BMI-1 activates hepatic stellate cells to promote EMT of colorectal cancer cells

BMI-1 activates hepatic stellate cells to promote EMT of colorectal cancer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article BMI-1 activates hepatic stellate cells to promote EMT of colorectal cancer cells Zhongyang Jiang, Ximei Ma, Xiaohui Luan, Zhenyu Liuyang, Yiyang Hong, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2247681/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Activated hepatic stellate cells (aHSCs) are the major source of cancer-associated fibroblasts (CAFs) in the liver. Though the crosstalk between aHSCs and colorectal cancer (CRC) cells supports liver metastasis (LM), the mechanisms remain largely unknown. Here, we investigated BMI-1, a polycomb-group protein family member, which is high-expressed in LM, in hepatic stellate cells (HSCs) activation and interacting with CRC cells while promoting colorectal cancer liver metastasis (CRLM). We found the positivity of BMI-1 expression in the liver of CRLM patients was 77.8%, and the expression level of BMI-1 continued to increase during CRLM in mice. We overexpressed BMI-1 in HSCs (LX2) by lentivirus infection, and HSCs were activated, accompanied by increased expression levels of α-SMA, Fibronectin, TGF-β1, MMPs, and IL-6. CRC cells (HCT116 and DLD1) were cultured in HSCs-conditioned medium (LX2 NC CM or LX2 BMI-1 CM), and CRC cells cultured in BMI-1 CM exhibited enhanced proliferation and migration ability, and EMT phenotype with activation of TGF-β/SMAD pathway. Besides, a TFG-βR inhibitor SB-505124 largely diminished the effect of the BMI-1 CM on Smad2/3 phosphorylation in CRC cells. A mouse subcutaneous xenotransplantation tumor model was established by co-implantation of HSCs (LX2 NC or LX2 BMI-1) and CRC cells, andBMI-1 overexpressed LX2 HSCs promoted tumor growth and epithelial-mesenchymal transition (EMT) phenotype in vivo . In conclusion, BMI-1 activates HSCs to promote the EMT of CRC cells partially through the TGF-β/SMAD pathway. These findings demonstrate BMI-1 activated HSCs might be a new target in CRC therapy. BMI-1 hepatic stellate cell colorectal cancer liver metastasis epithelial-mesenchymal transition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In the world, colorectal cancer (CRC) is 3rd most common cancer and ranks 2nd in mortality from cancer (Bray et al. 2018 ; Siegel et al. 2020 ). In CRC patients, liver metastasis (LM) is the primary cause of death. Liver plays a major role in survival as the liver is often the only site of metastasis for CRC. In CRC patients, about 14 to 35% have LM at diagnosis, and about 70% have LM by the end of the disease (Valderrama-Treviño et al. 2017 ). In colorectal cancer liver metastasis (CRLM) patients, liver resection is the primary curative treatment option, with the 5-year survival rate of 20–50% (Tsilimigras et al. 2021 ). Though treatments have advanced in the past years, the rate of intrahepatic recurrence is still high. Hepatic microenvironment has numerous signaling factors from liver cells which consist of parenchymal and non-parenchymal cells, the interaction between cancer cells and stromal cells facilitates development of metastasis in liver (Milette et al. 2017 ; Brodt 2016 ). Cancer-associated fibroblasts (CAFs), comprising the major stromal cell type, are fibroblast populations found in primary and metastatic cancers and related to tumor initiation, progression and metastasis by regulating the extracellular matrix (ECM) remodeling and immune response (Mao et al. 2021 ; Kalluri 2016 ; Sahai et al. 2020 ). Hepatic stellate cells (HSCs) are the major non-parenchymal liver cells which store retinol and play a role in liver repair, liver fibrosis, and hepatocellular carcinoma (HCC) (Higashi et al. 2017 ; Friedman 2008 ; Barry et al. 2020 ; Shiraha et al. 2020 ). HSCs contribute 85–95% of fibroblasts in liver (Mederacke et al. 2013 ). HSCs are activated to CAFs by cancer cells to support tumor growth and metastasis (Makino et al. 2018 ; Zhao et al. 2022 ). Hepatic CAFs are thought to come primarily from HSCs and to a lesser extent from bone marrow-derived precursors, portal fibroblasts and endothelial cells (Biffi et al. 2021). In intrahepatic cholangiocarcinoma, HSCs-derived CAFs are the main tumor-interacting population (Affo et al. 2021 ). Although CRC is the most common primary cancer that metastasizes to the liver, the mechanisms by which HSCs interact with CRC cells to promote LM remain largely unclear. BMI-1, one of the polycomb-group (PcG) protein family members, plays a critical role in negatively regulating the Ink4a/Arf locus which encodes p16 INK4a and p19 ARF (Jacobs et al. 1999 ). BMI-1 regulates self-renewal of stem cell and participate in carcinogenesis of human cancers, including CRC (Jiang et al. 2009 ; Tateishi et al. 2006 ; Gil et al. 2005 ). We have previously reported that BMI-1 was overexpressed in CRLM and contributes to the LM by regulating epithelial-mesenchymal transition (EMT) of CRC cells in vitro and in vivo (Xu et al. 2021 ). Interestingly, we observed BMI-1 positive staining in liver cells both in human and mice liver specimens of CRLM. Studies have showed that quiescent HSCs can be activated by cancer cells and transformed into CAFs, and in turn activated HSCs (aHSCs) promote cancer cells invasion and EMT (Makino et al. 2018 ; Zhao et al. 2022 ). Thus, we speculated that BMI-1 plays a role in crosstalk between HSCs and CRC cells during LM. In this study, CRLM patients and mice showed increased BMI-1 expression in liver cells. BMI-1 overexpressed HSCs were activated and transformed into CAFs. CRC cells cultured in conditioned medium (CM) from BMI-1 overexpressed HSCs showed enhanced proliferation, migration and EMT ability. Experimental subcutaneous xenotransplantation tumor model in vivo also showed increased tumor proliferation and EMT of CRC cells co-cultured with BMI-1 overexpressed HSCs. These findings may provide a potential new target for treating CRLM. Materials And Methods Immunohistochemistry (IHC) analysis of CRLM patients LM and adjacent normal liver tissues from 18 clinical diagnosed patients with CRLM were collected from the Pathology Department (from 2013 to 2020), Sir Run Run Shaw Hospital, Hangzhou, China. The expression of BMI-1 was investigated with IHC staining. Anti-BMI-1 (1:100; Cell Signaling Technology, USA) was used as Primary antibody before secondary antibody incubation. Cell culture and treatment High metastatic human CRC cell line (HCT116), low metastatic human CRC cell line (DLD1), mouse CRC cell line (CT26) and human hepatic stellate cell line (LX2) were purchased from the American Type Culture Collection (Manassas, USA). Cells were cultured in DMEM medium (Gibco, USA) with 10% FBS and 1% penicillin/streptomycin in a 5% CO 2 humidified incubator at 37°C. BMI-1 overexpressed lentiviral construct (pGC-FU-GFP- BMI-1 ) and negative control (pGC-FU-GFP) were obtained from Genechem (Shanghai, China). The control (LX2 NC) and stable BMI-1 overexpressed LX2 (LX2 BMI-1) were established using lentivirus transfection under the manufacturer’s instructions. Besides, to make conditioned medium (CM), we firstly cultured transfected LX2 in a 10cm plate with 1×10 6 cells. Then we used 6mL serum-free DMEM medium to culture cells for 24h. We collected the supernatants, centrifuged at 1000× rpm for 10 min and filtrated through a 0.22mm filter unit (Millex, USA). CM from NC LX2(NC CM) and BMI-1 LX2(BMI-1 CM) were made by mixing the different supernatant with complete medium (1:1). After that, we cultured CRC cells with CM for 24 h. All CM would be used in 2 d. To inhibit Smad2 phosphorylation, after SB-505124 (0.05 µM) was utilized to pretreat CRC cells for 1 hour, the cells were then incubated with BMI-1 CM for 24 hours. Western blot Whole protein from the tissue and cell samples were extract using RIPA buffer with a 1% protease/phosphatase inhibitor. After centrifugation of the extraction solutions, BCA Protein Concentration Assay Kit (Solarbio, China) was utilized to quantify the proteins. After SDS-PAGE, proteins were migrated to PVDF membranes. Antibodies against BMI-1, GAPDH, β-actin, Vimentin, Smad2/3, phosphorylated Smad3 (1:1000; Cell Signaling Technology, USA), α-SMA, TGF-β1, phosphorylated Smad2 (1:1000; Abcam, USA), Fibronectin (1:1000; BD Biosciences, USA), E-cadherin, ZEB1, Twist-1 (1:1000; Novus Biologicals, USA), Snail (1:1000; Proteintech Group, USA) were utilized as primary antibodies. After washing with TBST buffer three times every 10 minutes, goat anti-rabbit/mouse IgG (Abcam, USA) was utilized as secondary antibodies. A Bio-Rad CD Touch detection system (USA) with ECL detection reagents was utilized to detect protein bands. qPCR Whole RNA from the tissue and cell samples were extract using Trizol reagent (Cwbio, China). Afterward, HiScript II Q RT SuperMix (Vazyme Biotech, China) was utilized for reverse transcription. ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, China) was utilized quantitative polymerase chain reaction (qPCR). A Bio-Rad CFX-96 Real-Time PCR system (USA) was utilized to analyze the results. The sequences of all primers were listed in Table S1. Immunofluorescent staining Immunofluorescent staining was performed on 96-well plates. Cells were fixed in paraformaldehyde and permeabilized with Triton X-100. Before incubating with a secondary antidody, antibodies against Vimentin (1:100; Cell Signaling Technology, USA) and Snail (1:100; Proteintech Group, USA) were used as primary antibodies. A Zeiss AXIO Observer A1 inverted fluorescence microscope system (Germany) was utilized to obtain images. Cell viability assay Cells were cultured with CM separately for 24, 48, 72, and 96 h. After treatments, CCK-8 reagent (Yeasen Biotechnology, China) was added and incubated for 2 h. A spectrophotometer (Thermo Fisher, USA) was utilized to detect the optical density (OD) value (450nm). Colony formation assay CRC cells were cultured with CM for 2 weeks. Cells were fixed in paraformaldehyde and stained by crystal violet. Colonies were viewed with an Olympus CKK53 microscope, and photos were taken. Image J was utilized to measure number of colonies. Wound healing assay CRC cells were cultured to reach 90% confluence. After creation of a linear wound, cells were cultured with CM for 48 h. Wounds were viewed with an Olympus CKK53 microscope, and photos were taken at 0, 24, 48 h. Image J was utilized to measure the migration rate. Transwell migration assay Transwells chambers were purchased form Corning. CRC cells were plated in the upper chambers with serum-free DMEM, while CM was in the lower chambers. After 48 h, cells in the upper chambers were removed, and cells on the lower membrane surface were fixed and stained. The migrated cells were viewed with an Olympus CKK53 microscope, and photos were taken. Image J was utilized to measure number of cells. IHC Tissue samples were fixed, dehydrated, embedded and sectioned. After deparaffinization, sections were blocked with goat serum and incubated with primary antibodies for 24 h. Then, the sections were incubated with secondary antibody for 30 min, followed by counterstaining with Mayer’s hematoxylin. At last, an Olympus CKK53 microscope was utilized to view and photograph the staining of the sections. Animal experiments All animal experimental procedures were approved by the Committee on the Ethics of Animal Experiments of Zhejiang University. Treatment protocol 1 5-week-old male C57 mice (19-20g) were randomly divided into five groups (n = 3). Isoflurane (inhal.) was utilized to anesthetized the mice. Then, the spleen was exteriorized through a left flank incision. CT26 cells (2×10 6 ) were intrasplenic injected to establish the tumor, and then injection site was pressed for 5 min. Surgical thread was used to close the peritoneum and skin. The mice were sacrificed at 7, 14, 21 and 28d after CT26 cells inoculation. Resected liver tissues were collected for qPCR, WB and IHC assay. Treatment protocol 2 5-week-old male BALB/c nude mice (17–18 g) were randomly divided into four groups (n = 5): (1) HCT116 cells (5×10 6 ) mixed with LX2 NC cells (1×10 6 ); (2) HCT116 cells (5×10 6 ) mixed with LX2 BMI-1 cells (1×10 6 ); (3) DLD1 cells (5×10 6 ) mixed with LX2 NC cells (1×10 6 ); (4) DLD1 cells (5×10 6 ) mixed with LX2 BMI-1 cells (1×10 6 ). The mixed cells were subcutaneously injected into the flanks to establish the tumor. Tumor sizes were estimated every two days after the tumors were measurable. The mice were killed at 28d after cell inoculation. Resected tumor tissues were collected for qPCR, WB and IHC assay. Total tumor volume (mm 3 ) = L× W 2 /2 (L = length and W = width). Statistical analysis In all experiments, the date is showed as the mean + SD based on three independent experiments. The difference between the groups was analyzed using the χ2 test, Fisher’s exact probability, Student’s t test or one-way Analysis of Variance (ANOVA) properly in GraphPad Prism 8 Software or SPSS 25.0. P < 0.05 was regarded as statistically significant. Results The expression of BMI-1 is up-regulated in liver cells during CRLM In our previous research, it was found that BMI-1 was overexpressed in LM of human CRC (Xu et al. 2021 ). Interestingly, though the normal liver cells were BMI-1 negative, 77.8% CRLM patients (14 among 18 samples, Fig. 1 A) was BMI-1 positive in liver cells. The positivity of BMI-1 expression was not highly related to clinic pathological characteristics (including tumor size and number in liver, differentiation degree, T stage, N stage) except tumor site (Table 1 ). BMI-1 expression positivity in liver cells of rectum cancer LM patients was significantly higher (100%) than colon cancer LM patients (60%). To investigate the BMI-1expression level in liver cells during CRLM, we checked BMI-1 expression at 0, 7, 14, 21, and 28 d after mice were intrasplenic injected with CT26 cells (Fig. 1 B). As shown in Fig. 1 C, compared with healthy control mice, the protein expression level of had no significant change at 7 and 14 d, but markedly increased to 4.58 fold at 21 d and continued increasing to 7.29 fold at 28 d (Fig. 1 D). The mRNA expression level of BMI-1 accordingly increased to 2.92 fold at 21 d and 6.71 fold at 28 d (Fig. 1 E). IHC result also showed that the nuclei of liver cells were BMI-1 positive in CRLM mice since 14 d, and BMI-1 positive rate and intensity increased markedly at 21 and 28 d (Fig. 1 F). These results suggest that up-regulated BMI-1 expression in liver cells may play a role in CRLM. Table 1 Correlation between CRCLM patient clinicopathological characteristics and BMI-1 expression in the liver. Characteristics Cases (n = 18) BMI-1 expression P value Negative (n = 4) Positive (n = 14) Age (yr) 0.051 < 60 12 4 8 ≥ 60 6 0 6 Gender 0.509 Male 11 3 8 Female 7 1 6 Primary tumor site 0.018 a Colon 10 4 6 Rectum 8 0 8 Tumor size (in Liver) 0.278 < 30mm 8 3 5 30-50mm 3 0 3 ≥ 50mm 5 1 5 Tumor numbers (in liver) 0.432 1–2 12 2 10 ≥ 3 6 2 4 Differentiation degree 0.881 Low 4 1 3 middle-high 14 3 11 T stage 1 3 18 4 14 N stage 0.248 N0 9 3 6 N1 + N2 7 1 8 Table 1 Correlation between CRCLM patient clinicopathological characteristics and BMI-1 expression in the livers. Characteristics Cases (n = 18) BMI-1 expression P value Negative (n = 4) Positive (n = 14) Age (yr) 0.051 < 60 12 4 8 ≥ 60 6 0 6 Gender 0.509 Male 11 3 8 Female 7 1 6 Primary tumor site 0.018 a Colon 10 4 6 Rectum 8 0 8 Tumor size (in Liver) 0.278 < 30mm 8 3 5 30-50mm 3 0 3 ≥ 50mm 5 1 5 Tumor numbers (in liver) 0.432 1–2 12 2 10 ≥ 3 6 2 4 Differentiation degree 0.881 Low 4 1 3 middle-high 14 3 11 T stage 1 3 18 4 14 N stage 0.248 N0 9 3 6 N1 + N2 7 1 8 BMI-1 activates hepatic stellate cells To investigate the effects of BMI-1 on liver cells, we overexpressed BMI-1 inLX2 HSC and confirmed the overexpression by western blotting (Fig. 2 A). High expression of α-SMA, a myofibroblast marker, is a key marker of aHSCs. aHSCs highly secrete a key cytokine TGF-β1 and promote hepatic fibrosis with Fibronectin expression (Tsuchida et al. 2017). Compared with control quiescent HSCs, BMI-1 overexpressed LX2 HSCs showed significant increase of α-SMA expression. Fibronectin and TGF-β1 expression were also markedly increased in BMI-1 overexpressed LX2 HSCs. Meanwhile, immunofluorescent staining also confirmed that the expression of α-SMA and Fibronectin were significantly increased in LX2 cells overexpressed BMI-1 (Fig. 2 B). Besides, we also observed the transcript level of matrix metalloproteinases (MMP1, MMP2, MMP3, MMP7, MMP9) and inflammatory cytokines (TGF-β1 and IL-6) were remarkably increased (Fig. 2 C). Thus, LX2 HSCs were activated by overexpression of BMI-1. BMI-1 overexpressed HSC CM enhances CRC cell proliferation and migration To explore the effect of BMI-1 on the interaction between HSCs and CRC cells, CRC cells (HCT116 and DLD1) were cultured in control LX2 CM (NC CM) or BMI-1 overexpressed LX2 CM (BMI-1 CM). We first checked the cell proliferation by CCK-8 assay. Compared with cells cultured in NC CM, the proliferation ability of CRC cells cultured in BMI-1 CM markedly enhanced at day 4 (Fig. 3 A), the colony formation ability of CRC cells cultured in BMI-1 CM also significantly increased (Fig. 3 B). Meanwhile, CRC cells cultured in BMI-1 CM showed higher wound healing rates (Fig. 3 C). Transwell migration assays showed the same results (Fig. 3 D). We then investigated EMT-related molecular changes. Consistent with the above results, the mRNA and protein expression levels of ZEB-1, Twist-1, Vimentin, and Snail in CRC cells cultured in BMI-1 CM were significantly increased while E-cadherin was decreased (Fig. 3 E). Immunofluorescent staining further demonstrated that Snail and Vimentin expression were obviously increased in CRC cells cultured in BMI-1 CM (Fig. 3 F). Therefore, BMI-1 maybe an important regulator in activated HSCs which promoted malignant phenotype of CRC cells. BMI-1 overexpressed HSC CM promotes CRC cell EMT via activating Smad2/3 pathway As mentioned before, TGF-β1 was upregulated in BMI-1 overexpressed LX2 HSC (Fig. 2 A). TGF-β/SMAD signal pathway plays an important role in metastasis by modulating EMT process (Xu et al. 2009 ; Hao et al. 2019 ), so we hypothesize BMI-1 CM induces CRC cells EMT through activating Smads. We first checked the expression levels of Smad2/3. As shown in Fig. 4 A and B, though the mRNA and protein expression level of Smad2 and Smad3 had no changes in CRC cells cultured inBMI-1 CM, the Smad2 phosphorylation and Smad3 phosphorylation were significantly increased compared with CRC cells cultured in NC CM. Then we used SB-505124 (a TFG-βR inhibitor which inhibit Smad2 phosphorylation) to further confirm the involvement of Smads on the pro-EMT effects of BMI-1 CM. SB-505124largely decreased BMI-1 CM-induced Smad2 phosphorylation and Smad3 phosphorylation in CRC cells (Fig. 4 C, D). Meanwhile, the downstream target proteins (Snail and Vimentin) of TGF-β/SMAD pathway accordingly decreased in SB-505124 treated CRC cells which cultured in BMI-1 CM (Fig. 4 C, D). These results confirmed that BMI-1 CM induces CRC cells EMT partially via activating Smad2/3. BMI-1 overexpressed HSC promotes tumor growth in vivo We further evaluated the effects of BMI-1 overexpressed LX2 HSC on CRC cell growth in vivo . A subcutaneous xenotransplantation tumor model by co-implantation of HSCs (LX2 NC or LX2 BMI-1) and CRC cells (Fig. 5 A). Compared with mice co-implantation with LX2 NC, tumor grew faster and larger in mice co-implantation with LX2 BMI-1 HSC and CRC cells as shown by increased tumor volume and weight (Fig. 5 B, C). Moreover, consistent with in vitro results, we found that Snail and Vimentin were upregulated in mice co-implantation with LX2 BMI-1 HSC and CRC cells according to the results of qPCR and Western blotting (Fig. 5 D, E). Furthermore, the increased expression of Snail and Vimentin in mice liver co-implantation with LX2 BMI-1 HSC and CRC cells were confirmed by IHC (Fig. 5 F). These results showed that the LX2 BMI-1 HSC promotes CRC tumor growth and EMT process in a mouse model. Discussion Liver is the most common target organ for CRC metastases. Accumulating evidences suggest that the interactions between cancer cells and liver microenvironment promote the progression of LM. In the liver microenvironment, HSC-derived CAFs are the main tumor-interacting population and play a vital role in cancer cell metastasis (Zhao et al. 2022 ; Kubo et al. 2016 ). BMI-1 has been proofed to promote tumorigenesis and tumor progression in different types of cancers including CRC and HCC (Tateishi et al. 2006 ; Xu et al. 2021 ; Effendi et al. 2010 ; Li et al. 2013 ; Xu et al. 2018 ). We previously reported that BMI-1 was abnormally high expressed in CRLM and inhibition of BMI-1 in CRC cells dramatically reduced LM in vivo (Xu et al. 2021 ). The normal liver cells were BMI-1 negative, we observed that BMI-1 positive liver cells were common in CRLM samples from humans and mice. Specially, the protein and mRNA expression levels of BMI-1 in liver cells were gradually increased in the process of CRLM in mice, suggesting that BMI-1 also participate in regulating liver cells during the course of CRLM. In this study, we found that BMI-1 has a novel function as a regulator in activating HSCs and plays an important role in crosstalk between HSCs and CRC cells. Quiescent HSCs normally resident in the space of Disse and become activated with a myofibroblast-like phenotype (α-SMA + ) in response to inflammatory stimuli and liver damage (Tsuchida et al. 2017). Studies showed that cancer cells also can activate HSCs to release cytokines and chemokines to promote LM (Zhao et al. 2022 ; Huang et al. 2019 ). aHSCs have been reported to be able to orchestrate pre-metastatic and pro-metastatic niche which accelerate CRLM (Eveno et al. 2015 ; Illemann et al. 2016 ; Schütte et al. 2017 ). In this study, we confirmed BMI-1 overexpressed HSCs were activated to α-SMA + myofibroblasts with increased expression of Fibronectin, TGF-β1, MMPs, and IL-6. These secreted components can maintain the activated status of HSCs by enhancing the autocrine signaling loop (Watanabe et al. 2011 ). aHSCs are the major source of ECM, aHSCs secreted factors play a pivotal role in remodeling the ECM during tumor invasion and metastasis (Ahmad et al. 2003 ). TGF-β1 is the most potent fibrogenic cytokine the fibrotic marker (Xu et al. 2016 ; Hellerbrand et al. 1999 ), Fibronectin and MMPs are important ECM regulators in liver fibrosis process (Klingberg et al. 2013 ). The ECM deposition by aHSCs provides pro-metastasis environment. In mice intrasplenic injected with CT26 cells, the small liver metastases had formed at day 7. But BMI-1 positive liver cells appeared at day 14 and increased at day 21 and 28 after CT26 cell injection. These results suggested that BMI-1 activated HSCs after CRC cells metastasized to liver. Thus, BMI-1 activates HSCs to secret factors to form pro-metastasis environment in liver. The interactions between cancer cells and aHSCs promote tumor growth and metastasis. CRC cells can stimulate aHSCs to release cytokines and chemokines which in turn promote CRC growth and invasion (Zhao et al. 2022 ; Huang et al. 2019 ). We found that high-metastatic HCT116 and low-metastatic DLD1 CRC cells cultured in CM from BMI-1 overexpressed HSCs showed same enhanced proliferation and migration ability, and both HCT116 and DLD1 cells co-implanted with BMI-1 overexpressed HSCs showed increased tumor growth in vivo . These results demonstrated that BMI-1 activated HSCs promote CRC cell proliferation and migration regardless of their ability to metastasize. TGF-β1 is a key inducer of EMT transcription factors which can promote EMT through TGF-β/SMAD pathway (Xu et al. 2009 ; Hao et al. 2019 ). The markers of EMT, including Vimentin, Snail, Zeb-1, Twist-1 were upregulated in CRC cells cultured in CM from BMI-1 overexpressed HSCs. The TGFβ/SMAD signaling was activated and the phosphorylate of Smad2/3 were induced in CRC cells cultured in CM from BMI-1 overexpressed HSCs, TGF-βR inhibitor SB-505124 treatment attenuated the effect of BMI-1. Moreover, CRC cells co-implanted with BMI-1 overexpressed HSCs showed increased expression of EMT markers in vivo . Taking together, BMI-1 overexpressed HSCs promote CRC cells growth and migration through activating TGF-β/SMAD pathway and enhancing EMT. Conclusion We proposed a novel mechanism of BMI-1 involvement in promoting CRLM. BMI-1 activated HSCs, the aHSCs released cytokines and chemokines (Fibronectin, TGF-β, MMPs et al) to form pro-metastasis environment in liver. aHSCs promote CRC cell proliferation, migration and EMT through activating TGF-β/SMAD pathway (Fig. 6 ). Our findings provide a potential target for treatment of CRLM. Abbreviations CRC Colorectal cancer HSCs Hepatic stellate cells aHSCs Activated hepatic stellate cells CAFs Cancer-associated fibroblasts LM Liver metastasis CRLM Colorectal cancer liver metastasis EMT Epithelial-mesenchymal transition ECM Extracellular matrix HCC Hepatocellular carcinoma PcG Polycomb-group Declarations Acknowledgments This work was funded by National Nature Science Foundation of China (No. 81472213), the Health Commission of Zhejiang Province (No. 2019ZD010, No. 2019ZD029), the Science Technology Department of Zhejiang Province (No. LGF20H220001), the Zhejiang Provincial Administration of Traditional Chinese Medicine (No. 2021ZA088). We thank Xiaoli Hong and Chao Bi from the Core Facilities, Zhejiang University School of Medicine for their technical support. Author contribution Z. J., Q. D., and G. W. conceived and designed the work. Z. J., X. M., Z. LY., Y. H., and Y. D. performed research, collected, and analyzed the data. Z. J., Q. D., and G. W. wrote the manuscript. All authors read and approved the final manuscript. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Declarations Funding This work was funded by National Nature Science Foundation of China (No. 81472213), the Health Commission of Zhejiang Province (No. 2019ZD010, No. 2019ZD029), the Science Technology Department of Zhejiang Province (No. LGF20H220001), the Zhejiang Provincial Administration of Traditional Chinese Medicine (No. 2021ZA088). 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Mao X, Xu J, Wang W, Liang C, Hua J, Liu J, et al. Crosstalk between cancer-associated fibroblasts and immune cells in the tumor microenvironment: new findings and future perspectives. Mol Cancer. 2021;201:131. Mederacke I, Hsu CC, Troeger JS, Huebener P, Mu X, Dapito DH, et al. Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology. Nat Commun. 2013;4:2823. Milette S, Sicklick JK, Lowy AM and Brodt P. Molecular Pathways: Targeting the Microenvironment of Liver Metastases. Clin Cancer Res. 2017;2321:6390–9. Sahai E, Astsaturov I, Cukierman E, DeNardo DG, Egeblad M, Evans RM, et al. A framework for advancing our understanding of cancer-associated fibroblasts. Nat Rev Cancer. 2020;203:174–86. Schütte M, Risch T, Abdavi-Azar N, Boehnke K, Schumacher D, Keil M, et al. Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors. Nat Commun. 2017;8:14262. Shiraha H, Iwamuro M and Okada H. Hepatic Stellate Cells in Liver Tumor. Adv Exp Med Biol. 2020;1234:43–56. Siegel RL, Miller KD, Goding Sauer A, Fedewa SA, Butterly LF, Anderson JC, et al. Colorectal cancer statistics, 2020. CA Cancer J Clin. 2020;703:145–64. Tateishi K, Ohta M, Kanai F, Guleng B, Tanaka Y, Asaoka Y, et al. Dysregulated expression of stem cell factor Bmi1 in precancerous lesions of the gastrointestinal tract. Clin Cancer Res. 2006;1223:6960–6. Tsilimigras DI, Brodt P, Clavien P-A, Muschel RJ, D'Angelica MI, Endo I, et al. Liver metastases. Nat Rev Dis Primers. 2021;71:27. Tsuchida T and Friedman SL. Mechanisms of hepatic stellate cell activation. Nat Rev Gastroenterol Hepatol. 2017;147:397–411. Valderrama-Treviño AI, Barrera-Mera B, Ceballos-Villalva JC and Montalvo-Javé EE. Hepatic Metastasis from Colorectal Cancer. Euroasian J Hepatogastroenterol. 2017;72:166–75. Watanabe T, Tajima H, Hironori H, Nakagawara H, Ohnishi I, Takamura H, et al. Sodium valproate blocks the transforming growth factor (TGF)-β1 autocrine loop and attenuates the TGF-β1-induced collagen synthesis in a human hepatic stellate cell line. Int J Mol Med. 2011;286:919–25. Xu F, Liu C, Zhou D and Zhang L. TGF-β/SMAD Pathway and Its Regulation in Hepatic Fibrosis. J Histochem Cytochem. 2016;643:157–67. Xu J, Lamouille S and Derynck R. TGF-beta-induced epithelial to mesenchymal transition. Cell Res. 2009;192:156–72. Xu Z, Tao J, Chen P, Chen L, Sharma S, Wang G, et al. Sodium Butyrate Inhibits Colorectal Cancer Cell Migration by Downregulating Bmi-1 Through Enhanced miR-200c Expression. Mol Nutr Food Res. 2018;626:e1700844. Xu Z, Zhou Z, Zhang J, Xuan F, Fan M, Zhou D, et al. Targeting BMI-1-mediated epithelial-mesenchymal transition to inhibit colorectal cancer liver metastasis. Acta Pharm Sin B. 2021;115:1274–85. Zhao S, Mi Y, Zheng B, Wei P, Gu Y, Zhang Z, et al. Highly-metastatic colorectal cancer cell released miR-181a-5p-rich extracellular vesicles promote liver metastasis by activating hepatic stellate cells and remodelling the tumour microenvironment. J Extracell Vesicles. 2022;111:e12186. Additional Declarations No competing interests reported. Supplementary Files TableS1docx.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2247681","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":159214817,"identity":"88ba26e2-2f41-4dec-890d-3732b2409f2a","order_by":0,"name":"Zhongyang Jiang","email":"","orcid":"","institution":"Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhongyang","middleName":"","lastName":"Jiang","suffix":""},{"id":159214818,"identity":"61ac7a71-caa5-4e06-9cd0-2389e7f542e9","order_by":1,"name":"Ximei Ma","email":"","orcid":"","institution":"The Second Affiliated Hospital of Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ximei","middleName":"","lastName":"Ma","suffix":""},{"id":159214819,"identity":"1cd34beb-88f4-4d84-9a3e-cdc75e417c35","order_by":2,"name":"Xiaohui Luan","email":"","orcid":"","institution":"Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaohui","middleName":"","lastName":"Luan","suffix":""},{"id":159214821,"identity":"fa855a8c-b304-4654-9d6d-d90b877d7f6b","order_by":3,"name":"Zhenyu Liuyang","email":"","orcid":"","institution":"Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhenyu","middleName":"","lastName":"Liuyang","suffix":""},{"id":159214823,"identity":"f3d511ba-e30d-4c95-ad6e-1d350caa1fb2","order_by":4,"name":"Yiyang Hong","email":"","orcid":"","institution":"Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yiyang","middleName":"","lastName":"Hong","suffix":""},{"id":159214824,"identity":"36dd1344-ea25-4572-85c0-7471f086d90b","order_by":5,"name":"Yuan Dai","email":"","orcid":"","institution":"Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Dai","suffix":""},{"id":159214825,"identity":"9d6098a8-3fde-4f03-8483-c4712b052878","order_by":6,"name":"Qinghua Dong","email":"","orcid":"","institution":"Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qinghua","middleName":"","lastName":"Dong","suffix":""},{"id":159214826,"identity":"30046438-0ffe-438b-b676-85833db64d74","order_by":7,"name":"Guanyu Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYHAD5gMMEiD6APFa2BJI1sJjAKEJaTE4fvbwy69tdnkGN3I+f7BsY5Dju5HA+LkAn5YzeWnWsm3JxQY3cjcYSLYxGEveSGCWnoFHi9mBHDNjyTbmxA1ALQlALUBGAhszDz4t59+AtNQDVeY8OADUUk9Yy40c44cf2w6DtDA2ALUkGBDSYn/jjRkzw7njiTPPPDNmkDgnYTjzzMNmaXxaJPtzjD/+KKtO7Due/PizRJmNPN/x5IOf8WkBAjawmQoHgPEvAY5Mxgb8GoAKP/4AkvJAdYwfCKkdBaNgFIyCEQkAVRlSbNmis6MAAAAASUVORK5CYII=","orcid":"","institution":"Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Guanyu","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2022-11-07 15:47:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2247681/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2247681/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":30273923,"identity":"4162110b-f528-4658-9d04-6c5cc7c6c2f3","added_by":"auto","created_at":"2022-12-13 16:56:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1916706,"visible":true,"origin":"","legend":"\u003cp\u003eBMI-1 expression increases in liver cells during CRLM. \u003cstrong\u003eA:\u003c/strong\u003e Immunohistochemistry analysis of BMI-1 in LM and paired liver tissues of CRLM patients; \u003cstrong\u003eB: \u003c/strong\u003eThe spleens and livers of mice were photographed after intrasplenic injected with CT26 cells at indicated times; \u003cstrong\u003eC:\u003c/strong\u003e Western blot analysis of BMI-1 expression in mice livers (n=3); \u003cstrong\u003eD: \u003c/strong\u003eQuantitative protein expression of BMI-1 normalized to GAPDH in mice livers (n=3); \u003cstrong\u003eE: \u003c/strong\u003eqPCR detection of BMI-1 expression in mice livers (n=3); *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003eF:\u003c/strong\u003e Immunohistochemistry confirmed that BMI-1 expression was increased in mice livers during CRLM process.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2247681/v1/415654ca34eedf59e1eefcff.png"},{"id":30273922,"identity":"35b07704-2e62-452a-b378-5d38f9d3e00b","added_by":"auto","created_at":"2022-12-13 16:56:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":526212,"visible":true,"origin":"","legend":"\u003cp\u003eBMI-1 activates LX2 HSC. \u003cstrong\u003eA: \u003c/strong\u003eWestern blot analysis of BMI-1 and aHSC related markers (α-SMA, Fibronectin and TGF-β1) in LX2 NC and LX2 BMI-1 HSCs; \u003cstrong\u003eB:\u003c/strong\u003e Quantitative PCR detection of MMPs and cytokines in LX2 HSC, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; \u003cstrong\u003eC:\u003c/strong\u003eImmunofluorescence analysis showed α-SMA and Fibronectin expression were increased in LX2 BMI-1 HSC.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2247681/v1/4de90b35183442477abc1333.png"},{"id":30273471,"identity":"d47594ea-9048-4fe6-9f12-6042915f5dc3","added_by":"auto","created_at":"2022-12-13 16:48:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1584593,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of BMI-1 overexpressed HSC CM on cell proliferation and migration of CRC cells. \u003cstrong\u003eA:\u003c/strong\u003e Cell viability of HCT116 and DLD-1 cells cultured in conditioned medium from negative control LX2 (NC CM) or conditioned medium from BMI-1 overexpressed LX2 (BMI-1 CM) was checked by CCK-8; \u003cstrong\u003eB: \u003c/strong\u003eColony formation assay of CRC cultured in NC CM or BMI-1 CM; \u003cstrong\u003eC: \u003c/strong\u003eWound healing assay of CRC cultured in NC CM or BMI-1 CM, the healing percentages were calculated at 24 h and 48 h; \u003cstrong\u003eD:\u003c/strong\u003e Transwell migration assay of CRC cultured in NC CM or BMI-1 CM, the migrated cell numbers were counted; \u003cstrong\u003eE:\u003c/strong\u003e Quantitative PCR assay of EMT related markers expression in CRC cells cultured in NC CM or BMI-1 CM; \u003cstrong\u003eF: \u003c/strong\u003eWestern blot assays of EMT related markers expression in CRC cells cultured in NC CM or BMI-1 CM; \u003cstrong\u003eG:\u003c/strong\u003e Immunofluorescence analysis showed Vimentin and Snail expression were increased in CRC cells cultured in BMI-1 CM.\u003csup\u003e \u003c/sup\u003e*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2247681/v1/2345690ca1342d585c24f594.png"},{"id":30273472,"identity":"f30e19b8-956b-42af-bddb-01a91ab7c4ed","added_by":"auto","created_at":"2022-12-13 16:48:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":531402,"visible":true,"origin":"","legend":"\u003cp\u003eBMI-1 CM induces activation of the TGF-β/SMAD pathway in CRC cells. \u003cstrong\u003eA: \u003c/strong\u003eQuantitative PCR analysis showed the expression of Smad2 and Smad3 had no difference in CRC cells cultured in NC CM or BMI-1 CM; \u003cstrong\u003eB:\u003c/strong\u003e Western blot assays of Smad2, Smad3, p-Smad2, p-Smad3expression in CRC cells cultured in NC CM or BMI-1 CM;\u003cstrong\u003e C: \u003c/strong\u003eWestern blot analysis of expression of Smad pathway members and EMT related proteins (Snail and Vimentin)in CRC cells cultured in NC CM or BMI-1 CM with/without 0.5 μM SB-505124 treatment, the relative expressions level of the proteins was calculated and showed in D. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2247681/v1/9bf0d5cddab074e150e8ff5d.png"},{"id":30273477,"identity":"c2fff172-182c-4212-a252-96496cb0c762","added_by":"auto","created_at":"2022-12-13 16:48:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1914813,"visible":true,"origin":"","legend":"\u003cp\u003eBMI-1 overexpressed LX2 promotes tumor growth of CRC \u003cem\u003ein vivo\u003c/em\u003e. \u003cstrong\u003eA: \u003c/strong\u003eCRC cells (HCT116 or DLD-1) and LX2 cells (negative control or BMI-1 overexpressed) were co-implanted subcutaneously into the flanks of nude mice. After 7 d, the volume of tumors were measured every 2 d from 8 d to 28 d; \u003cstrong\u003eB:\u003c/strong\u003eTumors were photographed at 28 d; \u003cstrong\u003eC: \u003c/strong\u003eTumors were weighted (n=5); \u003cstrong\u003eD: \u003c/strong\u003eWestern blot assay of Vimentin and Snail expression in mice tumors of different groups, the fold changes were calculated; \u003cstrong\u003eE: \u003c/strong\u003eQuantitative PCR assay of EMT related markers expression in mice tumors of different groups; \u003cstrong\u003eF: \u003c/strong\u003eImmunohistochemistry analysis of Vimentin and Snail expression in mice tumors. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-2247681/v1/580ec0e4e95ca15d40cc32bc.png"},{"id":30273474,"identity":"0c982468-6dd5-495f-8880-21e52c5aeb86","added_by":"auto","created_at":"2022-12-13 16:48:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":576702,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic summary of BMI-1 activates HSCs to promote CRC cells EMT and LM.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-2247681/v1/e52a5cd17e593972401ddb0c.png"},{"id":30544578,"identity":"a6315d38-effb-4657-9618-527cde7a7a85","added_by":"auto","created_at":"2022-12-20 03:29:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4398662,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2247681/v1/17496050-9b97-4152-992a-a357170ce859.pdf"},{"id":30273924,"identity":"b74464b3-d6a9-45a7-ad47-c47bc8455c97","added_by":"auto","created_at":"2022-12-13 16:56:02","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":17749,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1docx.docx","url":"https://assets-eu.researchsquare.com/files/rs-2247681/v1/64f5ebab0da262386cb3246e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"BMI-1 activates hepatic stellate cells to promote EMT of colorectal cancer cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the world, colorectal cancer (CRC) is 3rd most common cancer and ranks 2nd in mortality from cancer (Bray et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Siegel et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In CRC patients, liver metastasis (LM) is the primary cause of death. Liver plays a major role in survival as the liver is often the only site of metastasis for CRC. In CRC patients, about 14 to 35% have LM at diagnosis, and about 70% have LM by the end of the disease (Valderrama-Trevi\u0026ntilde;o et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In colorectal cancer liver metastasis (CRLM) patients, liver resection is the primary curative treatment option, with the 5-year survival rate of 20\u0026ndash;50% (Tsilimigras et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Though treatments have advanced in the past years, the rate of intrahepatic recurrence is still high.\u003c/p\u003e \u003cp\u003eHepatic microenvironment has numerous signaling factors from liver cells which consist of parenchymal and non-parenchymal cells, the interaction between cancer cells and stromal cells facilitates development of metastasis in liver (Milette et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Brodt \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Cancer-associated fibroblasts (CAFs), comprising the major stromal cell type, are fibroblast populations found in primary and metastatic cancers and related to tumor initiation, progression and metastasis by regulating the extracellular matrix (ECM) remodeling and immune response (Mao et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kalluri \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sahai et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Hepatic stellate cells (HSCs) are the major non-parenchymal liver cells which store retinol and play a role in liver repair, liver fibrosis, and hepatocellular carcinoma (HCC) (Higashi et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Friedman \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Barry et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Shiraha et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). HSCs contribute 85\u0026ndash;95% of fibroblasts in liver (Mederacke et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). HSCs are activated to CAFs by cancer cells to support tumor growth and metastasis (Makino et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Hepatic CAFs are thought to come primarily from HSCs and to a lesser extent from bone marrow-derived precursors, portal fibroblasts and endothelial cells (Biffi et al. 2021). In intrahepatic cholangiocarcinoma, HSCs-derived CAFs are the main tumor-interacting population (Affo et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although CRC is the most common primary cancer that metastasizes to the liver, the mechanisms by which HSCs interact with CRC cells to promote LM remain largely unclear.\u003c/p\u003e \u003cp\u003eBMI-1, one of the polycomb-group (PcG) protein family members, plays a critical role in negatively regulating the Ink4a/Arf locus which encodes p16\u003csup\u003eINK4a\u003c/sup\u003e and p19\u003csup\u003eARF\u003c/sup\u003e (Jacobs et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). BMI-1 regulates self-renewal of stem cell and participate in carcinogenesis of human cancers, including CRC (Jiang et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Tateishi et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Gil et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). We have previously reported that BMI-1 was overexpressed in CRLM and contributes to the LM by regulating epithelial-mesenchymal transition (EMT) of CRC cells \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e (Xu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Interestingly, we observed BMI-1 positive staining in liver cells both in human and mice liver specimens of CRLM. Studies have showed that quiescent HSCs can be activated by cancer cells and transformed into CAFs, and in turn activated HSCs (aHSCs) promote cancer cells invasion and EMT (Makino et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Thus, we speculated that BMI-1 plays a role in crosstalk between HSCs and CRC cells during LM.\u003c/p\u003e \u003cp\u003eIn this study, CRLM patients and mice showed increased BMI-1 expression in liver cells. BMI-1 overexpressed HSCs were activated and transformed into CAFs. CRC cells cultured in conditioned medium (CM) from BMI-1 overexpressed HSCs showed enhanced proliferation, migration and EMT ability. Experimental subcutaneous xenotransplantation tumor model \u003cem\u003ein vivo\u003c/em\u003e also showed increased tumor proliferation and EMT of CRC cells co-cultured with BMI-1 overexpressed HSCs. These findings may provide a potential new target for treating CRLM.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC) analysis of CRLM patients\u003c/h2\u003e \u003cp\u003eLM and adjacent normal liver tissues from 18 clinical diagnosed patients with CRLM were collected from the Pathology Department (from 2013 to 2020), Sir Run Run Shaw Hospital, Hangzhou, China. The expression of BMI-1 was investigated with IHC staining. Anti-BMI-1 (1:100; Cell Signaling Technology, USA) was used as Primary antibody before secondary antibody incubation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and treatment\u003c/h2\u003e \u003cp\u003eHigh metastatic human CRC cell line (HCT116), low metastatic human CRC cell line (DLD1), mouse CRC cell line (CT26) and human hepatic stellate cell line (LX2) were purchased from the American Type Culture Collection (Manassas, USA). Cells were cultured in DMEM medium (Gibco, USA) with 10% FBS and 1% penicillin/streptomycin in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator at 37\u0026deg;C. BMI-1 overexpressed lentiviral construct (pGC-FU-GFP-\u003cem\u003eBMI-1\u003c/em\u003e) and negative control (pGC-FU-GFP) were obtained from Genechem (Shanghai, China). The control (LX2 NC) and stable BMI-1 overexpressed LX2 (LX2 BMI-1) were established using lentivirus transfection under the manufacturer\u0026rsquo;s instructions. Besides, to make conditioned medium (CM), we firstly cultured transfected LX2 in a 10cm plate with 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells. Then we used 6mL serum-free DMEM medium to culture cells for 24h. We collected the supernatants, centrifuged at 1000\u0026times; rpm for 10 min and filtrated through a 0.22mm filter unit (Millex, USA). CM from NC LX2(NC CM) and BMI-1 LX2(BMI-1 CM) were made by mixing the different supernatant with complete medium (1:1). After that, we cultured CRC cells with CM for 24 h. All CM would be used in 2 d. To inhibit Smad2 phosphorylation, after SB-505124 (0.05 \u0026micro;M) was utilized to pretreat CRC cells for 1 hour, the cells were then incubated with BMI-1 CM for 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eWhole protein from the tissue and cell samples were extract using RIPA buffer with a 1% protease/phosphatase inhibitor. After centrifugation of the extraction solutions, BCA Protein Concentration Assay Kit (Solarbio, China) was utilized to quantify the proteins. After SDS-PAGE, proteins were migrated to PVDF membranes. Antibodies against BMI-1, GAPDH, β-actin, Vimentin, Smad2/3, phosphorylated Smad3 (1:1000; Cell Signaling Technology, USA), α-SMA, TGF-β1, phosphorylated Smad2 (1:1000; Abcam, USA), Fibronectin (1:1000; BD Biosciences, USA), E-cadherin, ZEB1, Twist-1 (1:1000; Novus Biologicals, USA), Snail (1:1000; Proteintech Group, USA) were utilized as primary antibodies. After washing with TBST buffer three times every 10 minutes, goat anti-rabbit/mouse IgG (Abcam, USA) was utilized as secondary antibodies. A Bio-Rad CD Touch detection system (USA) with ECL detection reagents was utilized to detect protein bands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eqPCR\u003c/h2\u003e \u003cp\u003eWhole RNA from the tissue and cell samples were extract using Trizol reagent (Cwbio, China). Afterward, HiScript II Q RT SuperMix (Vazyme Biotech, China) was utilized for reverse transcription. ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, China) was utilized quantitative polymerase chain reaction (qPCR). A Bio-Rad CFX-96 Real-Time PCR system (USA) was utilized to analyze the results. The sequences of all primers were listed in Table S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescent staining\u003c/h2\u003e \u003cp\u003eImmunofluorescent staining was performed on 96-well plates. Cells were fixed in paraformaldehyde and permeabilized with Triton X-100. Before incubating with a secondary antidody, antibodies against Vimentin (1:100; Cell Signaling Technology, USA) and Snail (1:100; Proteintech Group, USA) were used as primary antibodies. A Zeiss AXIO Observer A1 inverted fluorescence microscope system (Germany) was utilized to obtain images.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assay\u003c/h2\u003e \u003cp\u003eCells were cultured with CM separately for 24, 48, 72, and 96 h. After treatments, CCK-8 reagent (Yeasen Biotechnology, China) was added and incubated for 2 h. A spectrophotometer (Thermo Fisher, USA) was utilized to detect the optical density (OD) value (450nm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eColony formation assay\u003c/h2\u003e \u003cp\u003eCRC cells were cultured with CM for 2 weeks. Cells were fixed in paraformaldehyde and stained by crystal violet. Colonies were viewed with an Olympus CKK53 microscope, and photos were taken. Image J was utilized to measure number of colonies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWound healing assay\u003c/h2\u003e \u003cp\u003eCRC cells were cultured to reach 90% confluence. After creation of a linear wound, cells were cultured with CM for 48 h. Wounds were viewed with an Olympus CKK53 microscope, and photos were taken at 0, 24, 48 h. Image J was utilized to measure the migration rate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTranswell migration assay\u003c/h2\u003e \u003cp\u003eTranswells chambers were purchased form Corning. CRC cells were plated in the upper chambers with serum-free DMEM, while CM was in the lower chambers. After 48 h, cells in the upper chambers were removed, and cells on the lower membrane surface were fixed and stained. The migrated cells were viewed with an Olympus CKK53 microscope, and photos were taken. Image J was utilized to measure number of cells.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eIHC\u003c/h2\u003e \u003cp\u003eTissue samples were fixed, dehydrated, embedded and sectioned. After deparaffinization, sections were blocked with goat serum and incubated with primary antibodies for 24 h. Then, the sections were incubated with secondary antibody for 30 min, followed by counterstaining with Mayer\u0026rsquo;s hematoxylin. At last, an Olympus CKK53 microscope was utilized to view and photograph the staining of the sections.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003e All animal experimental procedures were approved by the Committee on the Ethics of Animal Experiments of Zhejiang University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTreatment protocol 1\u003c/h2\u003e \u003cp\u003e5-week-old male C57 mice (19-20g) were randomly divided into five groups (n\u0026thinsp;=\u0026thinsp;3). Isoflurane (inhal.) was utilized to anesthetized the mice. Then, the spleen was exteriorized through a left flank incision. CT26 cells (2\u0026times;10\u003csup\u003e6\u003c/sup\u003e) were intrasplenic injected to establish the tumor, and then injection site was pressed for 5 min. Surgical thread was used to close the peritoneum and skin. The mice were sacrificed at 7, 14, 21 and 28d after CT26 cells inoculation. Resected liver tissues were collected for qPCR, WB and IHC assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTreatment protocol 2\u003c/h2\u003e \u003cp\u003e5-week-old male BALB/c nude mice (17\u0026ndash;18 g) were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;5): (1) HCT116 cells (5\u0026times;10\u003csup\u003e6\u003c/sup\u003e) mixed with LX2 NC cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e); (2) HCT116 cells (5\u0026times;10\u003csup\u003e6\u003c/sup\u003e) mixed with LX2 BMI-1 cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e); (3) DLD1 cells (5\u0026times;10\u003csup\u003e6\u003c/sup\u003e) mixed with LX2 NC cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e); (4) DLD1 cells (5\u0026times;10\u003csup\u003e6\u003c/sup\u003e) mixed with LX2 BMI-1 cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e). The mixed cells were subcutaneously injected into the flanks to establish the tumor. Tumor sizes were estimated every two days after the tumors were measurable. The mice were killed at 28d after cell inoculation. Resected tumor tissues were collected for qPCR, WB and IHC assay. Total tumor volume (mm\u003csup\u003e3\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;L\u0026times; W\u003csup\u003e2\u003c/sup\u003e/2 (L\u0026thinsp;=\u0026thinsp;length and W\u0026thinsp;=\u0026thinsp;width).\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eIn all experiments, the date is showed as the mean\u0026thinsp;+\u0026thinsp;SD based on three independent experiments. The difference between the groups was analyzed using the χ2 test, Fisher\u0026rsquo;s exact probability, Student\u0026rsquo;s t test or one-way Analysis of Variance (ANOVA) properly in GraphPad Prism 8 Software or SPSS 25.0. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was regarded as statistically significant.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe expression of BMI-1 is up-regulated in liver cells during CRLM\u003c/h2\u003e \u003cp\u003eIn our previous research, it was found that BMI-1 was overexpressed in LM of human CRC (Xu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Interestingly, though the normal liver cells were BMI-1 negative, 77.8% CRLM patients (14 among 18 samples, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) was BMI-1 positive in liver cells. The positivity of BMI-1 expression was not highly related to clinic pathological characteristics (including tumor size and number in liver, differentiation degree, T stage, N stage) except tumor site (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). BMI-1 expression positivity in liver cells of rectum cancer LM patients was significantly higher (100%) than colon cancer LM patients (60%). To investigate the BMI-1expression level in liver cells during CRLM, we checked BMI-1 expression at 0, 7, 14, 21, and 28 d after mice were intrasplenic injected with CT26 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, compared with healthy control mice, the protein expression level of had no significant change at 7 and 14 d, but markedly increased to 4.58 fold at 21 d and continued increasing to 7.29 fold at 28 d (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The mRNA expression level of BMI-1 accordingly increased to 2.92 fold at 21 d and 6.71 fold at 28 d (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). IHC result also showed that the nuclei of liver cells were BMI-1 positive in CRLM mice since 14 d, and BMI-1 positive rate and intensity increased markedly at 21 and 28 d (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These results suggest that up-regulated BMI-1 expression in liver cells may play a role in CRLM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between CRCLM patient clinicopathological characteristics and BMI-1 expression in the liver.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCases (n\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eBMI-1 expression\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePositive (n\u0026thinsp;=\u0026thinsp;14)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (yr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.051\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.509\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimary tumor site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.018\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor size (in Liver)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.278\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;30mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30-50mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;50mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTumor numbers (in liver)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.432\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u0026ndash;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDifferentiation degree\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.881\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiddle-high\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.248\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN1\u0026thinsp;+\u0026thinsp;N2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between CRCLM patient clinicopathological characteristics and BMI-1 expression in the livers.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCases (n\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eBMI-1 expression\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePositive (n\u0026thinsp;=\u0026thinsp;14)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (yr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.051\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.509\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimary tumor site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.018\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor size (in Liver)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.278\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;30mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30-50mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;50mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTumor numbers (in liver)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.432\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u0026ndash;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDifferentiation degree\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.881\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiddle-high\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.248\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN1\u0026thinsp;+\u0026thinsp;N2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eBMI-1 activates hepatic stellate cells\u003c/h2\u003e \u003cp\u003eTo investigate the effects of BMI-1 on liver cells, we overexpressed BMI-1 inLX2 HSC and confirmed the overexpression by western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). High expression of α-SMA, a myofibroblast marker, is a key marker of aHSCs. aHSCs highly secrete a key cytokine TGF-β1 and promote hepatic fibrosis with Fibronectin expression (Tsuchida et al. 2017). Compared with control quiescent HSCs, BMI-1 overexpressed LX2 HSCs showed significant increase of α-SMA expression. Fibronectin and TGF-β1 expression were also markedly increased in BMI-1 overexpressed LX2 HSCs. Meanwhile, immunofluorescent staining also confirmed that the expression of α-SMA and Fibronectin were significantly increased in LX2 cells overexpressed BMI-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Besides, we also observed the transcript level of matrix metalloproteinases (MMP1, MMP2, MMP3, MMP7, MMP9) and inflammatory cytokines (TGF-β1 and IL-6) were remarkably increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Thus, LX2 HSCs were activated by overexpression of BMI-1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eBMI-1 overexpressed HSC CM enhances CRC cell proliferation and migration\u003c/h2\u003e \u003cp\u003eTo explore the effect of BMI-1 on the interaction between HSCs and CRC cells, CRC cells (HCT116 and DLD1) were cultured in control LX2 CM (NC CM) or BMI-1 overexpressed LX2 CM (BMI-1 CM). We first checked the cell proliferation by CCK-8 assay. Compared with cells cultured in NC CM, the proliferation ability of CRC cells cultured in BMI-1 CM markedly enhanced at day 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), the colony formation ability of CRC cells cultured in BMI-1 CM also significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Meanwhile, CRC cells cultured in BMI-1 CM showed higher wound healing rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Transwell migration assays showed the same results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). We then investigated EMT-related molecular changes. Consistent with the above results, the mRNA and protein expression levels of ZEB-1, Twist-1, Vimentin, and Snail in CRC cells cultured in BMI-1 CM were significantly increased while E-cadherin was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Immunofluorescent staining further demonstrated that Snail and Vimentin expression were obviously increased in CRC cells cultured in BMI-1 CM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Therefore, BMI-1 maybe an important regulator in activated HSCs which promoted malignant phenotype of CRC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eBMI-1 overexpressed HSC CM promotes CRC cell EMT via activating Smad2/3 pathway\u003c/h2\u003e \u003cp\u003eAs mentioned before, TGF-β1 was upregulated in BMI-1 overexpressed LX2 HSC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). TGF-β/SMAD signal pathway plays an important role in metastasis by modulating EMT process (Xu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hao et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), so we hypothesize BMI-1 CM induces CRC cells EMT through activating Smads. We first checked the expression levels of Smad2/3. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B, though the mRNA and protein expression level of Smad2 and Smad3 had no changes in CRC cells cultured inBMI-1 CM, the Smad2 phosphorylation and Smad3 phosphorylation were significantly increased compared with CRC cells cultured in NC CM. Then we used SB-505124 (a TFG-βR inhibitor which inhibit Smad2 phosphorylation) to further confirm the involvement of Smads on the pro-EMT effects of BMI-1 CM. SB-505124largely decreased BMI-1 CM-induced Smad2 phosphorylation and Smad3 phosphorylation in CRC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). Meanwhile, the downstream target proteins (Snail and Vimentin) of TGF-β/SMAD pathway accordingly decreased in SB-505124 treated CRC cells which cultured in BMI-1 CM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). These results confirmed that BMI-1 CM induces CRC cells EMT partially via activating Smad2/3.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eBMI-1 overexpressed HSC promotes tumor growth in vivo\u003c/h2\u003e \u003cp\u003eWe further evaluated the effects of BMI-1 overexpressed LX2 HSC on CRC cell growth \u003cem\u003ein vivo\u003c/em\u003e. A subcutaneous xenotransplantation tumor model by co-implantation of HSCs (LX2 NC or LX2 BMI-1) and CRC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Compared with mice co-implantation with LX2 NC, tumor grew faster and larger in mice co-implantation with LX2 BMI-1 HSC and CRC cells as shown by increased tumor volume and weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). Moreover, consistent with \u003cem\u003ein vitro\u003c/em\u003e results, we found that Snail and Vimentin were upregulated in mice co-implantation with LX2 BMI-1 HSC and CRC cells according to the results of qPCR and Western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). Furthermore, the increased expression of Snail and Vimentin in mice liver co-implantation with LX2 BMI-1 HSC and CRC cells were confirmed by IHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). These results showed that the LX2 BMI-1 HSC promotes CRC tumor growth and EMT process in a mouse model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLiver is the most common target organ for CRC metastases. Accumulating evidences suggest that the interactions between cancer cells and liver microenvironment promote the progression of LM. In the liver microenvironment, HSC-derived CAFs are the main tumor-interacting population and play a vital role in cancer cell metastasis (Zhao et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kubo et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). BMI-1 has been proofed to promote tumorigenesis and tumor progression in different types of cancers including CRC and HCC (Tateishi et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Effendi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). We previously reported that BMI-1 was abnormally high expressed in CRLM and inhibition of BMI-1 in CRC cells dramatically reduced LM \u003cem\u003ein vivo\u003c/em\u003e (Xu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The normal liver cells were BMI-1 negative, we observed that BMI-1 positive liver cells were common in CRLM samples from humans and mice. Specially, the protein and mRNA expression levels of BMI-1 in liver cells were gradually increased in the process of CRLM in mice, suggesting that BMI-1 also participate in regulating liver cells during the course of CRLM. In this study, we found that BMI-1 has a novel function as a regulator in activating HSCs and plays an important role in crosstalk between HSCs and CRC cells.\u003c/p\u003e \u003cp\u003eQuiescent HSCs normally resident in the space of Disse and become activated with a myofibroblast-like phenotype (α-SMA\u003csup\u003e+\u003c/sup\u003e) in response to inflammatory stimuli and liver damage (Tsuchida et al. 2017). Studies showed that cancer cells also can activate HSCs to release cytokines and chemokines to promote LM (Zhao et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). aHSCs have been reported to be able to orchestrate pre-metastatic and pro-metastatic niche which accelerate CRLM (Eveno et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Illemann et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sch\u0026uuml;tte et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this study, we confirmed BMI-1 overexpressed HSCs were activated to α-SMA\u003csup\u003e+\u003c/sup\u003e myofibroblasts with increased expression of Fibronectin, TGF-β1, MMPs, and IL-6. These secreted components can maintain the activated status of HSCs by enhancing the autocrine signaling loop (Watanabe et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). aHSCs are the major source of ECM, aHSCs secreted factors play a pivotal role in remodeling the ECM during tumor invasion and metastasis (Ahmad et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). TGF-β1 is the most potent fibrogenic cytokine the fibrotic marker (Xu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hellerbrand et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), Fibronectin and MMPs are important ECM regulators in liver fibrosis process (Klingberg et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The ECM deposition by aHSCs provides pro-metastasis environment. In mice intrasplenic injected with CT26 cells, the small liver metastases had formed at day 7. But BMI-1 positive liver cells appeared at day 14 and increased at day 21 and 28 after CT26 cell injection. These results suggested that BMI-1 activated HSCs after CRC cells metastasized to liver. Thus, BMI-1 activates HSCs to secret factors to form pro-metastasis environment in liver.\u003c/p\u003e \u003cp\u003eThe interactions between cancer cells and aHSCs promote tumor growth and metastasis. CRC cells can stimulate aHSCs to release cytokines and chemokines which in turn promote CRC growth and invasion (Zhao et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We found that high-metastatic HCT116 and low-metastatic DLD1 CRC cells cultured in CM from BMI-1 overexpressed HSCs showed same enhanced proliferation and migration ability, and both HCT116 and DLD1 cells co-implanted with BMI-1 overexpressed HSCs showed increased tumor growth \u003cem\u003ein vivo\u003c/em\u003e. These results demonstrated that BMI-1 activated HSCs promote CRC cell proliferation and migration regardless of their ability to metastasize. TGF-β1 is a key inducer of EMT transcription factors which can promote EMT through TGF-β/SMAD pathway (Xu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hao et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The markers of EMT, including Vimentin, Snail, Zeb-1, Twist-1 were upregulated in CRC cells cultured in CM from BMI-1 overexpressed HSCs. The TGFβ/SMAD signaling was activated and the phosphorylate of Smad2/3 were induced in CRC cells cultured in CM from BMI-1 overexpressed HSCs, TGF-βR inhibitor SB-505124 treatment attenuated the effect of BMI-1. Moreover, CRC cells co-implanted with BMI-1 overexpressed HSCs showed increased expression of EMT markers \u003cem\u003ein vivo\u003c/em\u003e. Taking together, BMI-1 overexpressed HSCs promote CRC cells growth and migration through activating TGF-β/SMAD pathway and enhancing EMT.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe proposed a novel mechanism of BMI-1 involvement in promoting CRLM. BMI-1 activated HSCs, the aHSCs released cytokines and chemokines (Fibronectin, TGF-β, MMPs et al) to form pro-metastasis environment in liver. aHSCs promote CRC cell proliferation, migration and EMT through activating TGF-β/SMAD pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Our findings provide a potential target for treatment of CRLM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eCRC\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eColorectal cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eHSCs\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHepatic stellate cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eaHSCs\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eActivated hepatic stellate cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eCAFs\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCancer-associated fibroblasts\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eLM\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLiver metastasis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eCRLM\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eColorectal cancer liver metastasis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eEMT\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpithelial-mesenchymal transition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eECM\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eHCC\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHepatocellular carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePcG\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolycomb-group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was funded by National Nature Science Foundation of China (No. 81472213), the Health Commission of Zhejiang Province (No. 2019ZD010, No. 2019ZD029), the Science Technology Department of Zhejiang Province (No. LGF20H220001), the Zhejiang Provincial Administration of Traditional Chinese Medicine (No. 2021ZA088). We thank Xiaoli Hong and Chao Bi from the Core Facilities, Zhejiang University School of Medicine for their technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZ. J., Q. D., and G. W. conceived and designed the work. Z. J., X. M., Z. LY., Y. H., and Y. D. performed research, collected, and analyzed the data. Z. J., Q. D., and G. W. wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was funded by National Nature Science Foundation of China (No. 81472213), the Health Commission of Zhejiang Province (No. 2019ZD010, No. 2019ZD029), the Science Technology Department of Zhejiang Province (No. LGF20H220001), the Zhejiang Provincial Administration of Traditional Chinese Medicine (No. 2021ZA088).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u0026nbsp; All procedures were performed in accordance with the guidelines of animal ethics and protocols of Sir Run Run Shaw Hospital of Zhejiang University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAffo S, Nair A, Brundu F, Ravichandra A, Bhattacharjee S, Matsuda M, et al. 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Highly-metastatic colorectal cancer cell released miR-181a-5p-rich extracellular vesicles promote liver metastasis by activating hepatic stellate cells and remodelling the tumour microenvironment. J Extracell Vesicles. 2022;111:e12186.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"BMI-1, hepatic stellate cell, colorectal cancer, liver metastasis, epithelial-mesenchymal transition","lastPublishedDoi":"10.21203/rs.3.rs-2247681/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2247681/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eActivated hepatic stellate cells (aHSCs) are the major source of cancer-associated fibroblasts (CAFs) in the liver. Though the crosstalk between aHSCs and colorectal cancer (CRC) cells supports liver metastasis (LM), the mechanisms remain largely unknown. Here, we investigated BMI-1, a polycomb-group protein family member, which is high-expressed in LM, in hepatic stellate cells (HSCs) activation and interacting with CRC cells while promoting colorectal cancer liver metastasis (CRLM). We found the positivity of BMI-1 expression in the liver of CRLM patients was 77.8%, and the expression level of BMI-1 continued to increase during CRLM in mice. We overexpressed BMI-1 in HSCs (LX2) by lentivirus infection, and HSCs were activated, accompanied by increased expression levels of α-SMA, Fibronectin, TGF-β1, MMPs, and IL-6. CRC cells (HCT116 and DLD1) were cultured in HSCs-conditioned medium (LX2 NC CM or LX2 BMI-1 CM), and CRC cells cultured in BMI-1 CM exhibited enhanced proliferation and migration ability, and EMT phenotype with activation of TGF-β/SMAD pathway. Besides, a TFG-βR inhibitor SB-505124 largely diminished the effect of the BMI-1 CM on Smad2/3 phosphorylation in CRC cells. A mouse subcutaneous xenotransplantation tumor model was established by co-implantation of HSCs (LX2 NC or LX2 BMI-1) and CRC cells, andBMI-1 overexpressed LX2 HSCs promoted tumor growth and epithelial-mesenchymal transition (EMT) phenotype \u003cem\u003ein vivo\u003c/em\u003e. In conclusion, BMI-1 activates HSCs to promote the EMT of CRC cells partially through the TGF-β/SMAD pathway. These findings demonstrate BMI-1 activated HSCs might be a new target in CRC therapy.\u003c/p\u003e","manuscriptTitle":"BMI-1 activates hepatic stellate cells to promote EMT of colorectal cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-13 16:47:57","doi":"10.21203/rs.3.rs-2247681/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ba136ce5-7af9-4f7f-9131-19a4ebd75106","owner":[],"postedDate":"December 13th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-12-20T03:29:11+00:00","versionOfRecord":[],"versionCreatedAt":"2022-12-13 16:47:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2247681","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2247681","identity":"rs-2247681","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}