The stromal-tumor amplifying STC1-Notch1 feedforward signal promotes the stemness of hepatocellular carcinoma

<jats:title>Abstract</jats:title>         <jats:p>Background  Cancer associated fibroblasts (CAFs), an important component of the tumor microenvironment (TME), play crucial roles in tumor stemness. Stanniocalcin-1 (STC1) was found secreted by CAFs in various cancers, but its main source and its role in hepatocellular carcinoma (HCC) was still unclear. Methods  The serum and intracellular expression levels of STC1 were detected by ELISA and western blot. The role of CAFs-derived STC1 in HCC stemness was probed by sphere formation, sorafenib resistance, colony formation, and transwell migration and invasion assays in vitro and orthotopic liver xenograft tumor model in vivo. An HCC tissue microarray containing 72 samples was used to identify the STC1 and the Notch1 in HCC tissues. Co-immunoprecipitation (CoIP) and dual-luciferase reporter assay were performed to further explore the underlying mechanisms. ELISA assays were used to detect the serum concentration of STC1 in HCC patients. Results  We demonstrated that CAFs were the main source of STC1 in HCC and that CAFs-derived STC1 promoted HCC stemness through the activation of the Notch signaling pathway. In HCC patients, the expression of STC1 was positively correlated with poor prognosis and the Nocth1 expression. Co-IP assay showed that STC1 directly bound to Notch1 receptors to activate the Notch signaling pathway, thereby promoting the stemness of HCC. Our data further demonstrated that STC1 was a direct transcriptional target of CSL in HCC cells. Furthermore, ELISA revealed that the serum STC1 concentration was higher in patients with advanced liver cancer than patients with early liver cancer. Conclusions  CAFs-derived STC1 promoted HCC stemness via the Notch signaling pathway. STC1 might serve as a potential biomarker for the prognostic assessment of HCC, and the stromal-tumor amplifying STC1-Notch1 feedforward signal could provide an effective therapeutic target for HCC patients.</jats:p>

section_content: As the fourth leading cause of cancer death, liver cancer causes a great burden on global health [1]. Hepatocellular carcinoma (HCC), which accounts for about 80% of all primary liver cancer, is a highly heterogeneous disease caused by multiple etiologies and has a low long-term survival rate [2, 3]. HCC generally develops with brosis or cirrhosis [4] and is usually accompanied by an accumulation of activated broblasts. The intricate cross-talk between the stroma and the tumor has a key impact on tumor progression in various cancers, including HCC [5] [6] [7] [8]. Further clari cation on the mechanisms of the tumor-stromal crosstalk is therefore needed to develop therapeutic strategies targeting both cancer cells and cancer-associated broblasts (CAFs) [9] [10] [11]. 

CAFs, an abundant stromal-activated broblast type of HCC TME [12], play a diverse role in the tumor progression of various cancers [13], including HCC [8, 14, 15]. Well-documented evidence links the multifaceted function of CAFs with the stemness phenotypes of cancer cells [5, 16]. Cancer stem cells, the special stem-like cell subpopulations of tumor cells, could fuel and maintain tumor growth at low cell numbers [17], and are responsible for the development, treatment resistance, and recurrence of tumors [18, 19]. 

CAFs not only exist around tumor cells but can also secrete various paracrine factors to interact with tumor cells. In prior reports, stanniocalcin-1 (STC1), a secreted glycoprotein hormone, was shown to be secreted by CAFs and could promote cancer progression in a variety of cancers, such as colorectal cancer, lung adenocarcinoma, and breast cancer [20] [21] [22]. STC1was also overexpressed in various cancers [23] [24] [25], and was reported to promote cancer stemness [26] [27] [28]. A recent study demonstrated that secretory STC1 promoted HCC metastasis via activation of the JNK pathway in HCC [29]. However, the main source of STC1 and its relationship with cancer stemness in HCC remains unclear. 

The Notch signaling pathway participates in various biological processes and is a major signaling pathway to regulate cancer stemness [30]. It is a highly conservative signaling pathway, and the core Notch signaling pathway consists of relatively few molecules. After Notch receptor activation, the Notch intracellular domain (NICD), is translocated into the nucleus and interacts with the DNA-bound protein CSL (RBP-J) to regulate the expression levels of downstream molecules [31, 32]. Our previous studies have demonstrated that Notch signaling is one key signal promoting HCC stemness [33] [34] [35] [36]. 

In this study, we con rmed that CAF was the main source of STC1 in HCC and provided evidence for an amplifying STC1-Notch1 axis in the HCC stromal-tumor crosstalk, which might be a new therapeutic target of HCC. In addition, STC1 might be a promising biomarker for the prognostic evaluation of HCC.
section_name: Background

section_name: Methods

section_content: We downloaded the transcriptomic data and the clinicopathological data of the TCGA-LIHC project from The Cancer Genome Atlas database at http://portal. gdc. cancer. gov/. Relevant survival data were gotten from previous literature [37]. Gene Set Enrichment Analysis (GSEA) and Gene Ontology (GO) enrichment analysis were performed on these data using "clusterPro ler" R packages [38]. For survival analysis, we excluded samples with an overall survival time of less than 30 days to exclude non-tumor causes of death. Kaplan-Meier survival analysis was performed with log-rank test.
section_name: Bioinformatics analysis

section_content: Samples of HCC tissue and the corresponding adjacent normal HCC tissues were collected from HCC patients who had undergone surgical resection and had not received radiotherapy and chemotherapy. We also collected 5 samples of hepatic hemangioma. The collection and usage of human specimens were approved by the Ethics Committee of Tongji Hospital, HUST, Wuhan, China (IRB ID: TJ-IRB20220562).
section_name: Clinical Liver Samples

section_content: IHC staining with the STC1 antibody (HPA 023918) and Notch1 (Abcam, ab8925) was performed to detect the protein expression level in HCC tissues. The IHC staining score was carried by ImageJ IHC pro ler (http://sourceforge. net/projects/ ihcpro ler).
section_name: Immunohistochemistry (Ihc)

section_content: The primary CAFs and NFs were isolated from the clinical HCC tissues and liver tissues adjacent to hepatic hemangioma respectively. The fresh liver tissues were washed with D-Hank's solution, cut into 2-3mm fragments and then plated in a culture dish with DMEM (GIBCO) comprising 15% fetal bovine serum (FBS) for attachment. Fibroblasts were allowed to grow out of tumor fragments for 1-2 weeks. 95% puri ed broblasts were obtained after 2-3 generations and veri ed by the broblast marker α-SMA.
section_name: Isolation And Puri cation Of Cafs And Normal Fibroblasts (Nfs)

section_content: MHCC-97H was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM comprising 10% FBS. SNU-398 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and cultivated in RPMI 1640 Medium (GIBCO) with 10% FBS.
section_name: Cell Culture

section_content: Cells were xed with 4% paraformaldehyde, washed with PBS for 3 times, permeabilized with 0. 1% Triton X-100 for 10 min, and incubated in 10% normal fetal sheep serum in PBS for 40 min at room temperature. Then the CAFs and NFs were co-incubated with α-SMA (Boster, BM0002) and STC1 (Santa Cruz, sc-293435) primary antibodies at 4°C for overnight. The MHCC-97H were incubated with Notch1 (Abcam, ab52627) and/or STC1 primary antibodies. Observed and photographed cells under uorescence microscope.
section_name: Immuno uorescence (If)

section_content: The culture medium supernatants from CAFs, NFs, and HCC cell lines (MHCC-97H and SNU-398) were collected and centrifuged at 1500 rpm for 5 min. For plasma samples, cells were removed by centrifugation at 3000 rpm for 10 minutes. Subsequently, the secretion of STC1 was detected according to the manufacturer's instructions using the ELISA kit (Boster, EK1404).
section_name: ELISA

section_content: The cells were given an appropriate amount of RIPA /PMSF/cocktail buffer and centrifuged to extract the protein. Then the total proteins were separated on SDS-PAGE and transferred onto the PVDF membrane (Merck Millipore, USA). After blocking with 5% milk for 1h, the PVDF membranes were incubated with primary antibodies overnight at 4°C. Anti-β-actin (Proteintech, 66009-1-ig) was used as a loading control. The primary antibodies were as follows: STC1 (Santa Cruz), NANOG (Cell Signaling Technology, 4903), OCT4 (CST, 2750), SOX2 (CST, 3579), Notch1 (CST, 3608), cleaved Notch1 (CST Cat# 4147), HES1 (CST, 11988), and HEY1 (ABclonal, A16110). The ImageJ software (NIH, America) was used to quantify the gray value of the protein bands. 

Trizol reagent (Invitrogen, Carlsbad, CA, USA) and HiScript® III Reverse Transcriptase (Vazyme, Jiangsu, China, R302-01) were used to extract the ribonucleic acid (RNA) and reversed to cDNA following the manufacturer's instructions. Then quantitative real-time PCR was carried out using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02). The data was analyzed by the 2 -ΔΔCt (Ct, cycle threshold) method. Primer sequences were provided in Table S1.
section_name: Western Blot And Qrt-pcr

section_content: The cells were seeded in 24-well low attachment plates (Corning, NY, USA) with tumor sphere medium, composed of DMEM/F12 medium with 1% penicillin/streptomycin, 1× B27 supplement (cat# 17504-044; GIBCO), 20 ng/ml EGF (cat# PHG0311; GIBCO), 10 ng/ml bFGF (cat# PHG0266; GIBCO), and 1% methyl cellulose (cat# M0262; Sigma-Aldrich). Fresh medium was added every 3-4 days. Spheres were visualized and counted by microscopy.
section_name: Sphere Formation Assay

section_content: The Cell Counting Kit (CCK-8) (40203ES92, Yeasen, Shanghai, China) was used to measure the sensitivity of the cells to Sorafenib. 1000 cells per well were seeded in 96-well plates, and after cell adhesion, treated with different concentrations (2. 5, 5, 10 or 20 µM) of Sorafenib (Sigma-Aldrich). After incubating for 24h at 37℃, CCK-8 reagent was added to each well. 2 hours later, absorption value at 450 nm was detected at the microplate reader (Thermo Fisher, Shanghai, China).
section_name: Cck8 Toxic Assay

section_content: Cells were seeded at a density of 1000 cells per well in 6-well plates. Approximately 10 days later, colonies were dyed with crystal violet (C0121, Beyotime, China) and counted after xing the cells with 4% methanol for 20 minutes. The results are presented as the mean ± SD.
section_name: Colony-formation Assay

section_content: For the migration assay, serum-free cell suspension was added into the upper transwell chambers (Millipore, Billerica, MA, USA) without FBS, and 600 ul of DMEM with 10% FBS was added as a chemoattractant. And cell invasion assay was performed with Matrigel (1:8 diluent, BD Biosciences, San Jose, CA, USA) coating the transwell chambers. After 24h (migration) or 32h (invasion) of incubation, the membranes were xed and imaged. The number of cells in 3 randomly selected was counted, and the experiments were repeated three times.
section_name: Transwell Migration And Invasion Assays

section_content: Viral particles expressed STC1 shRNA, STC1, or Notch1 shRNA were obtained from DesignGene Biotechnology(shanghai, China) and Genechem Corporation (Shanghai, China). The sequence for virusbased RNAi were provided in Table S1. CAFs or HCC cells were infected with lentivirus. 48h after infection, puromycin was added to screen cells for establishing stable cell lines.
section_name: Virus Infection

section_content: Cells were collected and lysed in an appropriate precooled IP buffer. The cell lysate was centrifuged after being frozen for 30min. Took 50ul lysate supernatant as input. And the rest was incubated with indicated antibody on a rotating device overnight at 4 ℃. The next day, protein A/G Magnetic Beads (MCE, HY-K0202) were washed 3 times with PBST and added to the cell supernatant-antibody system. After incubation on a rotating device at 4 ℃ for 4h, the magnetic beads were washed 6 times and boiled. Samples were conducted by Western blot analysis. The primary antibodies had been listed in Western blot.
section_name: Co-immunoprecipitation (Co-ip)

section_content: The STC1 promoter fragment was inserted into the pGL3-Basic vector. The stable lines overexpressed NICD and the control cells were co-transfected with STC1 reporter-gene plasmid and pGMLR-TK. Following a 48h incubation, a Dual-Luciferase Reporter Assay Kit (Yeasen, 11402ES60) was employed to measure re y luciferase activity, and Renilla luciferase activity was implemented for normalization.
section_name: Dual-luciferase Reporter Assay

section_content: Four-week-old male nude mice were bought from GemPharmatech company and raised in a pathogenfree barrier environment. HCC cells with or without CAFs were resuspended in PBS, and slowly injected into the left lobe of the liver. Animals were sacri ced about 4-5 weeks after implantation. Bioluminescence was measured after an intraperitoneal injection of 100ul of potassium D-luciferin salt (30 mg/mL, per animal). All experiments were approved by Tongji Hospital Institutional Review Board (IRB ID: TJH-202206023)
section_name: Mouse Orthotopic Liver Xenograft Tumor Model

section_content: Each assay was carefully performed with three independent experiments, and the data were shown as the mean and standard deviation (mean ± SD) unless otherwise indicated. Statistical analysis was processed using R (v4. 0. 2) and GraphPad Prism 9. 0 software. We chose appropriate statistical tests according to the types of data. For the data approximately normally distributed, student t-test and one-way ANOVA were performed. Wilcoxon signed-rank test was applied to analyze the nonnormal distribution data. Survival analysis was evaluated using a log-rank test. P < 0. 05 was regarded as statistically signi cant.
section_name: Statistical analysis

section_content: CAFs -derived STC1 promotes HCC stemness. 

CAFs were isolated from HCC patient tumor tissue samples, and NFs were puri ed from liver tissues adjacent to hepatic hemangioma. To check whether CAFs were the main source of STC1 in HCC, we rst analyzed the published single-cell sequencing data of HCC [39], and found that STC1 was higher expressed in broblasts than in HCC cells (Fig. 1A ). IF showed more α-smooth muscle actin (α-SMA) (green) and STC1 (red) colocalization in CAFs than those in NFs. α-SMA (green) and STC1 (red) colocalized in the cytoplasm and nucleus (Fig. 1B ). Next, we detected the secreted and intracellular levels of STC1 in the CAFs, NFs, MHCC-97h, and SNU-398 HCC cell lines, individually. ELISA assay showed that the CAF secreted the highest level of STC1 (Fig. 1C ). And the protein levels of STC1 were dramatically upregulated in CAFs relative to the levels in other cells (Fig. 1D ). Collectively, these data indicated that CAFs were the main source of STC1 in the HCC TME. 

Our previous study demonstrated that CAFs could promote or maintain stem-like properties in HCC [40]. To investigate whether CAF-derived STC1 affects HCC stemness, MHCC-97h and SNU-398 were cultured in different media: DMEM, the conditional medium of CAFs (CAF-CM), CAF-CM supplemented with 1µg/ml IgG (CAF-CM + IgG), CAF-CM supplemented with 1µg/ml STC1 neutralizing antibody (CAF-CM + STC1-Ab), and DMEM supplemented with human STC1 recombinant protein (rhSTC1, 20ng/ml). Then a series of assays were performed to test the stem-like properties of HCC. Sphere formation assay showed that CAF-CM or rhSTC1 signi cantly promoted the self-renewal ability of MHCC-97h and SNU-398 cells, while STC1 neutralizing antibody (STC1-Ab) dramatically reversed the increase in the self-renewal ability induced by CAF-CM (Fig. 1E ). Different concentrations of sorafenib were used to treat HCC cells for 24 h. The data indicated that CAF-CM, CAF-CM + IgG, and rhSTC1 groups possessed higher sorafenib resistance than DMEM and CAF-CM + STC1-Ab groups (Fig. 1F and S1A ). Colony formation assays demonstrated that CAF-CM or rhSTC1 could promote the proliferation ability, while STC1 neutralizing antibody attenuated the promoting effects of CAF-CM (Fig. 1G ). Moreover, transwell migration and invasion assays showed that after being treated with CAF-CM or rhSTC1 (20ng/ml), MHCC-97h and SNU-398 owned higher migration and invasion abilities than treated with DMEM. And compared with the CAF-CM group, the CAF-CM + IgG group still could promote migration or invasion, while the CAF-CM + STC1-Ab group had diminished migration and invasion abilities (Fig. 1H and S1B ). Western blot analysis revealed that the cancer stemness markers, including NANOG, SOX2, and OCT4, were upregulated in CAF-CM, CAF-CM + IgG, and rhSTC1 groups compared with DMEM. Meanwhile, compared with the DMEM group, cells exposed to CAF-CM + STC1-Ab showed no signi cant changes in the levels of NANOG, SOX2, and OCT4 (Fig. 1I ). These results suggested that CAFs were the main source of STC1 in the HCC TME, and the CAFsderived STC1 could promote HCC stemness. 

Knocking out of STC1 in CAFs attenuated its ability to promote HCC stemness. 

Primary CAFs were used to establish stable cell lines, CAFs-shcontrol, CAFs-shSTC1-1, and CAFs-shSTC1-2, with lentivirus infection (Fig. 2A ). The data showed that down-regulation of STC1 in CAFs signi cantly decreased CAFs-enhanced spheroids ability in HCC cells (Fig. 2B ). The resistance ability to sorafenib of HCC cells was reduced when cultured in CAFs knocking out STC1 conditioned medium, comparing with cultured in CAF-CM (Fig. 2C and S1C ). In addition, cells treated with CAF-shSTC1-1-CM and CAF-shSTC1-2-CM possessed lower proliferation, migration, and invasion abilities than those treated with CAFshcontrol -CM (Fig. 2D-F ). Western blot revealed that down-regulation of STC1 in CAFs signi cantly reversed CAFs-upregulated the expression of NANOG, SOX2, and OCT4 in MHCC-97H and SNU-398 (Fig. 2G ). Additionally, we explored the effect of CAFs-derived STC1 in vivo by the mouse orthotopic liver xenograft tumor model. SNU-398 luciferase-tagged (SNU-398-LUC) HCC cells mixed with or without CAFs of different treatments were injected into the livers of nude mice. Mice were randomly divided into four groups: SNU-398-LUC cells group, SNU-398-LUC cells + CAFs-shcontrol group, SNU-398-LUC cells + CAFs-shSTC1-1 group, SNU-398-LUC cells + CAFs-shSTC1-2 group. 28 days after cell injection, the tumor size was measured by bioluminescence imaging. Interestingly, we observed that that luminescence intensity from SNU-398-LUC cells + CAFs-shcontrol group was higher than that derived from SNU-398-LUC cells group, while knockdown of STC1 in CAFs attenuated the luminescence intensity (Fig. 2H ). Taken together, these data indicated STC1 played a vital role in CAFs-mediated promotion of HCC stemness in vitro and in vivo. 

CAFs -derived STC1 promoted HCC stemness in a Notch1-dependent manner Our previous studies have demonstrated that the Notch1 signaling pathway is important for promoting the stem-like properties in HCC [33] [34] [35]. To explore the underlying mechanism of how CAFs-derived STC1 promoted HCC stemness, we performed GSEA pathway enrichment analysis and GO analysis among the HCC samples in the TCGA database. The results indicated that high expression of STC1 was signi cantly related to positive regulation of the Notch signaling pathway (Fig. 3A and 3B ). IF showed more Notch1/NICD (green) expression in CAF-CM or rhSTC1 treated MHCC-97h cells than DMEM treated cells (Fig. 3C ). Next, we detected the expression of downstream molecules of the Notch1 signaling pathway and we found that in the CAF-CM, CAF-CM + IgG, and rhSTC1 group cells, the NICD, HES1, and HEY1 expression levels were signi cantly higher than that of the DMEM and CAF-CM + STC1 Ab groups (Fig. 3D ). The western blot assay was carried out to further verify the effect of STC1 on the Notch signaling pathway. We found that the expression of NICD, HES1 and HEY1 were signi cantly decreased in MHCC-97H and SNU-398 cells treated with CAF-shSTC1-1-CM and CAF-shSTC1-2-CM relative to those treated with CAF-shcontrol-CM (Fig. 3E ). These data suggested that the Notch signal pathway is downstream of STC1. 

To explore whether Notch1 was involved in CAFs-derived STC1-medicated HCC stemness, we knocked down the expression of Notch1 in MHCC-97H and SNU-398 with lentivirus transfection. Our previous research has proved that CAF-induced promotion of HCC stemness was weakened after Notch1 was knocked out, so in this part, we focused our attention mainly on the effect of Notch1 on CAFs-derived STC1 induced stem-cell like properties. The data revealed that the self-renewal ability of shNotch1 cells was attenuated regardless of being treated with rhSTC1 (Fig. 3F ). Similarly, after Notch1 knockdown, despite being treated with rhSTC1, the resistance to sorafenib ability of HCC cells were diminished (Fig. 3G and S2A ). And also, knockdown of Notch1 in MHCC-97H and SNU-398 cells decreased proliferation, migration, and invasion abilities despite being pretreated with rhSTC1 (Fig. 3H, 3I, and S2B-D). In addition, the expression of cancer stemness markers NANOG, OCT4, and SOX2 were decreased regardless of whether treated with or without rhSTC1 (Fig. S2E ). The in vivo orthotopic liver xenograft tumor model showed that knockdown of Notch1 lowered the luminescence intensity of the CAF-STC1 group (Fig. 3J ). Taken together, these data suggested that the effect of CAFs-derived STC1 on HCC stemness was prominently restrained through Notch1 knockdown, and the CAF/STC1/Notch1 axis might play a crucial role in the HCC stemness.
section_name: Results

section_content: To investigate the underlying mechanism of STC1-mediated activation of Notch1 signaling, we rst analyzed the correlation between STC1 and Notch1 in the HCC TCGA database at the GEPIA website (http://gepia. cancer-pku. cn/). IF showed the co-localization of STC1 (green) and Notch1 (red) in MHCC-97H (Fig. 4B ). The co-IP experiments were performed in MHCC-97H and SNU-398. The results proved there is a physical interaction between the STC1 and Notch1 (Fig. 4C-D ), and were consistent with the results of a previous study, which con rmed that STC1 could directly bind to Notch1 as a non-canonical Notch ligand to activate the Notch1 signaling pathway [28]. To evaluate the possible correlation of STC1 and Notch1 in human HCC tissues, protein levels of STC1 and Notch1 were detected by immunohistochemical (IHC) staining on a tissue microarray (Fig. 4E ). Importantly, we observed that STC1 expression was positively correlated with Nocth1 expression in HCC patients (Fig. 4F ; n = 72, r = 0. 6443, **** P < 0. 0001, Pearson's correlation). These data implied that CAFs-secreted STC1 could activate the Notch signaling pathway by directly binding to Notch1 in HCC cells, and in the HCC samples we collected, STC1 was positively correlated with Notch1.
section_name: CAFs-secreted STC1 directly bound to Notch1 receptors and activated the Notch signaling pathway

section_content: In the previous experiment, we surprisingly found that CAF-CM treated MHCC-97H had a higher STC1 expression than DMEM-treated cells (Fig. 4B ). In addition, we observed that Notch1 depletion reduced the protein expression of STC1 in MHCC-97H and SNU-398 (Fig. 5A ). This nding drove us to examine whether the STC1 gene was potentially regulated by NICD at the transcriptional level in HCC cells. STC1 protein and mRNA levels were up-regulated by overexpressing NICD with lentivirus transfection in MHCC-97H and SNU-398 (Fig. 5B and 5C ). NICD, the activated composition of the Notch receptor, is inserted into the nucleus as a transcriptional coactivator and interacts with CSL (RBP-J), the DNA-binding protein. We got the CSL binding motif from the JASPAR database (http://jaspardev. genereg. net/) (Fig. 5D ). Then, the STC1 promoter region for possible CSL binding sites was screened with the JASPAR, and we chose 3 most possible CSL binding sites for the mutation (Fig. 5E ). Dual-luciferase reporter assays suggested the mutation of R1 (binding region 1) in STC1 promoter abolished the promoter inducibility that was mediated by NICD overexpression (Fig. 5F and 5G ). Collectively, these ndings indicated that STC1 was regulated by Notch1 at the transcriptional level, which formed an amplifying STC1-Notch1 feedforward signal in tumor-stromal crosstalk. STC1 was correlated with poor prognosis of HCC patients and might serve as a biomarker. 

The effect of CAF-derived STC1 on HCC cells' stem-cell properties motivated us to explore the importance of STC1 expression in patients with HCC. By using TCGA public databases (GEPIA), we found that the HCC tumor tissues had a higher expression level of STC1 than normal liver samples (Fig. 6A ). In addition, high expression of STC1 predicted poor survival of HCC patients by Kaplan-Meier analysis (Fig. 6B ). During our analysis, samples with an overall survival time of fewer than 30 days were excluded to exclude non-tumor causes of death. As STC1 is a secretory protein, we further investigated whether the concentration of STC1 in serum could serve as a clinical biomarker for HCC. We measured the level of STC1 in the serum of patients with advanced liver cancer (n = 17) and patients with early liver cancer (n = 42) by ELISA. Patients with advanced liver cancer possessed higher serum STC1 concentration than patients with early liver cancer (Fig. 6C ), which suggested that STC1 could be a biomarker in HCC with poor prognosis. The STC1 expression level was assessed by IHC staining in a total of 72 HCC tissues and matched adjacent normal liver tissues (Fig. 6D ). The data indicated that HCC patients with high STC1 expression had shorter median overall survival than that of patients with low STC1 expression (19 months vs. 37 months, HR = 2. 63, 95% CI: 1. 23-5. 64, P = 0. 0127) (Fig. 6E ). We next examined the correlations between STC1 expression and multiple clinical-pathologic characteristics. Our data indicated that high STC1 expression was signi cantly correlated with the advanced TNM stage (Table 1 ; P = 0. 024; χ2 test). Together, these ndings suggest that STC1 was up-regulated and correlated with poor survival of HCC patients, and might be a biomarker for strati ed analysis of HCC patient prognosis. 

Table 1 The expression of STC1 related to clinicopathological features in HCC patients.
section_name: Notch1 directly regulated STC1 expression to establish a paracrine amplifying STC1-Notch1 feedforward signal

section_content: CAFs, one of the most important activating components of TME, differs from the normal resident broblasts and directly regulates tumor progression, metastasis, therapy resistance, tumor immune, and the cancer stem-like properties [41]. Previous studies had shown that CAFs were closely related to the stemlike properties of HCC cells, but the mechanism by which CAFs in regulating HCC stemness was not fully clari ed. Our previous studies indicated the impact of HCC stemness on HCC progression and the effect of the Notch signaling pathway on the stem-like properties of HCC [34]. However, the impact of CAFs on HCC stemness is not well understood yet. Herein, we focused on understanding the impact and the underlying mechanism of the CAFs in stemness facilitation and their association with the poor prognosis of HCC. 

In this study, we demonstrated that CAFs secreted STC1 and facilitated the stem-like properties of HCC. In the process of tumor progression, CAFs secreted cytokines, chemokines, secreted proteins, exosomes, and other media in the TME and promoted cancer stemness. CLCF1, which was secreted by CAFs, could promote tumor stemness in HCC [14]. Our previous studies demonstrated that CAFs promoted the stem-cell like properties of HCC through the secretion of IL6 and HGF [40]. Through the analysis of the single-cell sequencing data of HCC [39], we found that STC1 was mainly derived from broblasts rather than HCC cells. ELISA and western blot assay respectively proved that STC1 had higher secretion and intracellular expression levels in CAFs. STC1 has been reported to distribute in multiple biological activities such as tumorigenesis [42], angiogenesis [43], wound healing, and tumor immune resistance [23]. Long-term exposure of HCC cells to CAF-CM or rhSTC1 resulted in an increase in HCC stemness, while STC1-Ab dramatically reversed the promotion. The results showed that CAFs-derived STC1 could promote the stem-cell like properties of HCC. In addition, STC1 has recently been reported to facilitate stem-like traits in glioblastoma cells [28]. Silencing STC1 in CAFs decreased CAFs-mediated promotion of HCC stemness in vitro and tumorigenicity in vivo. These ndings strongly suggested the role of STC1 in the CAFs-induced promotion of HCC stemness. 

The Notch signaling pathway plays a crucial role in the regulation of stem cell properties in HCC [44],

colorectal cancer [45], and other tumors [46, 47]. Here, our data indicated that CAFs-derived STC1 could activate the Notch signaling pathway in HCC. GSEA pathway enrichment analysis and GO analysis implied that the Notch signaling pathway was positively enriched in tumor tissues highly expressing STC1. We observed that CAFs-derived STC1 activated the Notch signaling pathway of HCC cells and knockdown of STC1 in CAFs signi cantly inhibited this effect. In breast cancer, the Notch signaling driven crosstalk between tumor cells and CAFs to promote the radioresistance of tumor [48]. In our previous study, the Notch signaling pathway participated in the CAFs mediating stem-cell like properties, which was reversed when Notch1 was knocked out [36]. In this study, we found that the silence of Notch1 in HCC cells signi cantly inhibited CAFs-derived STC1 induced stemness in vitro and in vivo. These data supported the important role of the Notch signaling pathway in facilitating CAF-derived STC1 induced stem-cell like properties in HCC. 

Ligands binding to Notch receptors leads to the activation of Notch signaling. During this process, NICD is released after receptors undergo three cleavages and is inserted into the nucleus to regulate the transcription of downstream genes [30]. Recently, some new molecules have been reported as noncanonical ligands, for example, OSM-11 [49]. In glioblastoma, STC1 could bind directly to 1-24 EGF like domains in the Notch extracellular domain (NECD), which are the binding sites of Notch receptors [28]. Our data showed the direct binding between STC1 and Notch1, which suggested that STC1 might be a noncanonical ligand of Notch1 receptors. Moreover, STC1 and Nocth1 were positively correlated in clinical HCC samples. In addition, we found that the Su (H) motif of CSL, responsible for DNA binding, could bind to the promoter of STC1. CSL recruits histone deacetylases (HDACs) to inhibit the transcription of downstream genes when there is no NICD binding, and promotes the transcription of target genes after NICD binding [32]. The data revealed that the STC1 was a direct target of Notch1 in HCC cells. Taken together, we demonstrated that the stromal-tumor amplifying STC1-Notch1 feedforward signal promoted the HCC stemness, and provided new therapeutic targets targeting both cancer cells and CAFs. Besides, we believed that more therapeutic targets pointed at both cancer cells and CAFs could be developed in cancer, which could have a strategic advantage over targeting tumor cells or CAFs only. 

STC1 was found to be a biomarker in many diseases [52] [53] [54] [55]. In this study, we found that STC1 was highly expressed in HCC and was closely correlated with poor prognosis in HCC patients. Furthermore, we looked into 59 serum samples of HCC patients and demonstrated that high serum concentration of STC1 correlated with advanced stage in HCC. These data supported the important role of STC1 in HCC and showed that STC1 might be a promising biomarker for the diagnostic and prognostic assessment of HCC.
section_name: Discussion

section_content: In summary, we identi ed that CAFs were the main source of STC1 and STC1 was positively related to
section_name: Conclusions

section_name: Acknowledgments

section_content: We sincerely thank Menghan Sha and Mengjia Jing from the Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology for their help of animal experiments and mechanism diagram.
section_name: Acknowledgments

section_name: Funding

section_content: This was supported by the National Natural Science Foundation of China [grant numbers 81974380, 82173318 and 81802418 ].
section_name: Funding

section_name: Availability of data and materials

section_content: Data to this paper may be requested from the corresponding author.
section_name: Availability of data and materials

section_name: Supplementary Files

section_content: feedforward signal, a new mechanism leading to cancer stemness and progression, could provide an effective therapeutic target for HCC patients.

section_content: This is a list of supplementary les associated with this preprint. Click to download. SupplementaryFigure1. tif SupplementaryFigure2. tif SupplementaryTable1. docx
section_name: Supplementary Files