catenin, respectively [We evaluated the effect of kinetin riboside on the levels of intracellular β-catenin protein in SW480 and HCT116 colon cancer cells by Western blot analysis. Upregulation of intracellular β-catenin in SW480 and HCT116 colon cancer cells occurs due to an inactivation mutation in the ectively . As depiSeveral studies have reported that a specific reduction of β-catenin suppresses the growth of colon cancer cells , 24. BecKinetin riboside displays antiproliferative and apoptogenic activities in various human cancer cell lines. It has recently been shown to suppress tumor growth in murine xenograft models of human leukemia and melanoma , 26. HowThe stability of intracellular β-catenin protein is typically controlled by a destruction complex (APC/Axin/CK1/GSK-3β)-dependent pathway. N-terminal phosphorylation of β-catenin within a destruction complex is a prerequisite for the degradation of β-catenin through a proteasome-dependent mechanism , 6. HoweNatural compounds that suppress the proliferation of colon cancer cells through attenuation of the Wnt/β-catenin pathway have been identified. Genestein, a leading representative of the isoflavone family, inhibits the Wnt/β-catenin pathway by increasing a Wnt antagonist sFRP2 gene expression in DLD-1 colon cancer cells . ResveraIn conclusion, we showed that kinetin riboside is a Wnt/β-catenin pathway antagonist and demonstrated its antiproliferative activity against colorectal cancer cells. Kinetin riboside promotes intracellular β-catenin degradation via a mechanism that does not involve the β-catenin destruction complex. It also inhibits the proliferation of colorectal cancer cells by inducing apoptosis. These findings suggest that kinetin riboside can potentially serve as a preventive or therapeutic agent against cancers involving abnormal β-catenin accumulation.http://jmb.or.kr.Supplementary data for this paper are available on-line only at


Background: Little research to date has examined the quality of data obtained from resident performance evaluations. This study sought to address this need and compared inter-rater reliability obtained from norm-referenced and criterion-referenced evaluation scaling approaches for faculty completing resident performance evaluations.Methods: Resident performance evaluation data were examined from 2 institutions , with 4 evaluation forms: 2 criterion-referenced  and 2 norm-referenced. Faculty inter-rater reliability was calculated with intraclass correlation coefficients (ICCs)  for each competency area within the form. ICCs were transformed to z-scores, and 95% CIs were computed. Reliabilities for each evaluation form and competency, averages within competency, and averages within scaling type were examined.Results: Inter-rater reliability averages were higher for all competencies that used criterion-referenced scaling relative to those that used norm-referenced scaling. Aggregate scores of all independent categories  for criterion-referenced scaling demonstrated higher reliability  than norm-referenced scaling . Moreover, examination of the distributions of composite scores  suggested that the criterion-referenced evaluations better represented the performance continuum.Conclusion: Criterion-referenced evaluation approaches appear to provide superior inter-rater reliability relative to norm-referenced evaluation scaling approaches. Although more research is needed to identify resident evaluation best practices, using criterion-referenced scaling may provide more valid data than norm-referenced scaling. Graduate medical education programs are required to collect faculty evaluations of resident trainees on a regular basis, but the mechanism by which residents are evaluated is left to the discretion of individual programs. Thus, programs have employed a wide variety of approaches. However, many programs seem to have been influenced by changes in accreditation reporting requirements. For example, in 2013, the Accreditation Council for Graduate Medical Education (ACGME) launched the Next Accreditation System (NAS) that transitioned the focus of resident performance reporting practices toward assessing specific behavioral criteria linked to an underlying competency continuum . Biannually, programs must indicate each trainee's performance on the milestones, and these milestone reports oftentimes heavily rely on faculty evaluations of resident performance. Some performance management systems facilitated milestone reporting by creating default evaluation scales  that likely encouraged programs to use these scales. Furthermore, many individuals may have elected to use the same scaling as the milestones to simplify mapping in general.3 or on the feasibility of reporting,4 or they have used data gathered through the NAS to stratify competency-based achievement by level of training.5 But these studies have not addressed the reliability of evaluation data gathered by programs to support the NAS. This research effort sought to provide some preliminary inferences in this regard.Thus, resident evaluations from many programs similarly transformed to mirror the biannual ACGME milestone reporting scales in which residents are mapped to where they fall on the ability continuum (criterion-referenced). Prior to this transformation, most programs focused on identifying stragglers or those excelling and used comparison-type (norm-referenced) scales. Little published evidence compares these 2 types of scaling approaches. Studies evaluating the NAS have focused on evidence of the validity or construct validity of milestones ratings6In the context of evaluation creation, there are many potential considerations. First, you need to make sure that evaluators will be able to interpret the content in the scales . You need to ensure that the numbers you ask raters to use—the scale you have selected—are appropriate for the content being assessed and the proposed application. Scaling drives the translation from an abstract construct  to a tangible number. But that number can be more or less accurate depending on several factors .6 Thus, resultant ratings indicate whether an individual is better, worse, or about the same as a normative referent (such as the average resident).Norm-referenced evaluation scales require raters to make assumptions regarding where individuals reside on average relative to other similar individuals without specific regard to the competency continuum.7 Certain standards have to be met, and only via aligning trainee performance to these standards can educators ensure that trainees are adequately prepared. Thus, criterion-referenced scales assume an underlying continuum of skill to which resident performance can be mapped .Glaser and Klaus proposed that norm-referenced scaling was not sufficient for educators’ needs because the actual proficiency level of trainees needs to be identified.8 Norm-referenced evaluations may also be easier for nontrained individuals to develop, and they may be more familiar to inexperienced raters.9Arguably, the criterion-referenced scales may remove some ambiguities regarding the comparison group ; however, making norm-referenced comparisons may be less cognitively demanding than making absolute (criterion-based) judgments.10 Effective criterion referencing necessitates an understanding of how individuals on average perform to develop the required standards of performance. Thus, norm referencing is inherent in developing the standards in criterion-referenced assessment. Interestingly, Lok et al also argued that norm-referenced scaling can allow for more freedom for evaluators. For example, some trainees can be forced to fail in a norm-referenced model regardless of the overarching quality of performance. Conversely, with a criterion-referenced model, as long as trainees achieve the standards, they pass. Thus, criterion-referenced approaches may increase grade inflation and passing and may not effectively identify the worst performers.10 However, others have pointed out that norm-based evaluation approaches may lead to standards being set below where they would with criterion-based approaches.11Guidelines to date have mixed inferences at best, with arguments supporting both sides. In the context of education in general, Lok et al suggested that the 2 approaches are to some extent intertwined.12 Using 1-way random effects intraclass correlation coefficients (ICCs), they found absolutely no agreement between residents and faculty on these ratings and low agreement between faculty. Even faculty did not highly agree when rating residents using milestones in an ideal testing context of a standardized simulation. Conversely, Scielzo et al found that resident self-reports and faculty reports of resident criterion-assessed competencies were consistent.13 This study, however, used a correlational design . Pereira and colleagues, arguing that undergraduate medical education needs to move away from norm-referenced assessment, suggested that current norm-based approaches are not predictive of later performance and that undergraduate medical education needed to transition to criterion-referenced assessment.14 Interestingly, they also noted the dearth of research assessing the impact of criterion-referenced vs norm-referenced assessment in graduate medical education.In the context of medical education, little research has examined the impact of scaling type on assessments. For example, Wittels et al examined the correlation of resident self-assessment and corresponding faculty ratings of clinical competence using milestone ratings in the context of a highly standardized training simulation.Thus, as criterion-referenced evaluations are now widely used in resident education, examining this form of evaluation in comparison to its predecessor is imperative. Given our duty to society to ensure that physician trainees are ready for independent practice upon graduation and to ensure that our inferences in this regard are as accurate as possible, examining reliability is an important first step.6 Reliability in evaluation refers to the reproducibility of data or scores over time, event, or items designed to measure the same constructs.15 Reliability is an important consideration in evaluating the utility of a given measurement; without reliability, inferences are not stable. In other words, without reliability, you cannot have validity.17 Imagine your home scale giving the following 3 readings in a 3-minute period: 87 lbs, 205 lbs, and 147 lbs. Because the scale cannot systematically infer a weight, the weight results clearly are not valid, and it is probably time to discard the scale. Appendix B provides general information about reliability.We elected to examine inter-rater reliability to make some initial inferences regarding the quality of the information gained from the evaluation scaling types. Inter-rater reliability, the consensus or degree of agreement among raters, provides one indicator of the psychometric utility of a scale.In the case of resident assessments, low inter-rater reliability might suggest that the evaluation questions were hard to understand, that raters did not actually have enough opportunities to observe rating behaviors, that the items did not provide a good representation of the underlying constructs, or that the scaling approach may not be ideal for the behaviors being measured.In this study, we sought to determine the effect of using norm-referenced vs criterion-referenced scales on the reliability of evaluation data. Specifically, we examined the impact of evaluation scaling type  on the inter-rater reliability of faculty members completing resident performance evaluations.We compared the inter-rater reliability of 4 different evaluation forms. These evaluations came from 3 graduate medical education residency training programs at 2 universities: Dartmouth-Hitchcock and the University of Texas Southwestern (UTSW). Two of the evaluations were criterion-referenced and 2 were norm-referenced. One of the criterion-referenced evaluations had an additional norm-referenced item that we also included in our analysis.Program I was the internal medicine program at Dartmouth-Hitchcock. Program II was the internal medicine program at UTSW. Program III was the general surgery program at UTSW. Program I had both norm-referenced  and criterion-referenced  evaluations. The norm-referenced evaluations were collected in 2012-2013 prior to the ACGME transition, and the criterion-referenced evaluations were collected in 2013-2014. Program II used an evaluation form (n=196) that had predominantly criterion-referenced items (referred to as criterion-referenced 2), but also had 1 norm-referenced item (referred to as norm-referenced 2). Program III had a norm-referenced evaluation . We elected to include all of these different evaluations to try to overcome some of the limitations of this study, such as temporal differences  and having the exact same raters and individuals being rated . Appendix C presents the specific items used in each form and how each was linked to competencies.This study was an archival data analysis, an examination of end-of-rotation data collected for educational purposes . Thus, faculty evaluation behaviors were not influenced by knowing that their scores would be examined. Ethical approval was obtained from the Dartmouth College Institutional Review Board that declared the study exempt from formal review.Data were obtained for residents in postgraduate years (PGY) 1 to 3 in Programs I and II, and from residents in PGY 1 to 5 in Program III. Evaluation items had been mapped during evaluation creation to their relevant ACGME competencies: Practice-Based Learning and Improvement, Patient Care, Professionalism, Medical Knowledge, Interpersonal and Communication Skills, and Systems-Based Practice. A few items assessed overall perceptions of a resident's standing .If multiple items were associated with a competency, an average was computed (see Appendix C for more detail). As an example, if we had 2 items assessing Patient Care, we averaged these 2 items to represent that construct for each evaluation administration .The evaluation for Program II was designed with the goal of minimizing the time to complete while retaining as much important information regarding performance as possible. After careful analysis from the development team, we decided that broader items tapping multiple competencies were preferred. Thus, this evaluation contained 2 items that each represented 2 constructs . Thus, the same reliability index was used in the analysis for these competencies  for Program II.18 This assessment enabled us to examine the correspondence of scores across random raters (any of our faculty members may make these ratings). Similarly, we needed to evaluate whether our raters were in agreement, not just consistent. We also wanted to make estimates for a larger sample of raters consistent with our most important decisions .Inter-rater reliability was assessed with a 1-way random effects model ICC.19 based on an average of 10 assessments per resident, the minimum annual number of evaluations per resident, to provide an estimate of the accuracy of the data used for milestone reporting. This formula allows us to make more accurate estimates of reliability based on applied utilization, rather than just relying on the lower reliability inferences from the 2 observations.The first 2 observations for each trainee were used, with a resultant ICC . These values were then applied to the Spearman-Brown prediction formulaWe examined the average for all criterion-referenced ICC values within a competency compared to the average for all norm-referenced values. Thus, all ICCs were averaged within competency and scaling type. To examine the average reliability based on scaling type, we averaged all independent reliability values within scaling type after removing duplicate values for Program II. Appendix B provides general information about ICCs.z-scores. The 95% CIs were computed around each z-score using 1/sqrt(N-3) for the standard error. Inter-rater reliability estimates were calculated using SPSS Statistics version 27.0 (IBM Corporation),20 and Spearman-Brown estimates and z-score confidence intervals were calculated in Microsoft Excel (Microsoft Corporation).To facilitate comparison and the interpretation of CIs, these effect sizes were translated to Finally, all items were averaged for each individual being rated to provide an estimate of their overarching standing based on these evaluations. Thus, for resident X, all items were averaged for both evaluators, creating a composite estimate of overall ability. Then, these composite scores were broken into 5 equal intervals and mapped back to the underlying scales used to rate the trainees, enabling us to examine the distribution of scores for each program with multiple competencies (excluding the 1 norm-referenced assessment item in Program II) and providing another indicator to examine the quality of the data obtained. Graphs were created for each scaling type in Microsoft Excel.z-score for the average reliability for each scaling type by competency, higher reliability averages were observed for criterion-referenced evaluation scales relative to norm-referenced scales for all competencies (z-score of 1.16 (CI 1.05-1.27) for our criterion-referenced evaluations vs an average z-score of 0.76 (CI 0.63-0.89) for the norm-referenced evaluations. Neither z-score falls within the CI of the other, and thus, with 95% confidence is statistically different. Furthermore, the criterion-referenced item that assessed overall competence was statistically higher than the average of the 2 items using norm-referenced scaling (z=1.83 vs 1.31).First, we examined the extent of the impact of scaling type (norm-referenced vs criterion-referenced) for each competency. Examining the etencies . For exaz=1.37, CI 1.26-1.48) had statistically higher reliability than norm-referenced scaling   plus the question assessing overall competence were averaged. For Program II, the 4 independent competencies  plus the question assessing overall competence were averaged. The norm-referenced average included all 6 competencies plus the question assessing overall competency for Program I, the item assessing overall competence for Program II, and all 6 competencies for Program III. Overall, criterion-referenced scaling , but more research is needed.Future research should examine other validity and reliability estimates, such as the correspondence of ratings with in-service/board examination scores and/or other indicators of performance. Future research needs to evaluate faculty satisfaction with these rating types . Given the importance of our evaluation efforts, much more research is needed to identify best practices for creating the most psychometrically and practically useful evaluation tools possible.We found that criterion-referenced scaling results in higher inter-rater reliability relative to norm-referenced scaling for faculty evaluations of resident performance. Although more research is needed, criterion-referenced scaling appears to provide better inter-rater reliability and, in turn, more valid inferences for deriving milestone data. The results of this study suggest that programs should consider adopting criterion-referenced scaling approaches to ensure that programs are making the most accurate inferences possible when assessing residents’ skills.


The authors apologise that a few typographical errors were included in the publication of the above article.In Table 1, the mean for the NR (i.e. needy recipient) condition is 0.278 (not 0.253), the median for the Pun (i.e. punishment) condition is 0.25 (not 0.275), and the SD for the NR condition is 0.212 (not 0.221). In Figure 2, one person is missing from the column representing the number of people in the nr + pun  condition who gave away 40% of their endowments. These errors have no impact on the article's findings or conclusions.


Roof plate-specific spondin 1  is a Wnt/β-catenin signaling pathway activator that binds with Wnt ligands to stimulate the Wnt/β-catenin signaling pathway, which is key to hair regeneration. However, it is not clear whether recombinant RSPO1 (rRSPO1) affects hair regeneration. Here, we treat C57BL/6 male mice with rRSPO1 and investigate the expression of the Wnt/β-catenin signaling pathway and the activation of hair follicle stem cells in the dorsal skin. The mouse skin color score and hair-covered area are determined to describe hair growth, and the skin samples are subjected to H&E staining, western blot analysis and immunofluorescence staining to evaluate hair follicle development and the expressions of Wnt/β-catenin signaling pathway-related proteins. We find that rRSPO1 activates mouse hair follicle stem cells (mHFSCs) and accelerates hair regeneration. rRSPO1 increases the hair-covered area, the number of hair follicles, and the hair follicle diameter and length. Moreover, rRSPO1 enhances the activity of Wnt/β-catenin signaling pathway-related proteins and the expressions of HFSC markers, as well as mHFSC viability. These results indicate that subcutaneous injection of rRSPO1 can improve hair follicle development by activating the Wnt/β-catenin signaling pathway, thereby promoting hair regeneration. This study demonstrates that rRSPO1 has the potential to treat hair loss by activating the Wnt/β-catenin signaling pathway. The hair follicle is the basic structure of hair and is not only a sensory organ but also a vector for social interaction in human society. Hair loss disorders, such as androgenetic alopecia and alopecia areatas, are some of the most common types of hair loss in both men and women, affecting approximately 0.2%–2% of the world’s population ,2 . HaiThe hair follicle is a skin appendage derived from ectoderm, a specific organ of epithelial and mesenchymal interactions. The dermal papilla is required for hair follicle development and promotes proliferation and differentiationEscherichiacoli Rosetta strain to express recombinant RSPO1 (rRSPO1) protein. rRSPO1 has approximately 83.92% and 84.48% base sequence identity and approximately 84.34% and 91.98% amino acid residue sequence identity in mice and humans, respectively. Moreover, the production of rRSPO1 offers the double advantage of higher yields and lower price. Therefore, we hypothesized that rRSPO1, as a potential hair loss treatment, may transition hair follicles from the telogen to anagen phase in mice.Roof plate-specific spondin 1 , a member of the R-spondin family of secreted proteins, acts as an agonist of the Wnt/β-catenin signaling pathway and plays essential roles in many processes, including cell proliferation and metabolic homeostasisHere, we assessed whether RSPO1 promotes hair regeneration in a mouse model and developed a novel recombinant protein, rRSPO1. We demonstrated that rRSPO1 induces the activation of the Wnt/β-catenin signaling pathway. Activation of the Wnt/β-catenin signaling pathway may be associated with mouse HFSC (mHFSC) proliferation and differentiation. Together, our study provides support for the effects of RSPO1 on mouse hair regeneration and a recombinant protein to treat hair loss.Escherichiacoli Rosetta strain, separated rapidly by Ni-chelating affinity chromatography  and further purified. The protein was lyophilized and then reconstituted in sterile phosphate-buffered saline (PBS) prior to use.As described previously by our laboratoryMale C57BL/6 mice were purchased from Guangdong Medical Laboratory Animal Center . At 8 weeks of age, when the dorsal skin was at the telogen stage, mice were anaesthetized by intraperitoneal administration of sodium pentobarbital and then shaved and depilated. The animal experiment procedures in this study were approved by the Animal Ethics Committee of South China Agricultural University  and guided by the Care and Use of Laboratory Animals of South China Agricultural University.A total of 9 mice were selected for the screening experiments and were randomly divided into three treatment groups with 3 mice per group. The different groups were injected with PBS, rRSPO1  or rRSPO1 . Each mouse received a multiple-point subcutaneous injection for 7 consecutive days at the center of the back after depilation.A total of 16 mice were selected for the formal trial and were randomly divided into two treatment groups with 8 mice per group. The day after depilation, the mice were injected subcutaneously at multiple points with PBS alone (control group) or rRSPO1  for 7 consecutive days. The mice were maintained under conventional conditions at 22–25°C during the hair depilation and photographing procedures. The mouse body weight was measured once a day after hair depilation. The mice were sacrificed using cervical neck dislocation before dorsal skin collection, and the skin samples were stored at –80°C or in 4% paraformaldehyde until further use.2. The culture medium was refreshed every other day. For subsequent experiments, the cells were digested with trypsin when the culture reached a confluency of 80% and subcultured in 6-well plates.mHFSCs were isolated and cultured following a previously published protocol ,19 . Br3 cells/well in 96-well plates  and treated with rRSPO1 or iCRT3  for 24, 48 and 72 h.Then, 20 μL of MTT solution  was added to the cells and incubated for 4 h. The crystals were dissolved in 150 μL of DMSO (Sigma-Aldrich) for 30 min and the absorbances were detected with a microplate reader  at a wavelength of 490 nm.MTT assay was used to detect cell viability. mHFSCs were seeded at 3×10Cell Counting Kit-8 assay was performed to determine cell proliferation activity. After treatment, 10 μL of CCK-8 solution (Sigma-Aldrich) was added to each well and incubated for 4 h. The absorbances were detected with the microplate reader at a wavelength of 450 nm.https://imagej.nih.gov).The dorsal skin samples were fixed with 4% paraformaldehyde overnight and then washed with PBS, dehydrated with alcohol, and embedded in paraffin blocks. Sections of 5 μm were cut with a microtome  and stained with H&E. Then, images were captured under a microscope , and the number of hair follicles, hair follicle diameter and hair follicle length were measured using ImageJ software  lysis buffer  containing 0.1% phenylmethylsulfonyl fluoride . The protein concentrations were quantified using a BCA protein assay kit (Thermo Fisher Scientific) and mixed with 5× SDS-PAGE loading buffer . The proteins and marker  were separated by 10% SDS-PAGE  and transferred to polyvinylidene fluoride (PVDF) membranes , which were washed with TBST and blocked in 5% skim milk. Then, the membranes were incubated with primary antibodies and the corresponding secondary antibodies. Finally, after the membrane was immunoreacted with an electrochemiluminescence reagent  and detected with a chemiluminescence system . The band densities were analysed with ImageJ software and relative expression levels are shown as fold changes to the control group (arbitrary unit). The primary antibodies used were as follows: LRP6 , LGR5 , active β-catenin , β-catenin , TCF4 , Cyclin D1 , PCNA , CD34 , cytokeratin 14 , SOX9 , and β-actin . HRP-conjugated anti-rabbit IgG  and anti-mouse IgG  were used as the secondary antibodies.The dorsal skin sections and mHFSCs were permeabilized with 0.1% Triton X-100 for 15 min and blocked in 5% bovine serum albumin (BSA) for 120 min. The samples were incubated with primary antibodies overnight at 4°C. Secondary staining was performed by incubation with a Cy3 or FITC-conjugated secondary antibody  at room temperature. Finally, the nuclei were stained with 4′,6-diamidino-2-phenylindole , and images of tissue sections were obtained using a fluorescence microscope . Relative expression levels are shown as fold changes to the control group (arbitrary unit).t test and among three groups by one-way analysis of variance (ANOVA) and Tukey’s test. Differences were considered statistically significant atP<0.05.Results were analysed using SPSS software  and data are presented as the mean±SEM. The comparisons between two groups were performed bySupplementary Figure S1), we treated mice with rRSPO1 and monitored the hair morphological changes  and Day 14 compared to that of the control mice  and Day 14 , and the levels of LRP6, LGR5, β-catenin and TCF4 were significantly increased , and the levels of LRP6, active β-catenin and TCF4 were significantly increased . mHFSCs were cultured in DMEM/F12 with 300 ng/mL rRSPO1 for 24, 48 and 72 h. The MTT assay and CCK-8 assay results showed that treatment with 300 ng/mL rRSPO1 for 24, 48 and 72 h significantly affected the viability and proliferation of mHFSCs . As shown inP<0.01). Apparently, iCRT3 inhibits the effect of rRSPO1 and reduces Wnt/β-catenin signaling activity (To further investigate the effect of rRSPO1 on the viability of mHFSCs, we isolated and cultured mHFSCs from mice. Base on our concentration screening results, we chose the optimal concentration of 300 ng/mL for further analysis ( mHFSCs A,B. The P<0.05;C,D. Nextctivity G,H. ThesThe hair follicle cycle consists of three phases: anagen, catagen and telogen. The differentiated melanocyte stem cell progeny synthesizes and transfers pigments to the events of the epithelial cells at the anagen phase, which can darken the skin. In contrast to the anagen phase, differentiated melanocyte apoptosis occurs in the telogen phase, which turns the skin pinkHair follicle formation and maintenance are regulated by multiple signaling pathways in strict temporal and spatial patternset al.et al. and indicate that increased LRP6 expression is important in promoting hair growth. This implies that the promotion of hair growth by RSPO1 may be associated with interference with DKK1-mediated internalization. Weberet al.Our results showed that the expression levels of LRP6, LGR5, active β-catenin, β-catenin and TCF4 were significantly higher in the rRSPO1 group than in the control group. Moreover, the expression level of the target protein Cyclin D1 was increased in the rRSPO1 group. This finding suggests that RSPO1 can activate the Wnt/β-catenin signaling pathway and further upregulate the expressions of Wnt target genes. Moreover, we found that DKK1, a Wnt/β-catenin signaling pathway antagonist, is induced by dihydrotestosterone from balding dermal papilla cells, and high expression of DKK1 in mice retards hair follicle cycle transformationWnt/β-catenin signaling pathway activators show promise as novel drug candidates. The inability of the hair follicle cycle to transition from the telogen phase to the anagen phase is one of the reasons why hair regeneration is hinderedIn summary, this study suggests that rRSPO1 could directly target the Wnt/β-catenin signaling pathway in HFSCs to induce HFSC proliferation and differentiation to promote hair regeneration, which may be beneficial for the treatment of hair loss in the future.Acta Biochimica et Biphysica Sinica online.Supplementary data is available at476FigS1-S2


CX3CL1 secreted in the tumor microenvironment serves as a chemoattractant playing a critical role in metastasis of CX3CR1 expressing cancer cells. CX3CR1 can be expressed in both cancer and immune-inhibitory myeloid cells to facilitate their migration. We generated a novel monoclonal antibody against mouse CX3CR1 that binds to CX3CR1 and blocks the CX3CL1-CX3CR1 interaction. We next explored the immune evasion strategies implemented by the CX3CL1-CX3CR1 axis and find that it initiates a resistance program in cancer cells that results in 1) facilitation of tumor cell migration, 2) secretion of soluble mediators to generate a pro-metastatic niche, 3) secretion of soluble mediators to attract myeloid populations, and 4) generation of tumor-inflammasome. The CX3CR1 monoclonal antibody reduces migration of tumor cells and decreases secretion of immune suppressive soluble mediators by tumor cells. In combination with anti-PD-1 immunotherapy, this CX3CR1 monoclonal antibody enhances survival in an immunocompetent mouse colon carcinoma model through a decrease in tumor-promoting myeloid populations. Thus, this axis is involved in the mechanisms of resistance to anti-PD-1 immunotherapy and the combination therapy can overcome a portion of the resistance mechanisms to anti-PD-1. Anti-PDNon-responders to anti-PD-1 in non-small cell lung cancer showed an increase in the plasma concentrations of the CX3CR1 ligand, CX3CL1 . An immuCX3CR1 binds to its ligand, CX3CL1 , which has membrane-bound and shed forms. The CX3CL1-CX3CR1 axis promotes chemotaxis of CX3CR1+ cells towards soluble CX3CL1 as well as adhesion of CX3CR1+ cells to membrane-bound CX3CL1 , 12. CX3Following interaction with CX3CL1, CX3CR1 signaling supports tumorigenesis through several mechanisms: 1. CX3CL1 in the tumor milieu promotes an influx of CX3CR1+ myeloid cells; a hallmark event of aberrant myelopoiesis, 2. CX3CL1 in the tumor milieu promotes migration of multiple CX3CR1+ tumor types such as breast cancer, prostate cancer, CLL, neuroblastoma, glioblastoma, pancreatic ductal carcinoma (PDAC), colon carcinoma, gastric cancer, skin cancer, lung cancer, osteosarcoma, melanoma, multiple myeloma, and bladder cancer –18, and CRISPR deletion of CX3CR1 or its blockade in human tumor lines of PDAC, breast cancer, prostate cancer, bladder cancer and glioblastoma results in a decrease in the ability of tumor cells to migrate and metastasize , 37. CX3We generated a novel monoclonal antibody that binds to CX3CR1 with high affinity, blocks its interaction with CX3CL1 and antagonizes the immune-suppressive signals of this axis. We observe an improvement in response to anti-PD-1 therapy in an immunocompetent syngeneic mouse model of colon carcinoma. Mechanistically, combined PD-1 and CX3CR1 blockade reduces the migration of tumor cells, decreases the abundance of immune suppressive myeloid cells in the tumor, increases mature macrophages in the tumor and reduces secretion of soluble mediators from the tumor. The CX3CR1 monoclonal antibody can target both tumor and myeloid cells; inhibit tumor signals that recruit myeloid populations and inhibit immune-suppressive pathways regulated through this axis. In summary, the CX3CR1 antibody can mitigate CX3CL1-CX3CR1 mediated pro-tumorigenic effects of aberrant myelopoiesis during cancer progression.22.1The monoclonal antibodies for mouse CX3CR1, clones 455.1C11, and 455.8H12 were made by immunizing CX3CR1 knockout mice of C57BL/6 background with mouse CX3CR1 cDNA expression plasmid DNA as described in Latchman et al., 2001  RRID: I. Hybrido2.22. Jurkat cells transfected with mouse CX3CR1 were made in our laboratory and maintained in RPMI-1640 media with 5 µg/ml puromycin. 300 cells transfected with mouse CX3CR1, and 300 cells transfected with mouse PD-L1 were made in our laboratory and maintained in RPMI-1640 media with 5 µg/ml puromycin and 50 µM 2-mercaptoethanol in 5% CO2. CHO cells transfected to express human CX3CR1 were purchased from Sigma Aldrich Cat #HTS015RTA.CT26 colon carcinoma was purchased from ATCC . CT26 were maintained in RPMI-1640 with 10% heat inactivated FCS, 1% glutamax, 1% pen-strep in 5% COAll cell lines used were tested and negative for mycoplasma.2.3The isotype controls MOPC21 (mIgG1), and C1.18.4 (mIgG2a) were purchased from BioXcell. Isotype mIgG2c, Goat anti-mouse IgG-PE, goat anti-human IgG-PE , and goat anti-human IgG-Alexa647 (multi species adsorbed)  were purchased from Southern Biotech. CX3CL1-human IgG1 Fc fusion protein for blocking assays was a kind gift from Dr. Ulrich von Andrian, Harvard Medical School. CX3CL1 recombinant protein was purchased from Novus Biologicals (cat NBP2-35038).2.4The capacity of anti-CX3CR1 mAbs to block binding of CX3CL1 to CX3CR1 was tested by incubating Jurkat-mCX3CR1 cells with anti-CX3CR1 mAb hybridoma supernatant for 30 min, followed by addition of CX3CL1-hFc (at 2 µg/ml). The binding of CX3CL1-Fc was detected using Goat anti human IgG-PE (mouse adsorbed)  at 5 µg/ml. Isotype control was mIgG1 (BioXcell Cat# BE0083 RRID:1107784). Acquisition was performed on BD FACS LSR Fortessa, and data was analyzed using Flow Jo version 10 (RRID : SCR_008520). Antibody concentrations of anti-mouse CX3CR1 antibodies in hybridoma supernatants were determined by indirect ELISA. ELISA plates (Costar #3369) were coated with 2 µg/ml unlabeled goat anti-mouse Ig(H+L) antibody overnight. The next day, plates were washed, blocked with 1% BSA in PBS for one hour and then washed. Hybridoma supernatants and dilutions were added to individual wells, incubated at 37°C for 1 hour and then washed three times. Goat anti-mouse IgG2c HRP-conjugated secondary antibody  was added to the wells, incubated at 37°C for 1 hour and then washed three times. TMB substrate was added to develop the color and plates were analyzed using a SpectraMax 190 microplate reader. Purified mouse IgG2c was used to construct a standard curve to calculate the antibody concentrations in supernatants.The capability of anti-CX3CR1 mAbs clones 1C11 and 8H12 to block binding of each other to CX3CR1 was tested by incubating Jurkat-mCX3CR1 cells with anti-CX3CR1 mAb clone 1C11 for 30 min, followed by addition of anti-CX3CR1 mAb clone 8H12-PE at 1 µg/ml. Isotype control was C1.18 mIgG2a. Acquisition was performed on BD FACS LSR Fortessa and data was analyzed using Flow Jo version 10 (RRID : SCR_008520).2.5Agonistic and antagonistic assays for CX3CL1-CX3CR1 axis activity were performed using the PathHunter eXpress mCX3CR1 CHO-K1 β-Arrestin GPCR Assay kit  following the manufacturer’s protocol. In the antagonistic assay, mouse CX3CR1 CHO-K1 reporter cells were incubated with anti-CX3CR1 mAb clone 1C11 for 30 minutes at 37°C followed by addition of agonist CX3CL1  at 500 ng/ml for 90 minutes at 37°C. Isotype control was mIgG2c. Detection reagent (assay buffer and substrate) was then added, and luminescence was measured on Spectra Max M3 instrument. In the agonistic assay, mouse CX3CR1 CHO-K1 reporter cells were incubated with agonist CX3CL1  or with anti-CX3CR1 mAb clone 1C11 for 90 minutes at 37°C. Detection reagent (assay buffer and substrate) was then added followed by luminescence measurement.2.6Tumor cell migration assays were performed using Abcam cell migration/chemotaxis assay utilizing a Boyden chamber with an 8 µm membrane  as per the manufacturer’s protocol. Briefly, CX3CR1 expressing CT26 tumor cells were cultured in serum free media for 24 hours. Tumor cells were incubated with CX3CR1 mAb (clone 1C11) at 10 µg/ml for 30 minutes at 37°C. The chemoattractant CX3CL1  (at 100 ng/ml) was added to the bottom chamber and mAb-treated or control tumor cells were added to the top of the Boyden chamber. The chamber was incubated for 24 hours at 37°C followed by addition of cell dissociation solution and fluorescence measurement using Spectra max M3 instrument.2.7The CT26 tumor cell line was assayed with anti-mouse antibodies; CX3CR1 antibody (clone 1C11) made in our laboratory, CX3CL1 antibody , PD-L1 antibody . The corresponding isotypes were used as controls.2.8CT26 tumor cell line was treated with mouse IFN-γ  and TNF-α  at 100 ng/ml for 24 hours. Cells were subsequently assayed for expression of CX3CR1 by flow cytometry.2.9limma was used to infer the significance between the fractions  in mm3. Mice were euthanized based on the end point criteria of a tumor diameter more than 20 mm or ulceration in the tumor with ulceration diameter of more than 10 mm. For memory re-challenge experiments, 0.2 million CT26 tumor cells were subcutaneously injected in the opposite (left) flank, and mice were monitored for tumor growth and survival. Here, mice aged 6-8 weeks with no prior antibody treatment were included as a control. Animal experiments were performed as per the IACUC guidelines of the Dana-Farber Cancer Institute.BALB/cJ mice aged 6-8 weeks were purchased from Jackson Laboratory (RRID : IMSR_JAX:000651) and housed in a barrier facility. CT26 tumor cells were subcutaneously injected at 0.2 million/mouse in the right flank of BALB/cJ mice. The antibody treatments with anti-mouse PD-1 (clone 29F.1A12) , anti-mouse CX3CR1 (clone 1C11), rat IgG2a (clone 2A3)  were started in randomized mice on day 7 post tumor cell injection for a series of 5 treatments once every 3 days with 200 µg antibody per mouse injected intraperitoneally per treatment. Mice were monitored for tumor growth using digital calipers, with tumor size calculated as volume (L X W2.11++/Mg++) and incubated at 37°C for 30 minutes with agitation. Cells were then subjected to mechanical disruption and filtered to obtain a single cell suspension. Red blood cell lysis was performed using ACK lysis buffer (Gibco) when needed. Spleens were isolated from tumor bearing mice. Splenocytes were harvested from spleen using gentle disruption of the tissue. Red blood cell lysis was performed on the cell suspension using ACK lysis buffer (Gibco) and the cells were filtered to obtain single cells. Cells were Fc blocked by incubating with 10 µg/ml of anti-mouse CD16/32 antibody  for 15 minutes and stained with fluorochrome-conjugated antibodies against cell surface markers for 30 min. After cell surface staining, the cells were fixed and permeabilized using FOXP3 transcription factor fixation and permeabilization kit  to determine the expression of intracellular markers. The fluorochrome antibodies used are detailed in For study of tumor immune infiltrates in the untreated cohort, tumors were harvested when the tumor diameter reached a size greater than 0.5-1 cm. For the study of antibody mediated mechanistic effects, tumors were harvested on day 12 after two antibody treatments. Tumors were pooled with two-three tumors to make one sample and increase the per sample cell yield as required for data acquisition. Once harvested, tumors were minced, followed by suspension in a disaggregation buffer , 200 U/mL DNase  in HBSS without Ca2.12Tumor cells were treated with CX3CL1  at 2 µg/ml for 8 hours and RNA was prepared using RNAeasy kit . Following incubation, total RNA was prepared using RNAeasy kit .RNA sequencing was performed at the Molecular Biology Core Facility, Dana-Farber Cancer Institute. Libraries were prepared using Roche Kapa mRNA HyperPrep strand specific sample preparation kits from 200ng of purified total RNA (tumor sample) or Takara SmartSeq v4 reagents from 1ng of RNA (MDSC sample) according to the manufacturer’s protocol on a Beckman Coulter Biomek i7. The finished dsDNA libraries were quantified by Qubit fluorometer and Agilent TapeStation 4200. Uniquely dual indexed libraries were pooled in an equimolar ratio and shallowly sequenced on an Illumina MiSeq to further evaluate library quality and pool balance. The final pool was sequenced on an Illumina NovaSeq 6000 targeting 40 million 150bp read pairs per library at the Dana-Farber Cancer Institute Molecular Biology Core Facilities.2.13Fastq files were generated using Illumina bcl2fastq v2.20 software. Sequenced reads were aligned to the UCSC mm10 reference genome assembly and gene counts were quantified using STAR (v2.7.3a) . Differe2.14Tumor cells were treated with or without CX3CR1 antibody (clone 1C11) at 10 µg/ml for 30 minutes at 37°C followed by CX3CL1  at 2 µg/ml for 24 hours. Supernatants were collected and analyzed for quantification of chemokines and cytokines using Discovery assays  and were performed by Eve Technologies  as per the manufacturer’s instructions. The data was acquired on the Luminex 200 system.2.15Tumor cell lines were treated with mouse IFN-γ  and TNF-α  at 100 ng/ml for 8 hours. Tumor cells were treated with or without CX3CR1 antibody (10 µg/ml) for 30 minutes at 37°C followed by CX3CL1  at 2 µg/ml for 8 hours. RNA was isolated using RNeasy kit  using manufacturer’s protocol and cDNA was made using Bio-Rad cDNA kits. mRNA was quantified using Taqman qPCR . The assay was run on Applied Biosystems Quant Studio 6 real time PCR system. Expression levels were normalized to GAPDH. Primers were purchased from Applied Biosystems; mouse PD-L1 (Mm03048248_m1), mouse IL-6 (Mm00446190_m1), mouse NLRP3 (Mm00840904_m1) and mouse GAPDH (Mm99999915_g1).2.16Statistical analysis was performed using Graph-pad Prism version 9 software (RRID : SCR_002798). Data was presented as mean averages with standard error of the mean (SEM). The statistical analysis between two groups was performed using the t test. Statistical analysis between more than two groups was performed using one-way ANOVA with multiple comparisons performed using Tukey’s test. Statistical analysis in the Kaplan Meier survival curves was performed using log-rank (Mantel-Cox) test. P values less than 0.05 were significant and were defined as *p<0.05, **p<0.01, ****p<0.001, ****p<0.0001.33.1We generated monoclonal antibodies (mAb) that recognize mouse (m) CX3CR1 by immunizing CX3CR1 knockout mice and screening for reactivity with mCX3CR1 transfected Jurkat and 300.19 cells and a lack of reactivity with un-transfected cells. We used mCX3CR1 transfected cells as a tool to determine the binding affinity and biological activity of our CX3CR1 mAb. The binding assay showed that the CX3CR1 mAb 455.1C11 recognized mCX3CR1 in a concentration dependent manner with an EC-50 value of 0.09 µg/ml indicating an apparent binding affinity of 0.6 nM while mIgG1 isotype control (MOPC21) did not show binding to 300 mCX3CR1 cells .3.2We tested whether the CX3CR1 antibody had agonist or antagonistic activity when bound to CX3CR1 on the cell surface using CHO cells transfected to express mouse CX3CR1 and a β-gal reporter. In these cells, CX3CR1 is coupled to the enzyme donor component of β-gal enzyme and β-arrestin is coupled to the enzyme acceptor component of β-gal enzyme (Eurofin Discovery X). Recruitment of β-arrestin to CX3CR1 generates active β-gal enzyme that emits luminescence as a surrogate of CX3CR1 activation. To confirm the fidelity of the assay, we first showed that CX3CL1 led to β-arrestin recruitment and induced a β-gal signal in a concentration dependent fashion  had the ability to block the binding of CX3CL1 to CX3CR1. Jurkat-mCX3CR1 cells were incubated with the CX3CR1 mAb followed by addition of CX3CL1-Fc fusion protein. The 1C11 mAb blocked the CX3CL1-CX3CR1 ligand-receptor interaction in a concentration dependent fashion  and observed that the 1C11 mAb could recognize and bind to human CX3CR1 with an EC-50 of 6.4 µg/ml, considerably weaker than its binding to mouse CX3CR1 (EC-50 of 0.09 µg/ml) . TAMs in the CT26 tumor express CX3CR1, and within the TAM subsets, M2 macrophages had higher CX3CR1 expression than M1 macrophages (in-silico approach and grouped human cancers from TCGA based on their CX3CR1 expression into high CX3CR1 (top 50th percentile) and low (bottom 50th percentile) groups. Analysis of the immune populations in human tumors using CIBERSORT showed that a higher M2 macrophage fraction was associated with higher CX3CR1 expression in multiple cancer types  on the cell surface that initiates cell signals upon binding to its ligand, CX3CL1 . MoreoveWhether the axis can directly mediate tumor immune evasion is not known. We treated CT26 tumor cells with CX3CL1 and determined which soluble mediators were upregulated in the tumor cells by mRNA and multiplex ELISA analysis. Interestingly, production of several cytokines that promote tumor immune resistance was upregulated when CT26 tumor cells were treated with CX3CL1 , were upCX3CL1 upregulated the secretion of multiple chemoattractants involved in the trafficking and differentiation of myeloid derived suppressor cells (MDSC) in the tumor including high levels of CXCL1, G-CSF, IL-6, LIF (another member of the IL-6 family), CXCL2 , and CCL5   is a second binding partner of CX3CR1 but has 10-20-fold lower affinity than CX3CL1 . CCL26 i3.9To investigate the expression of the CX3CL1-CX3CR1 axis in human cancers, we used the TCGA and GTEx databases and examined the expression of CX3CR1 and CX3CL1 in human tumors versus their expression in normal tissue Figure 6Our results support a role for the CX3CL1-CX3CR1 axis in tumor immune evasion. Though it is well known that the axis is important in chemotaxis, its functions in immune evasion should be further explored.4MDSC represents a major resistance mechanism that limits the response to anti-PD-1. A strategy that targets MDSC in combination with anti-PD-1 immunotherapy can improve responses , 5. HereWe observe higher survival and response in the syngeneic immunocompetent CT26 tumor model when anti-CX3CR1 is combined with anti-PD-1. Both anti-PD-1 and anti-PD-1 + anti-CX3CR1 long-term survivor mice show immunological memory. Generation of effector memory may be the central mediator of immunological memory based on previous studies . CX3CR1 The CX3CL1-CX3CR1 axis activates multiple oncogenic programs in the tumor including PI3K/Akt, ERK, EGFR, p38, β-integrin, MMP2/9/14, ICAM, VCAM, JAK/STAT, MAPK, ICAM and p38 –36. Our Secretion of soluble CX3CL1 can be part of a feedback loop to maintain a CX3CR1 driven immune-suppressive signaling program in the tumor. Adaptive resistance to immune attack can be mediated by tumor cells increased expression of PD-L1 on their cell surface in response to IFN-γ. CX3CL1 expression is also known to increase in response to IFN-γ and TNF-α  due to SSmall molecule antagonists to CX3CR1 such as JMS-17-2 bind to both CX3CR1 and CCR1 . Thus, aIn summary, our results show that the CX3CL1-CX3CR1 axis can promote tumorigenesis and immune-suppression by 1) Signaling through CX3CR1 that recruits myeloid populations and enhances secretion of soluble mediators by the tumor; 2) Increase of NLRP3 inflammasome component in the tumor; 3) Increase of pro-tumorigenic programs in the tumor. By reducing these activities, the CX3CR1 monoclonal antibody can disrupt recruitment, facilitate remodeling, and alter the MDSC compartment in the tumor microenvironment  can be found below: The animal study was approved by Dana-Farber Cancer Institute Institutional Animal Care and Use Committee. The study was conducted in accordance with the local legislation and institutional requirements.AC and GF: Conception and design. AC, XB, YW, MG, PHu, and S-HH: Data acquisition. AC, YW, and MG: analyzed the data and produced the figures. AC, XB, YW, MG, JT, GL, MD, UvA, PHw, and GF contributed to the interpretation of the data. AC and GF wrote the manuscript. AC, XB, YW, MG, PHu, S-HH, GL, MD, UvA, PHw, and GF edited the manuscript. All authors contributed to the article and approved the submitted version.