Cancer-Secreted Exosomal HSPC111 Promotes Colorectal Cancer Liver Metastasis By Reprogramming Lipid Metabolism in Cancer-Associated Fibroblasts

doi:10.21203/rs.3.rs-708870/v1

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Cancer-Secreted Exosomal HSPC111 Promotes Colorectal Cancer Liver Metastasis By Reprogramming Lipid Metabolism in Cancer-Associated Fibroblasts

https://doi.org/10.21203/rs.3.rs-708870/v1

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Background: Metastasis and metabolic deregulation are two of the major hallmarks of cancer. Recent studies have revealed the critical driving role of metabolic reprogramming of tumor cells to promote colorectal cancer (CRC) metastasis. However, little is known about the metabolic alterations of cancer-associated fibroblasts (CAFs) in the pre-metastatic niche and how these changes facilitate CRC metastasis.

Methods: Liquid chromatography-mass spectrometry (LC-MS) and Isobaric Tags for Relative and Absolute Quantitation (i-TRAQ) method were performed to identify the comparative metabolites and proteins expression in CAFs treated with exosomes derived from CRC cells, respectively. Tissue Microarray (TMA) was used to evaluate the level of HSPC111 in patient’s primary CRC tissues with or without liver metastasis. Co-immunoprecipitation (Co-IP), RNA-seq, chromatin immunoprecipitation (ChIP) migration and wound healing assay and immunofluorescence staining were employed to explore the expression regulation mechanism of exosomal HSPC111 in CAFs. Xenograft models were used to determine whether exosomal HSPC111 can remolding pre-metastatic niche of CAFs to promote CRC liver metastasis (CRLM) in vivo.

Results: Here, we demonstrate that CRC cell-secreted exosomal HSPC111 induces a lipid metabolism reprogramming process in CAFs. Importantly, our results indicate that CRC patients with liver metastasis had significantly high level of HSPC111 in CRC tissues than CRC patients without liver metastasis. Mechanistically, HSPC111 upregulate the level of acetyl-CoA and histone acetylation by phosphorylating of ATP-citrate lyase (ACLY) in CAFs. This lipid metabolism reprogramming in CAFs facilitates CXCL5 secretion in vitro and pre-metastatic niche formation in the liver to promote CRLM in an exosomal HSPC111-dependent manner in vivo. In addition, conditioned medium (CM) from CAFs induce EMT of CRC cells by down-regulating E-cadherin levels and up-regulating Vimentin and Snail levels, which could be abolished by CXCL5-neutralizing antibody and CXCR2 inhibitor navarixin. Moreover, the HSPC111-ACLY association in CAFs was reinforced by CXCL5-CXCR2 axis, further promoting exosomal HSPC111 secretion from CRC cells to form a feedforward regulatory loop.

Conclusion: Our present study reveals a novel insight into the pro-metastatic role of lipid metabolism reprogramming in CAFs and suggests the CXCL5-CXCR2 axis may be a promising target for halting CRLM.

Cancer Biology

Colorectal cancer liver metastasis

Cancer-associated fibroblasts

Lipid metabolism

Exosome

HSPC111

Colorectal cancer (CRC) is the third most common malignancy and has risen to the second leading cause of cancer-related death worldwide ^{[1, 2]}. Distant metastasis is the major cause of death in CRC patients, and the liver is the most common organ for CRC metastasis ^[3]. Approximately 25% of CRC patients have synchronous colorectal cancer liver metastasis (CRLM) when diagnosed, and more than 50% of CRC patients develop liver metastasis eventually ^[4]. Although surgical techniques and systemic therapies are advanced, the prognosis of CRLM patients remains poor. It is urgent to explore the mechanism of CRLM and exploit effective therapeutic strategies for CRLM patients.

Metabolic reprogramming is a widely existing process in all living organisms to fulfill the bioenergetic and biosynthetic needs of various cellular activities ^{[5, 6]}. In the past decades, metabolic reprogramming has been recognized as one of the essential hallmarks of cancer, which is necessary for cancer cell proliferation, adaption to tumor microenvironment and distant metastasis ^{[7, 8]}. In recent years, accumulating evidence indicate that metabolic perturbation also exists in stromal cells, whose activation also participates in the process of tumor metastasis ^[9]. Prior to macroscopic metastases occurring, CRC cells can facilitate the formation of a pre-metastatic niche in the liver for their colonization ^[10-12]. So far, most studies have focused on the metabolic changes of stromal cells resident in the primary tumor ^{[13, 14]}, and there remains little known about the metabolic reprogramming of stromal cells in the pre-metastatic niche.

Exosomes are small membranous vesicles ranging from 50-160nm in diameter, containing proteins, nucleic acids and lipid, which could be transferred to recipient cells ^{[15, 16]}. In recent years, there has been accumulating evidence demonstrating that exosomes play a critical role in the formation of pre-metastatic niches, and mainly function as a communication medium ^{[17, 18]}. Cancer-associated fibroblasts (CAFs) are the dominant component of stromal cells in the pre-metastatic niche ^{[19, 20]}, and recent studies show that cancer cells are capable of transforming normal hepatic stellate cells to CAFs through exosomes ^{[21, 22]}. Therefore, whether cancer-derived exosomes could remodel CAFs metabolism to promote metastatic colonization deserves further investigation.

In the present study, we uncover an intriguing pathway that promotes CRLM development via exosome-mediated intercellular communication between CRC cells and CAFs in the liver pre-metastatic niche. CRC cells-secreted exosomal HSPC111 induces a lipid reprogramming process in CAFs, and promotes CRLM via the CXCL5-CXCR2 axis. Taken together, our results reveal a novel insight to explain how CAFs promote CRC cells liver colonization and provide a new therapeutic strategy for targeting CRLM.

Cell lines and cell culture

The human colorectal cancer cell line HCT116 was purchased from the China Center for Type Culture Collection (Wuhan, China); human colorectal cancer cell lines HT29 and SW480 were purchased from the American Type Culture Collection (Rockville, MD, USA); and the HEK293T cell line, human colorectal cancer cell line SW620 and human hepatic stellate cell line LX-2 were purchased from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai Institute of Cell Biology). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin and streptomycin (Beyotime Biotech Co. Ltd., China). The cells were cultured at 37 ℃ in a humidified incubator containing 5% CO₂.

Exosome isolation and characterization

Exosomes in medium were isolated by using ExoQuick-TC Exosome Isolation kit (System Biosciences, Palo Alto, CA, USA) according to the manufacturer’s instructions. Briefly, HCT116, HT29, SW620 and SW480 cells (3 × 10⁶ cells) were seeded in 100-mm dishes in culture medium with 10% FBS. After 24 h, the culture medium was changed to a culture medium without FBS. The culture supernatant was collected after 48h incubation and centrifuged at 4000 g for 30 minutes to remove cell debris. The supernatant was transferred to a sterile vessel, and one-fifth of ExoQuick-TC was added to supernatant. The samples were mixed before incubation overnight at 4 ℃. Exosomes were isolated by centrifugation at 1500 g for 30 minutes. Each pellet was resuspended with PBS and the protein concentration of exosome was determined by BCA assay (TAKARA, Japan) before further experiments.

For exosome transmission electron microscopy (TEM) observation, exosome samples were fixed with 2.5% glutaraldehyde at 4℃ overnight. Then, exosomes were added onto formvar/carbon-coated grids, stained with 2% uranyl acetate for 1 h. Exosome samples were analyzed by TEM (FEI, Tecnai F20) in 120kV.

The density and size of exosomes were tracked by using Nanosight NS300 system (NanoSight technology, Malver, UK). Exosomes were resuspended and diluted in PBS to achieve between 20 and 100 objects per frame. Exosome samples were injected into the sample chamber and measured in triplicate. Data was analyzed using the nanoparticle tracking analysis analytical software (version 2.3).

Exosome labeling and tracing

To assess transfer of exosomes into LX-2, CRCs-derived exosomes were labeled with DiO dye (Rengen Biosciences, China) according to the manufacturer’s instructions. Briefly, exosomes were incubated with DiO dye for 30 minutes at 37 ℃. Excessive DiO was removed with Spin Column. The LX-2 cells were seeded in 35 mm glass-bottom dishes (Cellvis, D35-14-1N) (1 × 10⁴ cells/dishes) and cultured for 24 h. Dio-labeled exosomes, after filtering by 0.22 μm filters, were added to the culture media of LX-2 cells and incubated for 8 h at 37 ℃. The LX-2 cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized with 0.1% Triton X-100 for 20 minutes at room temperature. The LX-2 cells were stained with 4',6-diamidino-2-phenylindole (DAPI) (Boster Biological Technology co., Ltd, China). The LX-2 cells were observed with confocal microscopy (Leica).

For exosome tracking experiments in vivo, purified exosomes were labeled by using DiR dye (KeyGEN BioTECH, China), followed by 120000 g ultracentrifugation for 60 minutes, and labeled exosomes were resuspended in PBS. Labeled exosomes (50 μg/mice) in 100 μL PBS were injected into BALB/c mice via the tail vein. As a control, mice were injected with the same volume of PBS. After 12 and 24 h, an IVIS imaging system (PerkinElmer) was used for tracking exosome distribution in vivo. To evaluate exosome uptake by liver cells, all mice were sacrificed after 24 h, and the liver tissues were harvested for immunofluorescence on frozen sections. Image capture and photography were performed using a confocal microscopy (SP8, Leica).

Animal model induction and treatment

In each animal experiment, mice were randomly allocated to each group. All animal experiments were performed in accordance with protocols approved by the institutional review board of Department of Laboratory Animal Science, Fudan University, and conformed to the National Guidelines for Animal Usage in Research. All animal experiments were performed on 5-7 weeks male nude mice (BALB/c nu/nu) (SLAC Laboratory Animal Co., Ltd, Shanghai, China).

To assess the changes of acetyl-CoA and citrate levels in CAFs in CRLM, the mice were injected with exosomes (10 μg in 100 μL PBS) derived from HCT116 cells via tail vein every other day for 3 weeks. Subsequently, HCT116 cells were injected into the spleens of the mice to induce liver metastasis model. Mice were then treated with exosomes via tail vein injection twice a week continuously. Four weeks after CRC cells injection, all mice were euthanized and liver tissues were collected for the isolation of primary fibroblasts.

To evaluate the roles of exosomes in fibroblasts, 10 μg of exosomes in 100 μL PBS were intravenously injected into mice through the tail vein every other day for 3 weeks. Mice in control groups were injected with equal volume of PBS. At the endpoints, mice were euthanized and liver tissues were collected for western blot assay.

For analyzing the roles of exosomal HSPC111 in CRLM, mice treated with exosomes (10 μg in 100 μL PBS) via tail vein injection every other day for 3 weeks. The same volume of PBS was injected into mice in control groups. Mice then were assigned to groups randomly. For the tracking assay in vivo, firefly luciferase (Hanbio, Shanghai, China) was transfected into HCT116 cells and SW480 cells, and tumor formation and metastasis were detected using in vivo bioluminescence imaging. Subsequently, 1 × 10⁷ luciferase-labeled HCT116 and SW480 cells were injected into the spleens of the mice to induce the liver metastasis model. Briefly, mice were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg). The spleen was exteriorized gently through a left lateral flank incision and injected with1 × 10⁷ luciferase-labeled HCT116 and SW480 cells suspension in 100 μL slowly using five-gauge needle. The spleen was replaced to the abdominal cavity, and 3-0 absorbable sutures were used for abdomen closure. The whole operation process followed the principles of aseptic surgery. Exosomes were injected into the tail vein twice a week continuously. The distribution of CRC cells in various organs was analyzed with IVIS Lumina LT series III (PerkinElmer, MA, USA). Fluorescence intensity within specific regions of individual mice was quantified using the region of the interest (ROI) tools in the Living Image 4.5 software (PerkinElmer). The ROI value of fluorescent area to liver surface area was used to reflect liver metastasis, and the metastasic lesions were determined by hematoxylin and eosin (H&E) staining. For studies of CXCR2 inhibitor in vivo, mice were treated with navarixin (CXCR2 inhibitor) 50 mg/kg body mass or vehicle (1% methyl cellulose) orally once daily for 4 weeks. Four weeks after CRC cells injection, mice were euthanized and liver metastasis were evaluated by HE staining.

Isolation and culture of primary fibroblasts

For the isolation of human primary liver fibroblasts, paired fresh CRLM and distant non-tumor liver tissues were obtained from six CRLM patients (male : female = 3 : 3, mean age 57 ± 13 years) and normal liver tissues were obtained from six hepatic hemangioma patients (male : female = 2 : 4, mean age 54 ± 12 years) undergoing partial liver resection at Department of General Surgery, Huashan hospital, Fudan University (Shanghai, China). None of the patients had received radiotherapy or chemotherapy prior to surgery. The specimens were used after obtaining written, informed consent from the patients and with the approval of the Ethical Committee of Huashan hospital, Fudan University (Shanghai, China). Tissues were finely minced with scalpel and enzymatically dissociated in 1mg/mL collagenase IV (Solarbio^® Life Sciences, China) at 37 ℃ for 2 hours with frequent shaking. The cell suspensions were filtered through a sterile 40 μm cell strainer (BD Biosciences, Milan, Italy). The cells were centrifuged at 300 g for 5 minutes and resuspended in DMEM with 10% FBS in 100 mm dish. Cells were maintained at 37 ℃ in a 5% CO₂ humidified atmosphere. After incubating for 24 hours, the cells were attached to the culture plate, and the unattached cells were removed. We used established primary liver fibroblasts at the passage number of 4-8. The primary cells from the marginal region of encapsulated CRLM were identified as CAFs while cells from distant non-tumor liver tissues were identified as non-tumoral fibroblasts (NTFs) and cells from normal liver tissues were identified as normal fibroblasts (NFs). The mice primary liver fibroblasts were isolated in the same manner as described above. The primary cells from CRLM nodules in mice were identified as CAFs while cells from distant non-tumor mice liver tissues were identified as non-tumoral fibroblasts (NTFs) and cells from normal mice liver tissues were identified as normal fibroblasts (NFs).

Immunofluorescence

For cell immunofluorescence, cells were plated in 35 mm glass-bottom dishes. After treatment, the cells were washed with PBS twice, fixed with 4% paraformaldehyde for 15 minutes, and then permeabilized in 0.1% Triton X-100 for 20 minutes. After being blocked with 5% bovine serum albumin, the cells were incubated with appropriate primary antibodies overnight at 4 ℃. Then the cells were incubated with Alexa Fluor 594- and Alexa Fluor 488-conjugated secondary antibody for 1 hour at room temperature followed by stained with DAPI before imaging under a fluorescent microscope.

For tissue immunofluorescence, tissues were fixed in a mix of 2% paraformaldehyde and 20% sucrose solution for 24 hours at room temperature, followed by embedding and section. Then tissue sections were stained with appropriate primary antibodies overnight at 4 ℃ in a humidified chamber. Then the sections were washed with PBS and incubated with secondary antibody conjugated to Alexa Fluor 594 and 488 for 1 hour at room temperature. Nuclei were stained with DAPI. Fluorescent images were taken with a fluorescent microscope. Information of the antibodies are listed in Additional file 1: Table S1.

Metabolomic analysis

LX-2 cells were seeded in 60 mm dishes (1 × 10⁵ cells) followed by treatment with 100 μg exosomes derived from HCT116 cells and incubated for 48 hours. After incubation, the LX-2 cells were washed twice with cold PBS and frozen in liquid nitrogen for quenching metabolism. To analyze metabolomics, samples were analyzed by Omicsolution Co., Ltd (Shanghai, China). Briefly, liquid nitrogen snap-frozen samples were thawed on ice, and metabolites were isolated in precooled 50% methanol (v/v). Samples were then vortexed for 5 minutes and incubated for 10 minutes at room temperature. The isolated mixture was placed at -20 ℃ overnight. After centrifugation at 4000 g for 30 minutes (4 ℃), the supernatant was used for metabolomic analysis by LC-MS. Chromatographic separations were performed by using of an ultra-performance liquid chromatography (UPLC) system (Shimadzu, Kyoto, Japan). An ACQUITY UPLC ZIC-HILIC column (150 mm × 2.1 mm, 3.5 μm, Merck, Germany) was used for reversed phase separation, and the injection volume of each sample was 5 μL. The flow rate was 0.25 mL/min, and the column oven was maintained at 30 ℃. The mobile phase consisted with solvent A (0.2% (v/v) ammonia in water) and solvent B (0.1% (v/v) formic acid in acetonitrile). Gradient curve conditions (concentration of solvent B) were designed as follows: 0-1.5 min, 90% B; 1.5-25 min, 90%-40% B; 25-28 min, 40%-90% B; 28-33 min 90% B. Metabolites eluted from the column were detected by using a TripleTOF5600plus mass spectrometer (Shimadzu, Kyoto, Japan). Principal component analysis was applied to reveal the relationship between samples and the expression of metabolites. Variable importance in projection (VIP) was used to measure the different expression of each metabolite in each group, and VIP values > 1 were considered as differential expression. All differentially expressed metabolites were selected for heat map and enrichment analysis.

Proteomic analysis

LX-2 cells were plated into 60 mm dishes and cultured with HCT116-derived exosomes for 48 hours. Then the cells were collected for proteomics analysis. Isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic analysis was conducted by Omicsolution Co., Ltd (Shanghai, China). Briefly, the cell lysate was extracted from each sample, and a total of 100 μg of each sample were removed and labeled using an iTRAQ-8plex kit (AB SCIEX, USA) according to the manufacturer’s instructions. Each set of labeled peptides was pooled and resuspended in strong cation exchange (SCX) prefractionation using SCX chromatography on an Ultremex SCX column (4.6 × 250 mm) with a 3000 high-performance liquid chromatography system (Thermo Fisher DINOEX, Waltham, MA, USA). Each fraction was analyzed using nano liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS); nano LC-MS was performed using a TripleTOF^® 5600 mass spectrometer for MS/MS (AB SCIEX) interfaced with Eksigent nano LC system. The functions of the multiple protein contents were obtained by searching the UniProt database. Proteins were filtered by using the following thresholds: p value < 0.05 and 1.2-fold change. All differentially expressed proteins were selected for heat map and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis.

Lentivirus-mediated knockdown or overexpression of gene expression

The short hairpin RNA (shRNA) targeting HSPC111 (shHSPC111 #1, shHSPC111 #2), shRNA targeting ACLY (shACLY) and negative control (shCtrl #1, shCtrl #2) were inserted into the lentiviral vector produced by Hanbio (Shanghai, China). HCT116 cells were transfected with lentivirus (pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO) targeting HSPC111 and LX-2 cells were transfected with lentivirus (pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO) targeting ACLY at a multiplicity of infection (MOI) of 20. The sequences of shRNAs are presented in Additional file 2: Table S2.

Lentiviral vectors that overexpress full-length human HSPC111 (pHBLV-CMV-MCS-6x-His-HSPC111-EF1-ZsGreen-T2A-puro) or ACLY (pHBLV-CMV-MCS-3x-Flag-ACLY-EF1-ZsGreen-T2A-puro), and corresponding negative control (pHBLV-CMV-MCS-EF1-ZsGreen-T2A-puro) were purchased from Hanbio (Shanghai, China). The overexpressed lentiviruses targeting HSPC111 were transfected with SW480 cells and those targeting ACLY were transfected with LX-2 at a MOI of 20. All stable cell lines were obtained by 6 μg/mL puromycin (Hanbio) selection for 1 weeks followed by subsequent assays.

Clinical specimens

A total of 66 human non-metastatic primary CRC tissues (defined as NMCT) and paired peritumoral normal colorectal tissues (defined as NCT), and 136 human primary CRC tissues with liver metastasis (defined as CTLM) were collected between 2014 and 2019 from Department of General Surgery, Huashan hospital, Fudan University (Shanghai, China). All procedures were performed with the approval of the Ethical Committee of Huashan hospital, Fudan University (Shanghai, China). Patient consent was obtained before the start of the study. The general clinical information of all patients was included in Additional file 3: Table S3. The follow-up data were obtained at two mouth intervals through telephone calls, outpatient visits and office visits. All patients were followed until February 2021. Overall survival (OS) was defined as the interval between surgery and either death or the last observation taken.

Tissue Microarray (TMA)

Representative tumor areas were microscopically selected on hematoxylin and eosin (H&E) stained sections and highlighted with a marker on the section and similarly on the respective donor block. Tissue cores were punched from the defined regions on the donor block by using 2-mm punch needles and inserted into the receiver block with the help of a needle of larger caliber. The receiver blocks were placed upside down, covered with melted paraffin in a 50 ℃ oven for 5 minutes, in order to ensure the inserted core had melted in to the receiver block. The receiver blocks, immediately, were frozen for 20 minutes followed by histological sectioning and mounting onto slides.

Immunohistochemistry (IHC)

Sections (3 μm) were taken from the TMA paraffin blocks and mounted on slide glasses and then air dried overnight. The sections were deparaffinized in xylene for 5 minutes and rehydrated in a descending series of alcohol (100%, 100%, 95%, 80%, 70%) for 5 minutes each. After heat-mediated antigen retrieval by using citrate buffer, the sections were washed in distilled water and PBS. Endogenous peroxidase activity was blocked by a 15 minutes incubation in 3% H₂O₂ in distilled water. The sections were again washed in distilled water and PBS, and then incubated with appropriate primary antibody for 1 hour at room temperature. After washing in PBS for 10 minutes, the sections were incubated with a horseradish peroxidase (HRP)-labeled polymer (Dako EnVision + System) for 1 hour and washed in PBS for 10 minutes, and then incubated in 3,3-diaminobenzidine chromogen solution (Dako EnVision). The sections were slightly counterstained with Harris hematoxylin (Solarbio^® Life Sciences, China), dehydrated in an ascending series of alcohol (95%, 100%, 100%, 100%), and cleared in xylene. Cover slides were placed on the section using a resinous mountant. Information of the antibodies are listed in Additional file 1: Table S1. The immunohistochemical analysis of HSPC111 in the TMA sections was performed using a semiquantitative scoring system ranging from 0 to 3 points. The presence of cytoplasmic staining was considered positive, and according to the staining intensity, the immunopositivity was graded as none (0), weak (1), moderate (2), and strong (3).

Acetyl-CoA and citrate measurement

The concentration of acetyl-CoA was detected by using an Acetyl-CoA assay kit (Sigma MAK039) according to manufacturer’s protocols. Briefly, cells or tissues were washed with cold PBS, harvested in RIPA buffer (Beyotime Biotech Co. Ltd., China), and then sonicated. The cellular or tissue protein was precipitated with PCA (BioVision K808-200). After centrifugation at 13000 g for 2 minutes, supernatant was recovered and neutralized by the addition of potassium bicarbonate until the pH of the sample was in the range of 6-8 and precipitates were removed by centrifugation. The supernatant was used to measure acetyl-CoA concentration in triplicate by using fluorescence assay method. Fluorescence intensity was measured (λ_ex= 535/ λ_em= 587 nm) in black 96-well flat-bottom plates with clear bottoms.

The concentration of citrate was measured by using a citrate colorimetric assay kit (BioVision K655-100) according to manufacturer’s protocols. Briefly, cells were rapidly homogenized with citrate assay buffer, then centrifuged at 15000 g for 10 minutes to remove cell debris. The cellular protein was precipitated with PCA (BioVision K808-200). The sample was added into duplicate wells of a 96-well plate to bring the volume to 50 μL with a citrate assay buffer. After incubation at room temperature for 30 minutes, optical density (OD) value was measured at 570 nm.

RNA sequencing

The total RNA of the LX-2 cells incubated with appropriate exosomes for 48 h in vitro was extracted using TRIzol reagent (Invitrogen) and subjected to Biomarker Technologies (Beijing, China) for RNA sequencing. RNA purity was assessed using Nanodrop 2000 (ThermoFisher) and Bioanalyzer 2100 (Aglient). The sequencing libraries were constructed using NEBNext UltraTM RNA Library Prep kit for Illumina (NEB, USA). The library products were sequenced on an Illumina Hiseq 2500 platform, and paired-end reads were generated. The depth of sequencing coverage was 10 × and at least 10 Gb clean data were obtained from each sample. All usable reads were uniquely mapped to a gene that was used to evaluate the expression level. The DEGseq R package was used to analyze differentially expressed genes in two samples based on the conditions of a fold change (FC) ≥ 1.5 and p-values < 0.05. All differentially expressed genes were used for heat map analysis.

Co-immunoprecipitation (Co-IP)

For Co-IP analysis, LX-2 cells were washed twice with PBS and lysed in NP-40 lysis buffer (Solarbio^® Life Sciences, China) supplemented with protease inhibitor cocktail (PIC) (Solarbio^® Life Sciences, China) for 30 minutes on ice. The lysates were clarified by centrifugation at 12000 rpm for 15 minutes at 4 ℃, and then incubated with antibodies (Additional file 1: Table S1) for overnight at 4 ℃ followed by mixing with protein A/G magnetic beads (MCE, China). Immunocomplexes were washed five times with NP-40 buffer and resuspended in 4 × protein SDS-PAGE loading buffer followed by boiling for 10 minutes. The supernatants were collected by centrifugation at 12000 rpm for 5 minutes, followed by western blot analysis.

HEK293T cells were co-transfected with His-HSPC111 and Flag-ACLY lentivirus were lysed with NP-40 lysis buffer. Then the cell lysate was mixed with a His antibody or Flag antibody (Additional file 1: Table S1) for overnight at 4 ℃ followed by incubation with Anti-His Magnetic Beads (Sango Biotech, China) and Anti-Flag Magnetic Beads (MCE, China), respectively. Immunocomplexes were washed and boiled for western blot analysis.

Chromatin immunoprecipitation (ChIP)

ChIP was performed using a SimpleChIP^® Enzymatic Chromatin IP kit (Cell Signaling Technology, 9003) according to the manufacturer’s instructions. Briefly, crosslinking of proteins-DNA was carried out using 37% formaldehyde for 10 minutes at room temperature. The crosslinking was quenched by adding glycine to a final concentration of 0.125M for 5 minutes at room temperature and followed by washing with cold PBS twice. Cells were scraped into PBS containing 1mM PIC. The suspension was centrifuged and the pellet was resuspended in buffer A + DTT + PIC followed by incubation on ice for 10 minutes. The pellet was mixed in 1 mL buffer B + DTT containing 5 μL micrococcal nuclease and incubated at 37 ℃ for 20 minutes. Nuclei were pelleted by centrifugation at 16000 g for 1 minute and resuspended in ChIP buffer. The nuclei pellet was further lysed by brief sonication (10 sets of 60 s pulses), and lysates were clarified by centrifugation at 9400 g for 10 minutes. The supernatant was incubated with ChIP-grade H3K27ac antibody (ab4729, Abcam) overnight at 4 ℃ with rotation. After the addition of ChIP-grade protein G magnetic beads, the samples were incubated for 2 hours at 4 ℃ with rotation. The protein G magnetic beads were pelleted by placing the tubes in a magnetic stand (MCE, China) and washed with three times low-salt washes and one time high-salt washes. Then DNA was eluted from the protein G magnetic beads by ChIP Elution Buffer, and the crosslinking was reversed by proteinase K digestion. The eluted DNA was purified using spin columns and detected by real-time PCR. The primer sets are listed in Additional file 4: Table S4. Real-time PCR amplification was performed using SimpleChIP^® Universal qPCR Master Mix (Cell Signaling Technology, 88989).

Western blot analysis

The harvested cells or tissues were lysed in RIPA lysis buffer, and the lysates were cleared by centrifugation at 12000 rpm for 20 minutes (4 ℃). Protein concentrations were detected by BCA assay. Protein was separated by 10% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes (Millipore, USA). Then the membranes were blocked with 5% nonfat milk and incubated with appropriate primary antibody overnight at 4 ℃. Subsequently, the membranes were incubated with HRP-conjugated secondary antibody. The protein complex was detected using enhanced chemiluminescence reagents (Millipore, USA). Quantitative analysis of western blot was performed with ImageJ software (ImageJ v1.8.0). Information of the antibodies are listed in Additional file 1: Table S1.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from cells with TRIzol reagent (Life Technologies, USA) according to the manufacturer’s instructions. The extracted total RNA was eluted in nuclease-free water and the concentration of the RNA was determined using NanoDrop 2000 (Thermo Fisher Scientific). The cDNA was synthesized with the PrimeScript RT Master Mix (Takara, Japan) using 500 ng total RNA as template. qRT-PCR was performed using TB Green Premix Ex Taq (Takara, Japan) on an ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Carlsbad, USA). The mRNA level of GAPDH was used as an internal control. The results of qPCR were defined from the threshold cycle (Ct), and relative expression levels of mRNA were calculated with 2^-ΔΔCt method. The primer sets for quantitative RT-PCR are listed in Additional file 4: Table S4.

Conditioned medium (CM) preparation

LX-2 cells were cultured with appropriate exosomes (20 μg/mL) from CRC cells for 48 h. Medium was collected and centrifuged at 300 g for 10 minutes to remove the cells, at 2000 g for 10 minutes to remove the dead cells and at 10000 g for 30 minutes to remove the cell debris (4 ℃). Then, the supernatant was filtered through a 0.025 μm filter (Millipore, USA) to remove exosomes and stored at -80 ℃ for further studies.

Enzyme-linked immunosorbent assay (ELISA)

In order to evaluate the protein level of CXCL5 in the CM, cells were cultured in the presence of appropriate exosomes (20 μg/mL) for 48 hours before supernatants were collected, and the CXCL5 level was detected by CXCL5 ELISA kit (EK0728, BOSTER) according to the manufacturer’s protocols.

Cell migration assay

HCT116 cells or SW480 cells (2.5 × 10⁵/well) were plated on the upper part of a 24-well transwell chamber (Corning, NY, USA) in 200 μL specific CM. The medium in the lower chamber was supplemented with DMEM containing 10% FBS. After 24 h, cells that migrated to the underside of the membrane were fixed using methanol, stained with 0.1% crystal violet, and imaged and counted with a microscope (Leica, Wetzlar, Germany). All experiments were performed in triplicate.

Wound healing assay

HCT116 cells or SW480 cells (1 × 10⁶/well) were plated into 6-well plates in 2 mL 10% DMEM medium. After cells were grown to a confluent layer, the cell monolayers were scratched using a pipette tip. The scratched cells were removed by gently washing with non-FBS DMEM gently. Then the cells were cultured with specific CM for 48 hours. Images were obtained at time point 0 and 48 hours.

Statistical analysis

All the data were presented as mean ± standard deviation of the mean (SD). SPSS software version 22.0 was used for statistical analyses. The differences between groups were assessed using the Student t-test, ANOVA test and χ² test. Kaplan-Meier analysis and log-rank tests were applied for survival analysis. A p value of < 0.05 was considered statistically significant.

CRC cell-secreted exosomes alters lipid metabolism of CAFs

To verify whether CRC cells-derived exosomes were involved in the metabolism reprogramming of CAFs, four CRC cell lines with different migration and invasion abilities were chosen, among which HCT116 and SW620 cells were high-metastatic tumor cells, versus HT29 and SW480 cells which were low-metastatic tumor cells. Firstly, we isolated and purified exosomes from cancer cell-conditioned medium. The cup-shaped structure, size and number of the isolated exosomes was confirmed by transmission electron microscopy and Nanosight particle tracking analysis (Additional file 5: Figure S1A). In addition, the presence of TSG101, CD63 and CD9 and the absence of Calnexin were further verified that the isolated particles were exosomes (Additional file 5: Figure S1B). LX-2, a human cell line of hepatic stellate cells (HSCs), was selected as the normal HSCs in our study. To evaluate the delivery efficacy of exosomes, we labeled the CRC cells-secreted exosomes with Dio (green). After incubation, immunofluorescence imaging indicated the presence of Dio spots in recipient LX-2 cells, suggesting that labeled exosomes derived by different CRC cells were delivered to HSCs (Additional file 5: Figure S1C).

To identify the different abilities of educating LX-2 cells among the exosomes secreted from the four CRC cells (Fig. 1A), western blotting analysis showed that the expression of α-SMA and FAP, biomarkers of CAFs, were most increased in HCT116-secreted exosomes (named Exo^HCT116) incubated LX-2 cells (Fig. 1B). To further evaluate the metabolite changes of CAFs, metabolomic analysis was conducted in Exo^HCT116incubated LX-2 cells, and we identified metabolites that were significantly altered after incubation with Exo^HCT116. KEGG pathway enrichment analysis showed that Exo^HCT116incubation most markedly influence metabolism of lipids and lipoproteins (Fig. 1C). Notably, the level of acetyl-CoA was increased and citrate level was reduced in Exo^HCT116incubated CAFs, compared with control group (Fig. 1D and Additional file 6: Table S5). In our previous studies, we have found that acetyl-CoA metabolism played a critical role in cancer metastasis ^[23].

Subsequently, to evaluate the levels of acetyl-CoA and citrate in primary CAFs, we isolated primary fibroblasts from human and mouse CRLM and distant non-tumor liver tissues, and normal liver tissues, which exhibited a spindle-like morphology (Additional file 7: Figure S2A). Immunofluorescence staining confirmed that the expression levels of α-SMA and FAP were higher in CAFs than those in NTFs and NFs (Additional file 7: Figure S2B), suggesting that CAFs are more activated than NTFs and NFs. We also excluded the presence of other cell types including endothelial cells, epithelial cells and immune cells by staining with the corresponding makers CD31, EpCAM and CD45 (Additional file 7: Figure S2B). Next, we examined the levels of acetyl-CoA and citrate in each type of primary fibroblasts, and found that acetyl-CoA level was elevated and citrate level was decreased in CAFs and NTFs compared with NFs (Fig. 1E and F). Collectively, these results revealed that CRC cells-derived exosomes mediated the activation of HSCs and enhanced lipid metabolism in CAFs.

CRC cell-secreted exosomal HSPC111 increases the level of acetyl-CoA in CAFs

In order to investigate the molecular mechanism responsible for promoting lipid metabolism at the protein level in CAFs, we conducted the iTRAQ proteomic analysis on LX-2 cells incubated with Exo^HCT116and control. The heat map of the expression levels of different proteins was shown in Fig. 2A, which indicates that 76 proteins were up-regulated and 110 proteins were down-regulated in Exo^HCT116incubation groups compared with control groups (Additional file 8: Table S6). Meanwhile, the KEGG pathway enrichment analysis confirmed that metabolic pathways were the most highly represented pathway (Fig. 2B). HSPC111 was most markedly elevated by more than 2.5-fold in LX-2 cells following Exo^HCT116incubation. Furthermore, western blot analysis indicated that HSPC111 protein levels were higher in HCT116 cells and lower in SW480 cells, which is consistent with their derived exosomes (Fig. 2C). Then, we generated two HSPC111-specific shRNAs to silence the HSPC111 expression, and the shRNA#1, which induced the most significant knockdown effect, was selected for knocking down the HSPC111 expression in HCT116 cells (HCT116 KD) that highly express HSPC111 (Additional file 9: Figure S3A). Meanwhile, we overexpressed HSPC111 in SW480 cells (SW480 OE) that minimally express HSPC111 (Additional file 9: Figure S3B). Subsequently, we found that the protein level of HSPC111 was decreased in HCT116 KD cells-derived exosomes (Exo^{HCT116 KD}) and increased in SW480 OE cells-derived exosomes (Exo^{SW480 OE}) (Fig. 2D). Next, we examined whether HSPC111 was involved in regulating acetyl-CoA levels. After incubation with Exo^{HCT116 KD} in CAFs, the cellular acetyl-CoA level was significantly decreased, whereas the cellular citrate level was significantly increased (Fig. 2E). When treated with Exo^{SW480 OE} in CAFs, the cellular acetyl-CoA level was significantly elevated, whereas the cellular citrate level was significantly reduced (Fig. 2F).

Subsequently, as ATP-citrate lyase (ACLY) activity was regulated at the posttranslational level by phosphorylation, which is known to increase its catalytic activity ^[24] (Fig. 2G), we reasoned that the changed cellular acetyl-CoA level might also be the result of elevated phosphorylation level of ACLY. To examine whether HSPC111 could activate ACLY, we detected the phosphorylation status of ACLY on Serine 455 in response to Exo^{HCT116 KD} or Exo^{SW480 OE} incubation in CAFs. Western blot showed that ACLY phosphorylation was triggered in Exo^{SW480 OE} incubation in CAFs, which was diminished in CAFs incubated with Exo^{HCT116 KD}(Fig. 2H). To further investigate whether HSPC111 and ACLY could directly interact, we co-transfected His-tagged HSPC111 and Flag-tagged ACLY into LX-2 cells and HEK-293T cells. Co-immunoprecipitation assays revealed the endogenous and exogenous interaction between HSPC111 and ACLY, respectively (Fig. 2I and J). Subsequently, to confirm that HSPC111 in exosomes derived from CRC cells affects acetyl-CoA level in LX-2 cells by interacting ACLY, we employed ACLY silencing (shACLY) LX-2 cells (Additional file 9: Figure S3C) and found that incubation with Exo^HCT116 significantly decreased cellular acetyl-CoA level and increased citrate level in shACLY LX-2 cells (Fig. 2K).

Next, as fluctuation of cellular acetyl-CoA levels affects histone acetylation ^[25-27] (Fig. 2G), we detected the levels of histone acetylation, and found that incubation with Exo^{HCT116 KD} in CAFs led to a dramatic reduction of histone H3 acetylation (H3ac) level, while incubation with Exo^{SW480 OE} resulted in a significant elevation of H3ac level in CAFs (Fig. 2L). However, a significant change in histone H2B acetylation and histone H4 acetylation (H4ac) levels was not observed (Fig. 2L). Then, we analyzed the levels of histone acetylation at six lysine residues of histone H3 tails by western blot. CAFs incubated with Exo^{HCT116 KD} or Exo^{SW480 OE}led to significant changes in the H3K27 acetylation (H3K27ac) level, but not at other five lysine residues (Fig. 2M). We also detected the general protein acetylation in CAFs incubated with Exo^{HCT116 KD} or Exo^{SW480 OE}and found that a specific band at the position of 17 kDa was altered, which is possibly the band of H3ac (Additional file 10: Figure S4A and B). Together, these results suggested that exosomal HSPC111 regulates the acetyl-CoA levels and H3 acetylation by interacting with ACLY in CAFs.

CRC cell-secreted exosomal HSPC111 promotes colorectal liver metastasis

In order to evaluate whether HSPC111 in CRC tissues is associated with CRLM, we compared a TMA-based IHC study of HSPC111 in 66 human non-metastatic primary CRC tissues (NMCT) and paired peritumoral normal colorectal tissues (NCT), and 136 human primary CRC tissues with liver metastasis (CTLM) (Additional file 3: Table. S3). These patients were divided into low (score 0-1) and high (score 2-3) HSPC111 expression group according to the immunostaining scores (Fig. 3A). We found that compared to the NCT, the protein level of HSPC111 significantly increased in CTLM and NMCT, especially in CTLM, and most CTLM had significantly high HSPC111 levels (Fig. 3B).

In the pre-metastatic niche or metastatic niche, CAFs have been demonstrated to participate in the process of cancer colonization ^[28]. To evaluate the distribution of exosomes in vivo, purified exosomes were labeled by DiR and then intravenously injected into mice via the tail vein. In vivo tracing of the injected exosomes showed that CRC cells-derived exosomes accumulated in liver, which also could be confirmed by immunofluorescence analysis (Fig. 3C). Importantly, western blot showed that the protein levels of α-SMA and FAP were increased in exosomes injection group, compared with vehicle group, which indicated that the increase of CAFs in liver tissues after exosome injection (Fig. 3D). These results demonstrated that exosomes secreted by CRC cells established a pre-metastatic niche formation in the liver. To further investigate whether exosomal HSPC111 affects CRLM in vivo, mice were pretreated with exosomes containing different levels of HSPC111 for three weeks, following by intrasplenic injection of luciferase-labeled HCT116 and SW480 cells as experimental metastasis models (Fig. 3E). In vivo imaging assay showed that purified HSPC111-enriched exosomes promoted liver metastasis, whereas silencing HSPC111 in exosomes markedly reduced the liver metastatic growth (Fig. 3F and H). Histological analysis further confirmed the metastatic promoting effect by HSPC111-enriched exosomes and silencing HSPC111 in exosomes decreased liver metastasis (Fig. 3G and I). Taken together, these results demonstrated that exosomal HSPC111 mediated the pre-metastatic role of cancer cells-derived exosomes during the process of CRLM.

CRC cell-secreted exosomal HSPC111 promotes CAFs to secrete CXCL5

It is well-known that histone acetylation is associated with transcriptional activation ^[29]. To elucidate the molecular changes elicited by exosomal HSPC111 that promote CRLM, we analyzed global gene expression changes by RNA sequencing (RNA-seq). By comparing the down-regulated genes in CAFs incubated with Exo^{HCT116 KD} (Additional file 11: Table S7) and up-regulated genes in CAFs incubated with Exo^{SW480 OE} (Additional file 12: Table S8), we observed some cancer-promoting genes, including CXCL5, TGFB, MMP2, SERPINE1, LOX and STC1 (Fig. 4A, B and C), suggesting that exosomal HSPC111 may promote the cancer-promoting effects of HSCs by epigenetic regulation of gene transcription. Since CXCL5, a subset of CXC chemokines, mediates various cellular behaviors including cancer cell migration and metastasis ^[30], and the CXCL5-CXCR2 axis regulates organ-specific metastasis, including liver metastasis of colorectal cancer ^{[31, 32]}, we selected CXCL5 as a prototype to examine our larger hypothesis and model. We first validated the RNA-seq data by qRT-PCR for the selected CXCL5 gene (Fig. 4D). Subsequently, western blot analysis demonstrated that incubation with Exo^{HCT116 KD} significantly decreased CXCL5 protein level in CAFs, whereas incubation with Exo^{SW480 OE} elevated CXCL5 protein level significantly in CAFs (Fig. 4E and F). Furthermore, the concentration of CXCL5 in culture medium was detected by ELISA assay, and the results were consistent with the results of western blot analysis (Fig. 4G and H). Meanwhile, consistent with these results, immunofluorescence staining revealed decreased α-SMA, FAP and CXCL5 expression in CAFs incubated with Exo^{HCT116 KD} and increased α-SMA, FAP and CXCL5 expression in CAFs incubated with Exo^{SW480 OE} (Fig. 4I and J). Accordingly, these results indicated that exosomal HSPC111 derived from CRC cells promoted CAFs to produce and release cancer-promoting gene CXCL5.

An increase in levels of acetyl-CoA and H3K27ac promotes CXCL5 secretion in CAFs

To validate whether the expression of CXCL5 was affected by altering the acetyl-CoA levels, we employed shACLY and ACLY overexpression (ACLY) LX-2 cells (Additional file 9: Figure S3D), and found that cellular acetyl-CoA levels were decreased in the shACLY group and increased in the ACLY group significantly, compared with control group (Fig. 5A and B). As a supplement of acetate can regulate the cellular acetyl-CoA levels ^[33], we then treated shACLY group and ACLY Ctrl group with different concentration of acetate, and found that cellular acetyl-CoA levels were elevated dose-dependently (Fig. 5A and B). Notably, western blot analysis also confirmed that ACLY silencing significantly decreased CXCL5 and H3K27ac protein levels, which increased in ACLY overexpression and acetate treatment group (Fig. 5C and D).

To verify whether HSPC111-dependent alteration of H3K27ac was required for the expression of CXCL5, we treated cells with Trichostatin A (TSA), a histone deacetylase inhibitor, or C646, a p300 acetyltransferase inhibitor, to regulate H3K27ac levels. We found that consistent with the changes of H3K27ac levels, TSA treatment significantly enhanced Exo^{HCT116 KD Ctrl} incubation induced CXCL5 protein levels, and hindered the effect of Exo^{HCT116 KD} incubation on decreasing CXCL5 protein levels in CAFs (Fig. 5E). On the other hand, C646 treatment hindered the effect of Exo^{SW480 OE} treatment on increasing H3K27ac and CXCL5 protein levels in CAFs (Fig. 5F). Subsequently, chromatin immunoprecipitation (ChIP) was used to examine histone acetylation on the CXCL5 gene promoter region. To this end, an antibody recognizing H3K27ac, a histone mark for active promoters, was used for ChIP, which found that Exo^{HCT116 KD} incubation decreased H3K27 acetylation on CXCL5 promoter, and TSA increased CXCL5 promoter acetylation in CAFs (Fig. 5G). Additionally, Exo^{SW480 OE} treatment elevated H3K27 acetylation on CXCL5 promoter, and C646 reduced CXCL5 promoter acetylation in CAFs (Fig. 5H). Thus, CRC cells-derived exosomal HSPC111 enhanced histone acetylation so as to induce an active chromatin structure for gene transcription of CAFs. Overall, these results suggested that increases in acetyl-CoA and H3K27ac levels are required for the expression of CXCL5 in CAFs.

CAFs-secreted CXCL5 reinforces exosomal HSPC111 excretion in CRC cells and promotes CRLM progression

Previous studies reported that CAFs are a key determinant in the malignant progression of cancer through the secretion of various chemokines, including CXCL5 ^[34]. The above results prompted us to determine whether CAFs-secreted CXCL5 exerts feedback regulation on HSPC111 expression in CRC cells. The conditioned medium (CM) from Exo^{HCT116 KD} and Exo^{SW480 OE} treated LX-2 cells (CM-Exo^{HCT116 KD} and CM-Exo^{SW480 OE}, respectively) was used to incubate HCT116 cells and SW480 cells, respectively. We observed that the protein levels of HSPC111 and CXCR2, a receptor of CXCL5, were decreased in the CM-Exo^{HCT116 KD} incubation group (Fig. 6A) and increased in the CM-Exo^{SW480 OE} incubation group significantly (Fig. 6B). To further explore whether CXCL5 regulates exosomal HSPC111 excretion in CRC cells, we stimulated CRC cells with recombinant human CXCL5, and found that the protein levels of HSPC111 were elevated dose-dependently in CRC cells and their derived exosomes, respectively (Additional file 13: Figure S5A and B). These results suggested a feedforward regulatory loop along with the CRC cells-HSCs-CRC cells trace. Additionally, CM-Exo^{HCT116 KD} treated HCT116 cells showed alleviated motility, whereas increased motility was shown in CM-Exo^{SW480 OE} treated SW480 cells (Fig. 6C and D).

It is well-known that the CXCL5-CXCR2 axis is involved in the process of epithelial-mesenchymal transition (EMT) and promoting tumor progression ^{[35, 36]}. Therefore, we hypothesized that the CXCL5-CXCR2 axis promotes CRC cells migration by inducing EMT. To verify our hypothesis, we detected changes in EMT markers in CRC cells, and found that CM-Exo^{HCT116 KD} incubation led to higher expression of E-cadherin and lower expression of Vimentin and Snail in HCT116 cells (Fig. 6A and E). However, down-regulation of E-cadherin, as well as up-regulation of Vimentin and Snail, were observed in SW480 cells incubated with CM-Exo^{SW480 OE} (Fig. 6B and F). Additionally, incubation of a CXCL5-neutralizing antibody with CM-Exo^{HCT116 KD} in HCT116 cells and CM-Exo^{SW480 OE} in SW480 cells not only reduced HSPC111 and CXCR2 expression (Fig. 6E and F), but also hindered the cell migration of CRC cells (Fig. 6G and Additional file 13: Figure S5C). Meanwhile, the expression of EMT-associated genes was also altered, leading to down-regulation of E-cadherin and up-regulation of Vimentin and Snail (Fig. 6E and F). Furthermore, consistent with these results, blocking CXCR2 using navarixin, a CXCR2 inhibitor, in CRC cells impaired HSPC111, CXCR2 and EMT-associated genes expression and cell migration of CRC cells as well (Fig. 6H, I, J and Additional file 13: Figure S5D). These results suggested that an important role for the CXCL5-CXCR2 axis as a signaling cascade from the tumor microenvironment to the cancer.

To further evaluate whether the CXCL5-CXCR2 axis is involved in CRLM in vivo, we generated a liver metastasis model using nude mice. We found that more metastatic lesions were observed in the Exo^{HCT116 KD Ctrl} group and fewer metastatic lesions were observed in the Exo^{HCT116 KD} group. Notably, nude mice treatment with navarixin inhibited tumor metastasis (Fig. 6K and L), which was also confirmed by histologic analyses. Collectively, these results demonstrated that CXCL5 secreted by CAFs exerts positive feedback on the expression of HSPC111 in CRC cells, resulting in further progression of CRLM.

Multiple studies have revealed the essential role of the pre-metastatic niche in CRC metastasis ^{[37, 38]}, and exosomal mediated cancer-stromal cell crosstalk has become one of the most accepted mechanisms for pre-metastatic niche formation. However, the specific metabolic change of stromal cells during the niche formation is largely undetermined. In our study, we found that lipid metabolism reprogramming occurs in CAFs induced by exosomes derived from high-metastatic CRC cells, which contained increased acetyl-CoA levels and decreased citrate levels. Then, we identified that HSPC111 was directly transferred from CRC cells to fibroblasts in the liver pre-metastatic niche via exosomes and subsequently converts fibroblasts to CAFs. In addition, CAFs promote CRC cells liver colonization by secreting CXCL5. The crosstalk between CRC cells and CAFs further illustrates the molecular mechanism of liver metastasis from CRC, and explains why diverse CRC cell types show different abilities to induce liver metastasis. Here, our data reveal a previously unappreciated lipid metabolism change in CAFs in pre-metastatic niche and provide a new insight into the mechanism of CRLM, which holds vital implications for efficient prevention and therapeutic strategies.

Previous studies revealed that as a major contributing factor, lipid metabolism promotes the metastatic potential of cancer cells ^[39]. Acetyl-CoA, an important metabolite of lipid metabolism, is involved in regulating the cell cycle and cell growth by promoting the acetylation of histones at genes ^{[27, 40]}. In the present study, we observed a close association between elevated acetyl-CoA levels in CAFs and CRLM. Mechanically, it is known that HSPC111 as a component of ribosomes regulates cell growth ^[41]. However, what is not known is how HSPC111 affects cancer metastasis. In our studies, we showed that exosomal HSPC111 silencing or overexpression derived from CRC cells led to altered acetyl-CoA levels and affected lipid metabolism in CAFs through phosphorylation of ACLY. Meanwhile, exosomal HSPC111 induced the pre-metastatic niche in liver to promote CRLM. A set of recent studies revealed that the elevation of ACLY expression and activity promotes cancer cell growth and metastasis ^{[42, 43]}, and phosphorylation of ACLY contributes to cellular acetyl-CoA production and then increases histone acetylation ^[44]. In our study, we observed exosomal HSPC111 treatment specifically affected the acetylation of H3K27 in CAFs without affecting acetylation of other histone protein. How the cellular acetyl-CoA specifically affects H3 acetylation needs further exploration.

Tumor cells recruit CAFs to establish a favorable pre-metastatic niche in the stromal microenvironment to support their colonization, growth and invasion in the liver ^[45]. In recent years, multiple studies have shown that CAFs crosstalk with cancer cells via secreting various cytokines and chemokines ^{[46, 47]}, and other studies have demonstrated that metabolic reprogramming of CAFs are important in cancer progress ^[48]. For example, TGF-β1 secreted by CAFs is associated with gastrointestinal stromal tumor migration and metastasis ^[49]. Moreover, lactate secreted from CAFs could be absorbed by prostate cancer cells and promote their growth ^[50]. In this study, exosomal HSPC111 derived from CRC cells induces a panel of cancer-promoting factors in CAFs, including CXCL5, TGFB, MMP2 and SERPINE1, so as to enhance their cancer-promoting effects. Previous studies have elucidated that CXCL5 produced by fibroblasts plays an important role in the progression, growth and metastasis of cancer ^[51]. Thus, we primarily concentrated on CXCL5 in our study. Since histone modifications have an effect on the gene promoter region, we performed additional experiments and found that TSA or C646 treatment indeed elevated or decreased cellular CXCL5 protein levels, respectively. Taken together, we assumed that CXCL5 may be an important target for inhibiting the pre-metastatic liver microenvironment to suppress CRLM.

We also observed that CM from exosomal HSPC111 incubated CAFs, containing a high concentration of CXCL5, can promote the migration of CRC cells. CXCL5 has been reported to recruit neutrophils in hepatocellular carcinoma to promote tumor growth and metastasis ^[52]. We pointed out that CXCL5 participated in CAFs-induced CRLM by interaction with CXCR2 in CRC cells and induced EMT phenotype. The CXCL5-CXCR2 axis has been shown to regulate neutrophil homeostasis ^[53] and facilitate migration and invasion of papillary thyroid carcinoma cells ^[35] and lung cancer cells ^[54], but no evidence has been shown that CXCL5 secreted by CAFs is linked with EMT to promote CRLM. EMT is a dynamic and reversible biological process in which cancer cells undergo a morphological conversion from an epithelial-like phenotype to a mesenchyme-like phenotype, and acquire the ability to invade and migrate ^[55]. This process is characterized by the protein of E-cadherin, vimentin and snail, of which E-cadherin controls epithelial genes, while vimentin activates mesenchymal genes and snail is a vital transcription factor that acts as a repressor of E-cadherin expression ^{[56, 57]}. In our study, we noticed that CM from exosomal HSPC111 incubated CAFs could downregulate the level of E-cadherin and upregulate the levels of vimentin and snail, which could be abolished by CXCL5 neutralizing antibody. To date, as CXCR2 is the only identified receptor for CXCL5, it is plausible that the pro-metastatic effects of CXCL5 secreted from CAFs will be abolished by inhibiting CXCR2. In the present study, we verified that inhibition of CXCR2 using navarixin was able to reverse the upregulated migration ability and EMT process of CRC cells in vitro, and inhibit CRLM process in vivo. Previous studies demonstrated that once reshaped by cancer cells, stromal cells in the tumor microenvironment exert their regurgitation-feeding activity on cancer cells via secreting cytokines and chemokines ^{[58, 59]}. In our study, we found that CXCL5 secreted by CAFs was involved in a feedforward regulatory loop to further increase the level of HSPC111 in CRC cells, which provide a new perspective on the function of HSPC111 in CRLM progression. These findings may extend our knowledge of the links between CAFs and CXCL5 and shed light on the mechanisms underlying how CAFs facilitate CRLM.

The present results indicate that exosomal HSPC111 mediated pre-metastatic niche formation is a critical feature of CRC liver metastasis. In this process, CAFs undergo a lipid metabolism reprogramming to facilitate CRC cells colonization via the CXCL5-CXCR2 axis (Fig. 7). Taken together, HSPC111 may become a novel potential marker for CRLM, and blockage of the CXCL5-CXCR2 axis may be a potential strategy to prevent the occurrences of CRLM.

CRC: Colorectal cancer; CRLM: Colorectal liver metastasis; CAFs: Cancer-associated fibroblasts; TEM: Transmission electron microscopy; iTRAQ: Isobaric tags for relative and absolute quantification; shRNA: Short hairpin RNA; KD: Knockdown; OE: Overexpression; NMCT: Human non-metastatic primary CRC tissues; NCT: Peritumoral normal colorectal tissues; CTLM: Primary CRC tissues with liver metastasis; CRLMT: Colorectal liver metastasis tissues; NLT: Peritumoral normal liver tissues; Non-tumoral fibroblasts: NTFs; Normal fibroblasts: NFs; TMA: Tissue microarray; H&E: Hematoxylin and eosin; IHC: Immunohistochemistry; Co-IP: Co-immunoprecipitation; ChIP: Chromatin immunoprecipitation; qRT-PCR: Quantitative real-time PCR; CM: Conditioned medium; ELISA: Enzyme-linked immunosorbent assay; ACLY: ATP-citrate lyase; TSA: Trichostatin A; EMT: Epithelial-mesenchymal transition

Ethics approval and consent to participate

Informed consent was provided by all patients, and the Ethical Committee of Huashan hospital, Fudan University (Shanghai, China). For animal studies, all of the experimental procedures were performed in accordance with protocols approved by the institutional review board of Department of Laboratory Animal Science, Fudan University.

Consent for publication

Not applicable.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article and its additional files.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the National Science and Technology Major Project of the thirteenth Five Year Plan (grant number 2017ZX10203207); the National Natural Science Foundation of China (grant number 82070655 and 81472677).

Authors’ contributions

CZ, XYW and JHC contributed to the design of the study. CZ, XYW and PZ performed most of the experiments, analyzed the data and wrote the manuscript. CZ, JL, JHH, RZ, JF, LL, WWZ and HLJ performed clinical data analysis, part of animal studies and pathology analysis. CZ and PZ conducted the isolation and characterization of exosomes. CZ and TCH conducted the immunofluorescence analysis. CZ, PZ and TCH performed co-IP and ChIP experiments. CZ and JHH conducted qRT-PCR measurement. CZ performed Acetyl-CoA and citrate measurement. CZ, XYW and PZ performed statistical analysis. JHC, LXQ and JBZ reviewed and edited the manuscript. The authors read and approved the final manuscript.

Acknowledgements

Our special acknowledgments to Dr. Laura Jordhen (Department of Family Medicine, Adventist Health Parkrose, 1350 NE 122nd Ave, Portland, OR 97230 USA) for revising the language of our manuscript.

Author details

¹Department of General Surgery, Huashan Hospital, Fudan University, 12 Wulumuqi Road (M), Shanghai 200040, China

²Institute of Cancer Metastasis, Fudan University, Shanghai, China

³Department of Pathology, Huashan Hospital, Fudan University, 12 Wulumuqi Road (M), Shanghai 200040, China

⁴Department of Infectious Diseases, Huashan Hospital, Fudan University, 12 Wulumuqi Road (M), Shanghai 200040, China

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Additionalfile1.xlsx
Additional file 1: Table S1. Information of the antibodies used in our research.
Additionalfile2.xlsx
Additional file 2: Table S2. Sequences of shRNA targeting HSPC111 and ACLY.
Additionalfile3.xlsx
Additional file 3: Table S3. Clinical characteristics of the CRC patients.
Additionalfile4.xlsx
Additional file 4: Table S4. Sequences of primer sets used in quantitative RT-PCR and Chromatin immunoprecipitation assay.
Additionalfile5.tif
Additional file 5: Figure S1. Exosome characterization and uptake assays. (A) Exosomes derived by different CRC cells were determined by transmission electron microscopy (TEM) and Nanosight particle tracking analysis (NTA). Scale bar = 50 nm. (B) Western blot confirmed the positive expression of TSG101, CD63 and CD9 and negative expression of Calnexin in exosomes, and conversely in different CRC cell lines. (C) Uptake of DiO-labeled exosomes was analyzed by immunofluorescence microscopy. Scale bar = 20 μm.
Additionalfile6.xlsx
Additional file 6: Table S5. Metabolites that were altered significantly after ExoHCT116 treatment.
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Additional file 7: Figure S2. Characterization of NFs, NTFs and CAFs isolated from patient and mouse liver tissues. (A) NFs, NTFs and CAFs isolated from patient and mouse liver tissues were photographed by light micrographs. Scale bar = 100 μm. (B) Representative immunofluorescence staining of α-SMA, FAP, CD31, EpCAM and CD45 in NFs, NTFs and CAFs isolated from patient and mouse liver tissues. Scale bar = 50 μm.
Additionalfile8.xlsx
Additional file 8: Table S6. Up-regulated and down-regulated genes in LX-2 cells after incubation with ExoHCT116.
Additionalfile9.tif
Additional file 9: Figure S3. Confirmation of HSPC111 knockdown and overexpression in CRC cells and ACLY knockdown and overexpression in LX-2 cells. (A and B) Confirmation of the knockdown efficiency of two HSPC111 shRNAs in HCT116 cells (A) and HSPC111 overexpression in SW480 cells (A). (C and D) Confirmation of the knockdown efficiency of ACLY shRNA (C) and ACLY overexpression (D) in LX-2 cells.
Additionalfile10.tif
Additional file 10: Figure S4. Total protein acetylation assays. (A and B) The effects of exosomes derived from HSPC111 KD or HSPC111 OE stably expressed HCT116 (A) or SW480 cells (B) on the levels of total protein acetylation in LX-2 cells.
Additionalfile11.xlsx
Additional file 11: Table S7. Up-regulated and down-regulated genes in LX-2 cells after incubation with ExoHCT116 KD.
Additionalfile12.xlsx
Additional file 12: Table S8. Up-regulated and down-regulated genes in LX-2 cells after incubation with ExoSW480 OE.
Additionalfile13.tif
Additional file 13: Figure S5. CAFs-secreted CXCL5 reinforces exosomal HSPC111 excretion in CRC cells and promotes CRLM progression. Relative to Fig 6. (A) Western blot showed HSPC111 protein levels in CXCL5 treated HCT116 and SW480 cells. (B) Western blot showed HSPC111 exosomal excretion levels in CXCL5 treated HCT116 and SW480 cells. (C) Migration of SW480 cells were analyzed by wound-healing assay after CAFs CM and CXCL5 neutralizing antibody treatment. Scale bar = 100 μm. (D) Migration of SW480 cells were analyzed by wound-healing assay after CAFs CM and CXCR2 inhibitor (navarixin) treatment. Scale bar = 100 μm. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

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Cancer-Secreted Exosomal HSPC111 Promotes Colorectal Cancer Liver Metastasis By Reprogramming Lipid Metabolism in Cancer-Associated Fibroblasts

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