CD90 is regulated by notch1 and hallmarks a more aggressive intrahepatic cholangiocarcinoma phenotype

<jats:title>Abstract</jats:title><jats:sec>                 <jats:title>Background</jats:title>                 <jats:p>Intrahepatic Cholangiocarcinoma (iCCA) is characterized by a strong stromal reaction playing a role in tumor progression. Thymus cell antigen 1 (<jats:italic>THY1</jats:italic>), also called Cluster of Differentiation 90 (CD90), is a key regulator of cell–cell and cell–matrix interaction. In iCCA, CD90 has been reported to be associated with a poor prognosis. In an iCCA PDX model, we recently found that CD90 was downregulated in mice treated with the Notch γ-secretase inhibitor Crenigacestat. The study aims to investigate the role of CD90 in relation to the NOTCH pathway.</jats:p>               </jats:sec><jats:sec>                 <jats:title>Methods</jats:title>                 <jats:p>THY1/CD90 gene and protein expression was evaluated in human iCCA tissues and xenograft models by qRT-PCR, immunohistochemistry, and immunofluorescence. Notch1 inhibition was achieved by siRNA. THY1/CD90 functions were investigated in xenograft models built with HuCCT1 and KKU-M213 cell lines, engineered to overexpress or knockdown <jats:italic>THY1</jats:italic>, respectively.</jats:p>               </jats:sec><jats:sec>                 <jats:title>Results</jats:title>                 <jats:p>CD90 co-localized with EPCAM, showing its epithelial origin. In vitro, <jats:italic>NOTCH1</jats:italic> silencing triggered <jats:italic>HES1</jats:italic> and <jats:italic>THY1</jats:italic> down-regulation. <jats:italic>RBPJ</jats:italic>, a critical transcriptional regulator of NOTCH signaling, exhibited putative binding sites on the <jats:italic>THY1</jats:italic> promoter and bound to the latter, implying CD90 as a downstream NOTCH pathway effector. In vivo, Crenigacestat suppressed iCCA growth and reduced CD90 expression in the PDX model. In the xenograft model, Crenigacestat inhibited tumor growth of HuCCT1 cells transfected to overexpress CD90 and KKU-M213 cells constitutively expressing high levels of CD90, while not affecting the growth of HuCCT1 control cells and KKU-M213 depleted of CD90. In an iCCA cohort, patients with higher expression levels of <jats:italic>NOTCH1/HES1/THY1</jats:italic> displayed a significantly shorter survival.</jats:p>               </jats:sec><jats:sec>                 <jats:title>Conclusions</jats:title>                 <jats:p>iCCA patients with higher NOTCH1/HES1/THY1 expression have the worst prognosis, but they are more likely to benefit from Notch signaling inhibition. These findings represent the scientific rationale for testing NOTCH1 inhibitors in clinical trials, taking the first step toward precision medicine for iCCA.</jats:p>               </jats:sec>

section_content: Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive tumor developing in the biliary tract and the second most common primary hepatic cancer after hepatocellular carcinoma (HCC) [1]. In recent years, the global trend for iCCA has risen, reaching 2-3/100,000 inhabitants in most countries [2]. 

Survival rates for iCCA patients at 1, 3, 5-years are only 52. 1, 21. 7, and 11. 2%, respectively [3]. The only curative treatment options for iCCA are partial hepatectomy or chemotherapy and radiotherapy. However, these therapeutic approaches are suitable only for patients with early-stage disease; in addition, the tumor frequently recurs. Treatments for advanced iCCA are limited, and the prognosis is poor [4]. The lack of more effective therapies is mainly due to the little knowledge of the carcinogenesis mechanisms underlying iCCA development and progression and the lack of reliable biomarkers, predictive tools, and proper molecular classi cation. It has been proposed that the stroma reaction, particularly the interaction between iCCA cells and extracellular matrix components (ECM) of the surrounding microenvironment, plays a crucial role in disease progression (Coulouarn, Hepatology 2013). In this context, Thymus cell antigen 1 (THY1), also called Cluster of Differentiation 90 (CD90), is a 25-37 KD glycophosphatidylinositol (GPI)-anchored protein expressed in numerous cell types, including T cells, neurons, endothelial cells, and cancer-associated broblasts (CAFs). THY1 is a critical regulator of cell adhesion and communication, cell-ECM interaction and the immune and nervous systems [10, 11]. 

The latest ndings report that in several tumors, including ovarian cancer, THY1 is overexpressed in cancer stem cells (CSC) and the tumor microenvironment, enhancing proliferation and metastasis abilities [16]. Furthermore, in HCC, THY1 expression is related to poor tumor differentiation, invasive properties, and dismal prognosis [17, 18]. In iCCA, CD90 was reported, in a single study by immunohistochemistry, to be associated with lymph nodes metastasis [19]. Nevertheless, no data explaining the role of CD90 at the molecular level in iCCA are yet available. Recently, performing a comprehensive transcriptomic analysis in a PDX model of iCCA, we discovered the downregulation of CD90 in mice treated with Crenigacestat (REF). This molecule is a selective NOTCH1 γ-secretase inhibitor (GSI), already tested in phase 1 clinical trial in patients with advanced or metastatic solid tumors including CCA (NCT02784795), and currently used in further clinical studies (https://clinicaltrials. gov/ct2/show/NCT02784795). NOTCH signaling plays an essential role in biliary development and regeneration, tubulogenesis, and survival of stem cell niches [5]. Aberrant and/or overexpression of NOTCH1 via HES1 has been related to the development of iCCA in a mouse model [6]. The NOTCH cascade is initiated by the proteolytic cleavage of the γ-secretase enzyme that releases the NOTCH Intracellular Domain (NICD), which engages ligands of the Jagged or Delta-like families. NICD binds to the Recombination Signal Binding Protein For Immunoglobulin Kappa J Region (RPBJ) transcription factor in the nucleus, thus regulating its activity [7]. Inhibition of RBPJ activity led to the total inhibition of the NOTCH signaling pathway and the consequent reduction of iCCA development in the

This study aims to investigate the function of CD90 in relation to the activated NOTCH signaling cascade and the potential clinical implications.
section_name: Background

section_name: Methods

section_content: The HuCCT1, RBE, KKU-M213, and KKU-M156 human iCCA cell lines were purchased from ATCC or RIKEN. Regular monitoring with the MycoFluor TM Mycoplasma Detection Kit (ThermoFisher Scienti c, Waltham, MA) was performed to obtain a mycoplasma-free cell culture environment. The rst two cell lines were cultured in DMEM (Dulbecco's Modi ed Eagle Medium), while the last two were cultured in RPMI, both supplemented with Sodium Pyruvate, Antibiotic-Antimycotic, Hepes, and 10% fetal bovine serum (FBS) (Thermo Fisher Scienti c, Waltham, MA). Crenigacestat (LY3039478, Selleckchem Chemicals, Houston, TX) was used in in vitro and in vivo studies as GSI. Stock solutions were prepared in dimethylsulfoxide (DMSO) (Thermo Fisher Scienti c, Waltham, MA), and aliquots were stored at -20 °C. 

Mycoplasma-free KKU-M213, RBE, and KKU-M156 iCCA cell lines, after validation (Genetica DNA Laboratories, Burlington, NC, USA), were used for the in vitro studies. Cell lines were maintained as monolayer cultures in Dulbecco's modi ed Eagle medium supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 g/mL streptomycin (Gibco, Grand Island, NY, USA). Cells were grown for 12 h, then serum-deprived for 24 h and treated with siRNA against NOTCH1 (# s453558, ThermoFisher Scienti c), following the manufacturer's recommendations. Effects at 48h after siRNA transfection are shown.
section_name: Cell lines and treatment

section_content: For loss of function studies, the KKU-M213 cell line was transduced with Human shRNA lentiviral particles carrying 4 THY1 speci c sequences (A to D) and scramble control-shRNA sequence (OriGene Technologies, Inc., Rockville, MD 20850, USA). Scramble control-shRNA sequence and THY1-shRNA sequence B were used in all the experiments involving THY1 downregulation at a multiplicity of infection (MOI) 100. 

For gain of function studies, the HuCCT1 cell line was transduced with human THY1-CMV-GFP and Lenti-CMV-GFP-2A-Puro-Blank Lentivirus (Aurogene, Italy) at MOI 40. 

Both genetically modi ed cell lines were selected with puromycin dihydrochloride (Thermo Fisher Scienti c, Waltham, MA) at 1 µg/ml and 0. 625 µg/ml for KKU-M213 and HuCCT1, respectively, to obtain stable THY1 silencing and THY1 overexpression, according to the manufacturer's instructions.
section_name: Stable THY1 silencing and overexpression

section_content: For immunohistochemistry (IHC), formalin-xed, para n-embedded (FFPE) tissues from 174 patients with iCCA, routinely collected for diagnosis purposes, were studied. Tumor sections of 4 μm were freshly cut and dried at 60 °C for 30 min. IHC analysis was carried out in sections after depara nization for 30 min and then rehydration in grades of alcohol. Antigen retrieval was performed at 90 °C for 20 min with Tris-borate-EDTA Buffer. An automated stainer (cat. K5007, Dako, Glostrup, Denmark) was used to stain the iCCA sections with the primary anti-CD90 antibody diluted 1:150 (Abcam, Cambridge, MA). The Real Envision DAB Substrate Kit (DAKO) was used according to the manufacturer's instructions. CD90 expression was scored for all staining patterns, according to the staining intensity and the percentage of positively stained cells, by two independent, blinded pathologists. 

Additionally, two groups (n=10) of KKU-M213 scramble shRNA tumors treated with vehicle or Crenigacestat and two groups (n=10) of HuCCT1 THY1++ tumors treated with vehicle or Crenigacestat were stained by immunohistochemistry with AKT and pAKT (1:100 Cell Signaling Technologies, Massachusetts, USA). 

For immuno uorescence (IF) staining, frozen tissues sections were xed in a 1:1 acetone:chloroform solution, blocked with 2% bovine serum albumin solution, and incubated with anti-CD90, anti-αSMA, and anti-EPCAM antibodies (1:500, Cell Signaling Technologies, Massachusetts, USA). After washing, slides were incubated with secondary goat anti-rabbit immunoglobulin G H&L (Alexa Fluor 488, Thermo Fisher Scienti c, Waltham, MA).
section_name: Immunohistochemistry and immuno uorescence

section_content: Two million stable CD90-silenced KKU-M213 cell lines or CD90 overexpressed HuCCT1 cell lines, or corresponding cells that received the empty vector, were subcutaneously injected into the anks of 4-5week-old females CD1 nude mice. Each mouse was monitored daily for clinical signs and mortality, and body weight was recorded twice a week. Tumor growth was assessed with a caliper once a week, evaluating tumor volume with the formula (mm 3 ) = [length (mm) × width (mm) 2 ]/2, where width and length are the shortest and the longest diameters. When the tumor masses volume reached approximately 70-100 mm 3, the mice were randomly divided into 8 experimental groups of ten animals and administered Crenigacestat (8 mg/kg) or vehicle by oral gavage every 2 days for 35 days. At the end of the study, mice were sacri ced by cervical dislocation, and tumor masses were collected and frozen to -80°C.
section_name: In vivo studies

section_content: Total RNA was extracted using TRIzol® (Thermo Fisher Scienti c, Waltham, MA) in combination with the TissueLyser homogenizer (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The RNA concentration was determined with the NanoDrop Spectrophotometer (Thermo Fisher Scienti c, Waltham, MA). 

Quantitative reverse-transcription real-time PCR (qRT-PCR) cDNA was obtained starting from 1 microgram of total RNA, using the iScript Reverse Transcription Supermix (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's instructions. 

Quantitative PCR reactions were performed using SsoAdvanced SYBR green (Biorad Laboratories, Hercules, CA, USA) and the primers THY1 Human PrimePCR™ SYBR® Green Assay ID: qHsaCED0036661 (Biorad) and Hs_GAPDH_1_SG QuantiTect Primer Assay ID: QT00079247 (Qiagen, Hilden, Germania). Gene Expression Assays for human NOTCH1 (Hs01062014_m1), HES1 (Hs00172878_m1), THY1 (Hs06633377_s1), and β-actin (4333762T) were purchased from Applied Biosystems (Foster City, CA, USA). Quantitative values for each gene were calculated by using the PE Biosystems Analysis software and expressed as number target (NT). NT = 2-ΔCt, wherein the ΔCt value of each sample was calculated by subtracting the average Ct value of the target gene from the average Ct value of the β-Actin gene. Experiments were repeated three times in triplicate. For the screening of AKT Signaling, PrimePCR arrays (Cat #10025059, Bio-Rad Laboratories) was used. Real-time PCR was performed on the CFX96 System (Biorad Laboratories, Hercules, CA, USA). Comparative real-time PCR was performed in triplicate, including no-template controls. Relative expression was calculated using the 2 -ΔΔCt method. The ΔCt value of each sample was calculated by subtracting the average Ct value of the target gene from the average Ct value of the GAPDH gene.
section_name: RNA extraction

section_content: Chromatin isolated from KKU-M213, KKU-M156, and RBE cells was subjected to immunoprecipitation using the MAGnify Chromatin Immunoprecipitation System (492024, Thermo Fisher Scienti c, Waltham, MA) according to the manufacturer's instructions. Chromatin was sonicated to a fragment length of about 500 bp and immunoprecipitated with 1 μg of RBPJ (Cell Signaling Technologies, Massachusetts, USA, Active Motif, Belgium, Germany), H3K4me3 (Abcam, Cambridge, MA), H3K27me3 (Abcam, Cambridge, MA), and IgG (Cell Signaling Technologies, Massachusetts, USA) antibodies. The set of primers used for ChIP allows the ampli cation of target regions, including RBPJ consensus motifs (1 and 2) sites at -1000bp (genomic position 119,424,535 -119,424,508) from TSS at the 5' UTR region of the THY gene locus. Primer sequences can be provided upon request.
section_name: Chromatin immunoprecipitation

section_content: All experimental results are expressed as mean ± standard error of the mean (SEM). GraphPad Prism version 5. 03 (GraphPad Software, San Diego, CA, USA) was used to calculate Unpaired Student's t-test and one-way analysis of variance (ANOVA). Differences were considered statistically signi cant at p values * P < 0. 05, ** P < 0. 01, and *** P < 0. 001. 

Each biomarkers distribution was compared using a graphical plot that shows the scatter distribution of the quantities across medians and interquartile ranges. Due to the non-normal distribution of the standard deviations of biomarker distribution, the Wilcoxon test on the median was used to compare the expression of the biomarkers in the tumor tissues vs. surrounding areas. Pearson correlation analysis was applied to reveal a linear association between each biomarker in the following combinations: CD90 vs. NOTCH1, CD90 vs. HES1, HES1 vs. NOTCH1. To assess the statistical signi cance of monotonic associations between the variables, a P<0. 05 was used as a cut-off to reject the null hypothesis. The whole patients' population was subdivided into two groups according to the median values of each biomarker, assayed both separately and summed. Then the non-parametric Kaplan-Meier method was used to explore the survival probability in months, comparing the two groups of patients (below median vs. above median) for each biomarker and all the biomarkers together. Log-rank test was applied to evaluate the equality of survival among categories, setting a statistical signi cance threshold of < 0. 05. RPBJ putative transcription factor binding motifs on the THY1 gene promoter were predicted using the Eukaryotic Promoter Database New (EPDnew [20], https://epd. ep. ch). 

The Ingenuity pathway analysis (IPA) software (Qiagen, Hilden, Germania) was used to uncover the canonical pathways and molecular networks in which modulated genes are involved.
section_name: Statistical and bioinformatic analysis

section_content: We have previously reported that in a PDX model of iCCA, Crenigacestat treatment reduces tumor growth. However, in some in vitro experimental models, Crenigacestat did not affect either proliferation, survival, migration, and adhesion, either in 2D or 3D experimental conditions [9]. This apparent inconsistency prompted us to make a deeper investigation on the cancer stemness involved in cancer responsiveness/resistance.
section_name: Results

section_content: In all frozen iCCA PDX tissues sections, CD90 was co-localized with EpCAM, a speci c molecular marker of epithelial cells (Figure 1A ) but not with alpha-smooth muscle actin (α-SMA), a marker of stromal cells such as myo broblasts (Figure 1B ). The distribution pattern of CD90 was intracellular, as observed in the microscopic elds. Similarly, in a collection of 174 human iCCA para n-embedded tissues, CD90 was predominantly expressed in the cytoplasm of the tumor epithelial cells, independently of moderate (Figure 1C-D ) or severe differentiation (Figure 1E-F ). These ndings suggest that in the PDX model and patients' tissues, CD90 is present on epithelial but not on stromal cells, showing a similar intracellular distribution.
section_name: THY1/CD90 is expressed by epithelial cells in iCCA tissues

section_content: In a PDX iCCA model that we have generated, THY1/CD90 was signi cantly downregulated at the transcriptional and protein level (p<0. 01 and p<0. 001, respectively), following treatment with Crenigacestat (Figure 2A-B ). These data suggest that THY1/CD90 expression in iCCA is regulated by the NOTCH1 inhibitor Crenigacestat. 

To con rm our previous observation, we rstly checked the presence of CD90 in different iCCA cell lines and detected a strong expression of THY1 mRNA in KKU-M213, RBE, and KKU-M156 versus a very weak expression in HuCCT1 (Figure 3A ). Then, to test the hypothesis that Crenigacestat inhibits THY1/CD90, we silenced NOTCH1 in the three cell lines highly expressing THY1. In all these cells, after knocking down NOTCH1, the canonical NOTCH target HES1 was also strongly decreased (p<0. 001), and as hypothesized, THY1 mRNA was also signi cantly (p<0. 001) downregulated (Figure 3B ). These results further support the role of NOTCH1 in modulating THY1 expression. Furthermore, in silico approaches revealed that RBPJ, an essential transcriptional regulator of the canonical NOTCH signaling, displays putative binding sites at the THY1 gene promoter (Figure 4A ). To verify and validate this prediction, we inhibited the NOTCH pathway in vitro by transient overexpression of the dominant-negative form of RBPJ (dnRBPJ) [8, 21] in the representative RBE cell line. As expected, signi cant overexpression of RBPJ and a consistent reduction of THY1 mRNA levels by qRT-PCR occurred (p<0. 001; Figure 4B ). 

To further investigate the eventual binding of RBPJ at the THY1 promoter, the in-silico analysis of the 5' UTR region of the THY locus was additionally analyzed. We found that the sequence-paired site (SPS) contains two RBPJ binding sites, at genomic positions 119,424,535 -119,424,508 (Figure 4C ). This region is marked by activating epigenetic modi cations typical of open chromatin, which is accessible to transcription machinery, as we identi ed an enrichment of the active marker H3K4me3 versus low amounts of the repressive marker H3K27me3. Of note, we discovered by chromatin immunoprecipitation that RBPJ binds to the detected SPS in iCCA cells, con rming our hypothesis that THY1 is a downstream target gene of the NOTCH1 pathway.
section_name: THY1/CD90 is a NOTCH target

section_content: To verify that Crenigacestat inhibits the NOTCH/HES1/CD90 axis in vitro, we used two different iCCA cell models, engineered for loss or gain of CD90 expression. In the rst case, we used KKU-M213 cells expressing constitutively high levels of THY1, as shown in Figure 5A. At different Multiplicity of Infection (MOI) levels, we introduced lentiviral particles with scramble-shRNA or CD90-shRNA expression vector. THY1/CD90 was signi cantly (p<0. 001) inhibited at transcriptional and protein levels, as shown in Figure 5B, in silenced transfected cells compared to scrambled transfected cells at MOI 100. Next, 10 µM Crenigacestat signi cantly (p<0. 01) inhibited CD90 levels in KKU-M213 scramble-shRNA but not in KKU-M213 THY1/CD90-shRNA cells (Figure 5C ). 

In the second case, we used HuCCT1 cells previously shown to express constitutively low levels of CD90. As described above, we tested different MOI gain of THY1/CD90 expression, validated by qRT-PCR and immuno uorescence staining. The mRNA and protein expression levels of THY1/CD90 were signi cantly increased after the transduction approach in HuCCT1_ THY1/CD90++ at MOI 40 compared to those transduced with empty vector (p<0. 05; Figure 6A-B ). Also in this case, Crenigacestat 10µM signi cantly (p<0. 001) reduced CD90 expression in the HuCCT1_CD90++ cells, but not in the HuCCT1 empty vector (Figure 6C ). These results suggest that Crenigacestat inhibits the NOTCH/HES1/CD90 axis in iCCA in vitro models.
section_name: Crenigacestat inhibits the expression of THY1/CD90 in iCCA cells

section_content: To further demonstrate the effectiveness of Crenigacestat on iCCA expressing CD90, we used two complementary xenograft models, built using the engineered iCCA cells with loss or gain of CD90 expression. First, stable KKU-M213 scramble-shRNA and KKU-M213 THY1/CD90-shRNA cells were engrafted in CD-1 nude mice. Then, after preliminary experiments to determine the growth curve, mice were treated 13 days after engraftment, when the tumor masses were sure to have grown, with vehicle or Crenigacestat at 8mg/Kg (twice a week) for four weeks, monitoring the tumor growth twice a week. KKU-M213 scramble shRNA tumors (constitutively expressing high levels of CD90) treated with vehicle grew faster than other experimental groups. However, Crenigacestat signi cantly (p<0. 001) inhibited the growth of KKU-M213 scramble shRNA tumors (expressing constitutively high levels of CD90) but not the growth of KKU-M213 THY1/CD90-shRNA. In controls, the vehicle did not affect the tumoral growth in either of the groups (Figure 7A ). 

In a complementary mirrored experiment, HuCCT1 empty vector and HuCCT1 THY1++ cells were engrafted in CD1 mice and treated with vehicle or Crenigacestat under the above described experimental conditions, after preliminary curve growth experiments. Consistently with the previous experiments, tumors originating from HuCCT1 THY1++ cells (constitutively expressing low levels of CD90 and therefore transfected for CD90 overexpression) treated with vehicle showed the most aggressive phenotype. Moreover, Crenigacestat signi cantly (p<0. 01) reduced the growth of the HuCCT1 THY1++ tumors, whereas it did not affect the growth of the HuCCT1 empty vector. In controls, the vehicle had no effect in either of the mice groups (Figure 7B ). 

In conclusion, the combination of both experimental models demonstrates that iCCA tumors expressing CD90 display a more aggressive phenotype, and Crenigacestat inhibits the growth of tumors expressing high levels of CD90.
section_name: Crenigacestat inhibits tumor progression in CD90-positive xenograft models

section_content: To identify the downstream targets implicated in inhibiting tumor progression of xenograft models CD90 positive after Crenigacestat treatment, we used a commercially PCR array with 40 genes involved in the AKT signaling, frequently coactivated with NOTCH signaling in human iCCA [22] [23] [24]. 

In the KKU-M213_scramble shRNA tumors (constitutively expressing high levels of CD90) treated with vehicle or Crenigacestat, we found 11 genes signi cantly downregulated (≤ -1. 5-fold change) in treated mice compared to vehicle (Additional Data1). Complementarily, in tumors originating from HuCCT1_CD90++ cells (transfected to overexpress CD90), treated with vehicle or Crenigacestat, we found 27 genes signi cantly downregulated (≤ -1. 5-fold change) in treated mice compared to vehicle (Additional Data2). Then, we focused our attention on genes commonly modulated in both models. The Venn diagram revealed 9 common genes in the two models (Figure 7C ). Crenigacestat treatment reduced the expression of these genes when compared with their corresponding vehicle group (Figure 7D ). 

As expected, Ingenuity pathway analysis (IPA) on these genes revealed that the PI3K/AKT signaling pathway was at the top of the signi cant pathways (Additional Figure 1A ). Moreover, the networks generated from the 9 genes were 2 and (Additional Figure 1B-C ), in both networks, downregulated genes were strongly interconnected and involved in cell proliferation and cancer. 

According to bioinformatics and gene expression analyses, we performed immunohistochemistry to detect AKT and pAKT expression in CD90 positive xenograft tumors treated with vehicle or Crenigacestat. The levels of AKT and pAKT were strongly (66,99%, p<0. 001) and moderately (48,55%, p<0. 001) reduced by Crenigacestat treatment, respectively, in HuCCT1_THY1++ (Figure 7E ) and KKU-M213_scramble shRNA (Figure 7F ) xenograft tumors compared to vehicle. 

Overall, we believe that the improvement response to Crenigacestat treatment in CD90 positive cells could be at least partly due to the inhibition of AKT signaling.
section_name: CD90 affects iCCA progression by regulating the AKT signaling

section_content: The analysis of RNA sequencing expression data on 31 iCCA patients' tissues and matched surrounding normal liver tissues from the GEO database (GSE107943) [25] showed that THY1 mRNA levels were signi cantly higher in tumors compared to adjacent non-neoplastic tissues (p= 1. 28*10 -7 ) (Additional Figure 2 ). In our population of 44 iCCA patients, for which clinicopathological data were available, with a mean age of 66. 36 years, the gender distribution was 61% (n=27) males and 39% (n=17) females; 41% (n=18) of the patients had evidence of cirrhosis, and in just 18% (n=8), tumor size was greater than 5 cm. Moreover, 50% (n=19) and 18% (n=8) of patients had lymph nodes and lung metastasis, respectively, as reported in Additional Table 1. To test differences in the concentration of each biomarker between the tumor tissue and the surrounding area, we plotted the median distribution differences (and their interquartile ranges) of NOTCH1, HES1, and THY1 in those areas in the whole population. The plots showed a statistically signi cant (p<0. 05) higher expression of each biomarker in the tumoral than in the peritumoral tissues (Figure 8A ). In addition, to assess the association between each biomarker present in the tumor tissue, we ran Pearson correlation analysis (Figure 8B ), demonstrating a positive, statistically signi cant association between any combination of them (p < 0. 01), in the whole sample. 

Finally, we investigated the prognostic power of NOTCH1, HES1, and THY1, alone or in combination, on iCCA patients' survival in months. As reported in Figure 8c, Kaplan Meier survival analysis demonstrated that patients with NOTCH1/HES1/THY1 expressions above the median values had worse survival than patients with values below the median (log-rank p <0. 05). In addition, survival was signi cantly shorter in months when considering the presence of each biomarker above the median (mean 17. 5 ± 10. 28, p<0. 05) than in patients without this condition (mean 30. 36 ± 12. 91). In conclusion, the presence of the NOTCH1/HES1/THY1 axis de nes a subset of iCCA patients with a more aggressive tumoral phenotype and a worse prognosis.
section_name: Prognostic role of the NOTCH1/HES1/CD90 axis in iCCA patients

section_content: The minimal effectiveness of currently used chemotherapy and fast growth make iCCA a very deadly cancer. The poor knowledge of the molecular mechanisms regulating tumor-host interactions hampers the development of target-oriented therapies. In this study, for the rst time, we show that CD90 is directly modulated by NOTCH and that a NOTCH1 γ-secretase inhibitor effectively reduces the progression of iCCA expressing high levels of CD90. We based this conclusion on the following data: 1) silencing of NOTCH1 leads to a substantial reduction of THY1 in iCCA cells constitutively expressing high levels of CD90; 2) inhibiting the NOTCH pathway by transient overexpression of the dominant-negative form of the RBPJ transcription factor, triggers a signi cant reduction of THY1 mRNA levels; 3) in a xenograft model, Crenigacestat inhibits tumoral growth of KKU-M213 iCCA cells constitutively expressing high levels of CD90 but is not effective on the same cells after CD90 knockdown; 4) in a second xenograft model, Crenigacestat inhibits tumor growth of CD90-transfected HuCCT1 iCCA cells, but is not effective on the control cells constitutively expressing low levels of CD90. 

Our results demonstrate that THY1 is regulated by the NOTCH pathway and not vice versa, as described in K. Wu et al [26] described in gastric cancer. In addition, as reported in several studies, DNA-binding protein RBPJ is a downstream transcription factor that occupies consensus DNA-binding sites while exchanging repressors for activators in response to NICD [27] [28] [29] [30]. In our work, inhibition of the NOTCH pathway by dnRBPJ overexpression decreased THY1 mRNA levels. Speci cally, RBPJ is recruited to the THY1 gene promoter via two RBPJ binding sites separated by spacer sequences of 15-17 base pairs (bp), enabling cooperative binding of NOTCH transcription complex dimers. Thus, the present data indicate that THY1 expression is downstream of the NOTCH1 pathway. Similarly, K. Wu and colleagues reported in their work that upregulation of miR-140-5p inhibits the NOTCH signaling pathway and consequently reduces THY1 expression in gastric cancer [26]. THY1/CD90 has been suggested as a microenvironment sensor in different cancers, including lung and glioblastoma [31, 32]. In HCC, CD45-CD90+ cells but not CD90-cells isolated from blood and human tumor tissues generate tumor nodules in Beige/SCID mice. 

The authors subsequently demonstrated that the additional stem cell marker CD44 on the CD90 + cells contributed to greater aggression and metastasis of HCC subpopulations of CSCs [33]. Moreover, we demonstrated that the response to a NOTCH1 γ-secretase inhibitor treatment in CD90 positive cells could be mediated by the AKT signaling, speci cally through AKT suppression. This nding agrees with Gao's study that proved the activation of the PI3K/AKT/HIF-1α signaling pathway in CD90 positive gastric cancer cell lines [24]. The mechanisms whereby CD90 regulates AKT remain to be addressed. 

In iCCA patients, CD90 has been reported in human surgical samples and correlated with lymph nodes metastasis, indicating poor prognosis [19]. In a GEO database cohort, as well as in our collection of iCCA human tissue samples, THY1 is more strongly expressed in tumoral compared to adjacent non-tumorous tissues. Herein, in a cohort of 44 patients, we demonstrate that NOTCH1, HES1, and THY1 mRNA levels are positively correlated and are more pronouncedly expressed in tumoral than in peritumoral tissue. In this context, the Kaplan Meier survival analysis of human iCCA tumors suggested that patients with high NOTCH1, HES1, and THY1 expression have the worst prognosis. Our ndings are concordant with an increasing body of evidence strongly suggesting that the NOTCH signaling pathway is a prominent driver of CCA progression and poor prognosis [23, 34, 35]. However, the importance of THY1 expression in human cancers is controversial. Indeed, except for human ovarian cancer and nasopharyngeal carcinoma, where CD90 seems to play a tumor suppressor role [36, 37], several works reported that CD90 overexpression in the tumor microenvironment enhances proliferation and metastasis, conferring the poorest outcome [19, [38] [39] [40]. Particularly, THY1 is highly expressed in cancer stem cells of gastric cancer, differentiated acute myeloid leukemia (AML) subtypes, lymph nodes metastasis in esophageal squamous carcinoma cells and also contributes to poor survival in HCC [17]. Here, we found an intriguing relationship between NOTCH signaling and THY1, closely associated with poor prognosis in patients with iCCA. According to the Luo et al. study, NOTCH1 signaling activated by JAGGED1 is positively related to CD90+ HCC CSCs with a rapid G1/S transition in the cell cycle phase [41]. Additionally, our results demonstrated that CD90 hallmarks a group of iCCA with a more aggressive phenotype responsive to Crenigacestat treatment. The identi cation of predictive biomarkers of response or survival such as NOTCH1, HES1, and CD90 offers hope to patients with iCCA eligible for and likely to bene t from Crenigacestat therapy.
section_name: Discussion

section_content: Overall, our results highlight that patients with higher NOTCH1/HES1/CD90 expression have the worst prognosis and shorter survival. Nevertheless, in preclinical experimental models, this newly identi ed axis is successfully druggable with a NOTCH γ-secretase inhibitor, suggesting CD90 as a promising molecular target for treatment in a subset of patients with this disease. To our knowledge, this is the rst demonstrated evidence for a molecular strati cation of iCCA predicting clinical outcomes. The successful use of the NOTCH1 γ-secretase inhibitor in preclinical models represents the scienti c rationale for using this molecule in future clinical trials. In addition, this study takes the rst step toward personalized medicine for patients currently lacking effective treatments.
section_name: Conclusions

section_content: Acknowledgements: The authors are grateful to Mary V. C. Pragnell, B. A., for English revision and Rodolfo Sardone for statistical analysis.

section_content: Availability of data and materials: All data generated or analysed during this study are included in this manuscript [and its supplementary information les].

section_name: Declarations

section_content: siRNAs: Small interfering RNAs, THY1: Thymus cell antigen 1.

section_content: Ethics approval and consent to participate: The animal experiments were approved by the Ethics Committee (Prot. N.
section_name: Declarations

section_content: This is a list of supplementary les associated with this preprint. Click to download.
section_name: Supplementary Files