Kupffer cells M2-like polarization increases liver metastatic burden via uptake of exosomal KRAS mutant protein from hypoxia colorectal carcinoma cells

Kupffer cells M2-like polarization increases liver metastatic burden via uptake of exosomal KRAS mutant protein from hypoxia colorectal carcinoma cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Kupffer cells M2-like polarization increases liver metastatic burden via uptake of exosomal KRAS mutant protein from hypoxia colorectal carcinoma cells Yu You, Zhihao FENG, Jiao LU, Jie XU, Ke YOU, Fuyao LIU, Tianzhu WU, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6300438/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective : This study aimed to investigate the metastasis-promoting effect of colorectal carcinoma cell-derived exosomes on liver metastasis, M2-like polarization of Kupffer cells, and the underlying mechanism. Methods : Mouse liver metastasis models were established to testify the involvement of CRC-derived exosomes on liver metastasis, and DIR and PKH26 fluorescent labeling strategies were used to trace the distribution of CRC-derived exosomes in vivo. GO and KEGG analyses of differentially expressed genes revealed the key cellular regulators and KRAS-induced signaling in CRC liver metastasis. The phenotype of Kupffer cells was determined using IHC and IF. In vitro model HMDMs were used to explore the polarization phenotype and therapeutic effects of AKT inhibition. Results : Exosome mutant KRAS induced AKT signaling in the process of kupffer cells (KCs) M2-like polarization, promoting CRC liver metastasis. AKT inhibitors may potentially be used as a therapeutic approach to prevent liver metastasis in CRC. KRAS exosome Kupffer cell M2 polarization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Colorectal cancer (CRC) is the world’s second-deadliest cancer after lung ccancer 1 . More than 90% of CRC-related deaths can be attributed to metastatic outgrowth in distant organs 2 . The liver is the most common organ for CRC metastasis, and patients with liver metastasis have a much poorer prognosis; the 5-year survival rate of untreated CRC patients with liver metastasis is 7–12% 3,4 . And approximately 60–80% of CRC patients eventually develop liver metastases 5 . Even in patients with early stage disease, intrahepatic metastases frequently occur after curative resection, and the underlying mechanisms are not well understood. Although colorectal cancer metastasis is influenced by multiple factors, the significance of hypoxia is well established 6 . Hypoxia is one of the hallmarks of the tumor microenvironment due to rapid tumor cell proliferation, unique tumor metabolism, and abnormal blood vessel 7 . Recent studies have revealed that, before the arrival of tumor cells, the formation of tumor-favoring microenvironments in distant organs can be forged 8 . These specific microenvironments are termed “pre-metastatic niches” (PMNs). PMNs are characterized by abnormal angiogenesis, extracellular matrix remodeling, and immunosuppression, which allows tumor cells to colonize and rapidly proliferate in distant organ 9 . A growing number of reports indicate that tumor-derived exosomes contribute to tumor metastasis by directly enhancing the tumor malignant phenotype and the formation of the tumor pre-metastatic niche 10 . Exosomes are small extracellular vesicles that are mainly distributed in size between 30–150 nm, secreted by all kinds of living cells. The phospholipid bilayer of exosomes protects their cargo from enzymatic degradation. Through direct fusion, phagocytosis, micropinocytosis, and receptor-mediated uptake, exosomes enter the recipient cell and transfer multiple nucleic acids, proteins, and lipids contained in the recipient cells to exercise long-distance cell-cell communication 11 . Our previous study revealed that hypoxia promotes exosome secretion and cancer metastasis. In this study, we found that colorectal carcinoma cells under hypoxia secrete exosomes containing mutated KRAS proteins, which are taken up by Kupffer cells and facilitate their M2-like polarization to modify the liver microenvironment and accelerate tumor metastasis via AKT signaling. 2. Materials and Methods 2.1 Cell lines and cell culture Mouse macrophage cell line RAW264.7, human colorectal cancer cell lines LoVo, HCT116, and COLO205 were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). Mouse colorectal cancer CT26 cells were purchased from Fenghui (Changsha, China). LoVo，HCT116，COLO205 and CT26 were maintained in RPMI1640 complete medium (Gibco, USA), RAW 264.7 were maintained in DMEM complete medium (Gibco, USA). The complete culture medium contained 10% fetal bovine serum (PAN, German) 4mM L-glutamine (Beyotime, China), penicillin(100u/ml) and streptomycin (100μg/ml). Before exosome isolation, regular culture media were removed and replaced with a complete culture medium supplemented with 10% exosome-depleted fetal bovine serum. Exosome-depleted FBS was prepared by 16h, 120,000g ultra-centrifugation at 4℃. CT26 cells were cultured in RPMI1640 containing 10% FBS to 70% confluence, washed twice with PBS, replaced with RPMI1640 exosome-depleted complete medium, transferred to a triple-gas incubator, and incubated for 48 h at 1% oxygen concentration and 5% CO 2 , and the cell culture supernatant was harvested. The RAB27 stable knockdown cell line was constructed using lentiviral transfection (Genchem Company Ltd., China). The antibodies and auxiliary reagents used are listed in Supplementary Table 1. 2.2 Isolation of exosomes Ultracentrifuge tubes were treated overnight with sodium hypochlorite solution prior to use to remove potential LPS contamination. The cell culture supernatant was centrifuged 3 times with different parameters to remove dead cells, cell debris, and large extracellular vesicles. First, the supernatant was centrifuged at 350 × g for 5 min and the precipitate was discarded. Secondly the supernatant was then centrifuged at 2000 × g for 10 min, and the precipitate was discarded. Thirdly the supernatant was centrifuged at 10000 g for 1 h and the precipitate was discarded. All centrifugations were performed at 4℃. At this point, the pretreated supernatant can be stored at -80℃ for 1 month. Exosomes were isolated by ultracentrifugation at 100,000g for 1h. The supernatant was carefully removed and the exosomes were washed by adding at least 15 mL of sterile PBS to resuspend the exosomes. After ultracentrifugation at 100,000g for 1h, the exosomes were resuspended in 50-200 μL sterile PBS for further experiments. Nanoparticle tracking analysis was performed using a Viva Cell (Shanghai, China). 2.3 Exosome fluorescent labeling DIR fluorescent labeling is suitable for in vivo imaging. DIR labeling was performed according to the manufacturer's instructions. Briefly, DIR dye was added to the exosome suspension to a final concentration of 25 μg/mL and incubated at 37°C for 20 min away from light. Sterile PBS (20 ml of sterile PBS was added to terminate the reaction and the exosomes were centrifuged and set aside. PKH26 provides a highly specific and long-lasting fluorescent signal. PKH26 labeling was performed according to the manufacturer's instructions by mixing equal volumes of exosome suspension and PKH working solution for 10 min and then adding 10 ml of sterile complete medium to terminate the reaction. The labeled exosomes were separated by ultracentrifugation. GFP-labeled exosomes have a fine microscopic morphology but are vulnerable to fluorescence bursts and are not fixable. After the overexpression of the CD63-GFP fusion protein in tumor cells, the corresponding exosomes were labeled with GFP. 2.4 Flow Cytometry To digest tissue or in vitro cultured cells into a single cell suspension, the cell density was adjusted to 1x10 6 /100μL, antibody titrated to optimize the amount of fluorescent antibody, allowed to stand on ice for 15-20 min away from light, washed three times with PBS to resuspend, and then detected by flow cytometry. When the target protein was phosphorylated, the cell samples were fixed with 4% paraformaldehyde at room temperature for 20 min and then permeabilized at -20°C for more than 2 h using a final concentration of 90% pre-cooled methanol. The subsequent steps are identical. 2.5 Western blot Cells were lysed in RIPA buffer (Beyotime, China) and subjected to ultrasound lysis for 30s to obtain whole-cell lysis. After 12000 g centrifugation for 15 min, the precipitate was discarded, and the protein concentration was determined by BCA assay. The protein sample was complete with 5X loading buffer (Beyotime, China) and boiled at 100℃or 10min. The protein samples were run on a 7.5-12.5% gel and transfer to a 0.45μM PVDF membrane (Millipore, USA). The membrane was blocked with 5% skimmed milk for 1h at room temperature. And the membrane was incubated with primary antibody at 4℃or overnight. The membrane was washed 3 times with TBST and incubated with a secondary antibody at room temperature for 1h. The results were visualized using a Bio-Rad ChemiDot Imagine system. 2.6 Enzyme-linked immunosorbent assay (Elisa) ELISA was used to detect exosomes (299-77603, WAKO, Japan) and TGF-beta1 (CHE0029,4A Biotech, China) in the cell culture supernatants. Briefly, cell culture supernatant was added to the ELISA plate, incubated for 2h, washed with wash buffer, and biotin-labeled primary antibody was added, followed by three washes and incubation with secondary antibody, followed by a 10 min reaction with color development solution. The reaction was terminated with a stop solution, and absorbance was measured immediately. The concentration of cytokines was calculated according to the standard curve and the dilution of the supernatant. Before using the kit for exosome concentration, dead cells, cell debris, and large extracellular vesicles were removed from the sample according to the method described above. Before testing for TGF-beta1, the cell supernatant was activated with HCl, as described in the kit instructions. 2.7 Animal experiments Male BALB/c and BALB/c nude mice (male, 4-5 weeks old, each weighing 23-28 g) were provided by the Experimental Animal Center of Chongqing Medical University (Chongqing, China). Humane care guided by the National Institutes of Health was provided to all animals. The protocols used in this study were evaluated and approved by the Animal Use and Ethics Committee of the 2nd Affiliated Hospital of Chongqing Medical University (2018–2021). (1) The spontaneous metastasis model simulates colorectal cancer metastasis under spontaneous conditions. CT26 cell masses were first prepared by subcutaneous injection of 10 6 CT26-NC or CT26 Rab27a KD cells into male nude mice. Approximately one week later, mice were euthanized, and the masses were dissected sterilely and divided to 0.1 cm 3 . After anesthetizing the nude mice, an incision was made in the abdomen, and the tumor mass was adhered to the sigmoid wall using biologic glue (3M, USA). Mice were stitched together and observed continuously. (2) An experimental liver metastasis model was used to assess the suitability of the liver environment for tumor growth. In brief, after anesthetizing the nude mice, an incision was made under the left rib, the spleen was dragged out, and 2 × 10 6 CT26-luc cells were injected along the long axis of the spleen using a 22G syringe for 5 min. The mice were stitched up, and their status was monitored continuously. (3) Vivo imaging: Preparation of luciferase substrate working solution: Dissolve D-luciferin potassium salt in sterile PBS at a final concentration of 15 mg/mL, mix well, and store immediately at -80°C for no longer than 6 min; or -20°C for no longer than 1 min. Intraperitoneal injection of luciferase substrate working solution in mice at a dose of 10 μL/g; wait 10 min. After 5 min of isoflurane gas anesthesia, the mice were placed in the prone position, exposed in bioluminescence mode on a small animal imager, and images were recorded. 2.8 mRNA sequncing and bioinformation analysis mRNA sequencing was performed using Majorbio (Shanghai, China). To identify DEGs (differentially expressed genes) between two different samples, the expression level of each transcript was calculated based on transcripts per million reads (TPM). The expression level of each transcript was calculated based on the transcripts per million reads (TPM). RSEM (http://deweylab.biostat.wisc.edu/rsem/) was used to quantify the gene abundance. Basically, differential expression analysis was performed using DESeq2/DEGseq/EdgeR with Q-values ≤ 0.05, log2FC|>1 for DEGs and Q-values <=0.05 (DESeq2 or EdgeR)/Q-value <= 0.001 (DEGseq) were considered significantly different (expressed genes). Functional enrichment analyses, including GO and KEGG analyses, were also performed. Functional enrichment analysis, including GO and KEGG, was performed to determine the DEGs that were significantly enriched in GO terms and metabolic pathways. DEGs were significantly enriched in GO terms and metabolic pathways at Bonferroni-corrected P-values ≤0.05 compared to the whole transcriptome background. GO functional enrichment and KEGG pathway analyses were performed using Goatools (https://github.com/tanghaibao/Goatools) and KOBAS (http://kobas.cbi.pku.edu.cn/home.do), respectively. Methods: Bioinformatics analysis and mapping were performed using the Majorbio online platform. 2.9 Immunohistochemistry Mice were euthanized by injecting an overdose of anesthetic. The right ear of the heart was immediately cut open and 20-30 mL of 4% paraformaldehyde was perfused into the apical part of the heart. The sections were then dewaxed three times in xylene, hydrated in gradient alcohol, placed in boiling sodium citrate buffer at pH 6.0, and maintained in a microwave oven at medium-low heat for 15 min in a subsoiling state for antigen retrieval. After the sections were cooled naturally in the buffer, they were washed three times with PBS, incubated in a 3% H2O2 solution for 25 min, and then washed three times with PBS. Subsequently, 5% normal goat serum was added dropwise for 1 h. After shaking off the goat serum, diluted primary antibody was added dropwise and incubated overnight at 4°C in a wet box. After incubation for at least 8 h, the secondary antibody was washed 3 times with PBS and incubated at room temperature for 1 h. After washing three times with PBS, freshly prepared DAB color development solution was added dropwise and observed under a microscope. The color development was terminated by timely rinsing with tap water. Hematoxylin was re-stained for 3 min and then rinsed with running water, and the blue color was returned by differentiation in hydrochloric acid ethanol differentiation solution. The sections were sequentially dehydrated in gradient alcohol, sealed with neutral resin after xylene transparency, and observed under a microscope. 2.10 Immunofluorescence Assay Tissue fixation, dehydration, embedding, sectioning, antigen retrieval, and sealing were performed in the same way as previously described for immunohistochemistry. Two primary antibodies of different species, CD163(1:100) and F4/80 (1:400), were simultaneously diluted in antibody diluent and incubated dropwise on the tissue overnight at 4°C. The secondary antibodies with different reactivities were diluted simultaneously in a secondary antibody diluent and incubated dropwise at room temperature for 90 min. After four washes with PBS, the sections were placed in 1% ethanolic solution of Sudan Black 3 B to remove tissue autofluorescence. The sections were then washed three times with PBS, sealed with an anti-fluorescent bursting agent containing DAPI, and observed under a fluorescence microscope. 2.11 Isolation of human monocytes and induction of HMDMs Peripheral blood collected from healthy donors was diluted 1:1 using the dilution solution in the kit, and cells were isolated according to the instructions in the Human Monocyte Isolation Kit (Boster, China). The cell layer on the lower side of the plasma was aspirated into serum-free 1640 basal medium in a CO2 incubator for 45 min. After washing thrice with cold PBS, the attached cells were human peripheral blood mononuclear cells. Human peripheral blood mononuclear cells were maintained in RPIM 1640+10% FBS with human recombinant M-CSF (NOVO protein, China) at a final concentration of 20ng/mL for 6-7days to differentiate into macrophages. 2.12 Statistical analysis The data were analyzed using GraphPad Prism 8.5. A two-tailed t-test was performed for two sets of normally distributed data. Data are expressed as mean ± standard deviation, unless otherwise specified. Statistical significance was set at P < 0.05. 3. Results 3.1 Identification of CRC derived exosomes Differential centrifugation followed by ultracentrifugation is the most common method for isolating exosomes from a large volume of the culture medium supernatant. To verify the purity of the exosomes, we used transmission electron microscopy to obtain the morphology of CRC cell line-derived exosomes. A typical morphology of exosomes was observed (Figure S1A). And the Nanoparticle tracking analysis (NTA) also confirmed that the particle size of these vesicles was mainly concentrated around 130 nm (Figure S1B)，which is in accordance with previous reports 12 . More importantly, we lysed these exosomes and examined their protein expression, while whole cell lysate (WCL) from CRC cell line served as a control, and our results confirmed that these vesicles expressed the exosome markers CD63, PDCD61P (ALIX), and TSG101, while the organelle marker protein calnexin ruled out organelle contamination 13 (Figure S1C). 3.2 CT26 derived exosomes promote CRC liver metastasis To confirm the involvement of exosomes in tumor metastasis in vivo, we utilized a mouse model of spontaneous colorectal cancer metastasis 14 . To regulate exosome production in CRC cells, we focused on RAB27a, which is a positive regulator of exosome biosynthesis 15 . A stable RAB27a knockdown CT26 cell line was established using lentivirus transduction. (Figure 1A). To minimize the inaccuracy associated with exosome isolation, we directly measured the concentration of exosomes in the culture supernatant of the CT26 cell line using NTA and ELISA. We found that RAB27a knockdown resulted in a 6-fold decrease in exosome secretion in CT26 cells. (Figure 1B and C). To determine the involvement of exosomes in liver metastasis and to avoid the rejection effect of xenotransplantation, we took advantage of a murine colorectal cancer cell line. CT26 and CT26 tumor masses were cultured and implanted into the sigmoid wall of BALB/c-nude mice. We found that CT26-Rab27a KD cells generated considerably less liver metastatic burden than CT26-NC cells, as demonstrated by vivo bioluminescence imaging and measurement of liver weight, the increased liver weight is due to the metastatic burden of tumors. (Figure 1D, E and F). This suggested that the effect of exosome-enhanced metastatic ability was partially abolished. Interestingly, Rab27-KD resulted in a larger primary tumor volume. To test whether exosomes promote metastasis by affecting the carcinoma cells themselves, we performed a series of classical tumor phenotype assays in vivo and in vitro and found that RAB27A knockdown affected the proliferation and apoptosis of CT26 cells (Figure S2A). The migration and invasion capacity in vitro were minimally affected. The tumorigenicity of CT26 cells also did not change significantly, as determined by the subcutaneous tumor assay (Figure S2B). Taken together, CT26-derived exosomes indeed contribute to colorectal cancer liver metastasis, possibly by modifying the hepatic microenvironment to make it more suitable for tumor cell growth. 3.3 Hypoxia CT26 exosomes increase liver metastatic burden in experimental liver To investigate the direct effect of CRC-derived exosomes on liver metastasis, we utilized an experimental liver metastasis model 16 , which assesses the suitability of the hepatic microenvironment for tumor growth, which generates immediate liver metastasis. We isolated exosomes from the CT26 culture supernatant and treated BALB/c-nude mice by injecting 10 μg CT26 derived exosomes into the tail vein once every other day, and after 10 times (21 d), we injected 2x10 6 CT26-luciferase cells into the mouse spleen under direct vision. After 14d (35d after the first exosome injection), we assessed the liver metastatic burden by in vivo bioluminescence imaging and measurement of liver weight (Figure 2A). Interestingly, we found no statistically significant difference between the liver metastatic burden of the CT26-N-EXO-treated and control groups (Figure 2B). Owing to the rapid proliferation of tumor cells and abnormal blood vessel structure within tumors, hypoxia is one of the most common features of the tumor microenvironment 6,7 . We hypothesized that the hypoxic microenvironment within the primary tumor may play a crucial role in this process. We further cultured CT26 cells under hypoxic conditions, isolated hypoxic CT26-derived exosomes (CT26-H-EXO) and treated BALB/c-nude mice with equal amounts of exosomes. Notably, we found that CT26-H-EXO treatment significantly increased the biofluorescence intensity in the liver region and the liver weight in the model of experimental liver metastasis (Figure 2C), suggesting that hypoxic CRC-derived exosomes had a greater ability to promote liver microenvironment remodeling and cause higher metastatic burden. Therefore, we used hypoxic exosomes in all subsequent experiments. 3.4 Kupffer cells are the main cellular component of exosome ingestion To further understand the biological function of hypoxic exosomes in colorectal cancer, we traced the distribution of CT26-H-EXO in vivo by optimizing multiple fluorescent labeling schemes. DIR is a far-IR lipophilic dye that offers low-background in vivo imaging 17 . To visualize the organ distribution of CRC-exosomes, we injected 50 μg of DIR-labeled CT26-H-EXO into the tail vein of BALB/c mice and performed in vivo imaging 24 h later. We found that the liver was the main organ uptake of CT26-H-EXO (Figure 3A). To exclude the influence of anatomical structures on exosome uptake, we injected equal amounts of DIR-labeled CT26-H-EXO into four different groups: tail vein injection (TV), retro-orbital injection (RO), and trans-spleen injection (SP) into three groups of BALB/c mice; the PBS group was used as a control. Among them, tail vein injection and retro-orbital injection allow exosomes to enter the body circulatory system, while trans-spleen injection causes exosomes to enter the portal vein first 18 , which is more similar to the process of in situ tumor release of exosomes. As shown in Figure 3B, irrespective of the injection method, the mouse livers in all groups exhibited the highest fluorescence intensity, demonstrating the tropism of CRC-derived exosomes in the liver. To determine which cellular component uptakes the CRC-derived exosomes, we took advantage of PKH26 labeling. Compared with DIR labeling, PKH26 labeling showed better specificity in previous reports 19 . After 24h of injection of 50 μg of PKH26-labeled CT26-H-EXO, we examined liver single-cell suspensions via flow cytometry, and F4/80 antibody was used to distinguish macrophages. As shown in Figure 3C, macrophages dominated the liver cellular components that engulf exosomes, consistent with F4/80+ and F4/80+ CD11b int cells 20,21 . Liver-resident macrophages, also known as Kupffer cells (KCs), are essential cellular components in liver homeostasis and have been proven to engulf tumor-derived exosomes and mediate the establishment of a pro-metastasis niche 16,22 . To obtain a direct morphology of KC uptake by CRC-derived exosomes, we overexpressed CD63-GFP recombinant protein in CT26 cells to obtain GFP-expressing CT26 exosomes 23 . After co-culture of GFP-expressing CT26 exosomes with primary mouse KCs for 24h, we confirmed to KC engulfment of CT26-H-exosomes by laser confocal imaging. Similarly, we found that human monocyte-derived macrophages also take up hypoxic exosomes derived from human CRC cell lines (Figure 3D). 3.5 CT26-H-EXO treatment increased Kupffer cells numbers and activated towards M2-like phenotype To investigate the underlying mechanism, mRNA sequencing was performed on livers derived from the PBS-treated group and CT26-H-EXO treated group derived livers (Figure 4A). Statistical analysis of differentially expressed genes showed that 21day of CT26-H-EXO treatment upregulated 1996 genes. GO analysis also indicated that macrophages were the most affected cellular component, and neutrophils and fibroblasts showed varying degrees of functional activation. Macrophages, including Kupffer cells, perform biological functions by secreting cytokines. Hence, we focused on the most obvious differential expression of various soluble cytokines associated with macrophage M2-like polarization (Figure 4B), such as IL-10, IL-8, TGFB3, and MIP-1, suggesting that the KCs phenotype may be skewed towards M2-like polarization. As shown by our IHC results (Figure 4C), the expression of the macrophage M2 polarization marker CD163 was significantly higher than that in the PBS-treated group. Consistently, as shown in Figure 4D, immunofluorescence confirmed that the number of F4/80-positive cells and expression of CD163 were elevated. 3.6 Hypoxia treatment increased KRAS abundance in CRC derived exosomes Next, we performed KEGG pathway analysis for the 2396 differentially expressed genes. As shown in Figure 5A, the AKT and MAPK pathways showed the most significant enrichment. Previous data suggested that mutated KRAS protein constitutively mediates AKT and MAPK activation signaling in carcinoma cells to induce a malignant phenotype. KRAS is reported to be one of the most frequent mutations in CRC (Figure 5B). We selected three human CRC cell lines, HCT116 and LoVo, harboring the KRAS G13D mutation, while colo205 was used as the wild-type control. We further confirmed KRAS G13D expression using western blotting with a KRAS G13D-specific antibody (Figure 5C). Similar to oxidative stress 24 , we found that KRAS G13D expression was considerably higher in tumor exosomes under hypoxic culture conditions, whereas KRAS G13D was rarely detectable in normoxic CRC-derived exosomes. To prove that KRAS-mutated proteins can be transmitted to macrophages via exosomes, we used KRAS G13D-FLAG fusion protein-coding plasmids. KRAS G13D-FLAG and CD63-GFP coding plasmids were co-transfected into CT26 cells to generate CD63-GFP and KRAS G13D-FLAG expressing CT26-H-EXO. As shown in Figure 5D, when the murine macrophage cell line RAW264.7 was co-cultured with CD63-GFP and KRAS G13D-FLAG expressing CT26-H-EXO, the confocal-laser images showed exosomes and KRAS mutated proteins in the RAW cells, indicating that KRAS G13D mutated protein can be transmitted to macrophages. 3.7 Human CRC hypoxia exosomes induce HMDMs M2-like polarization To address the effects of human CRC exosomes on macrophages. We used an in vitro macrophage model, human monocyte-derived macrophages (HMDMs). Human monocytes were differentiated into macrophages by incubating with 20ng/mL M-CSF for 7d, confirmed by the morphology and ICC results of CD68 expression. HMDMs were co-cultured with equal doses of HCT116 or LoVo-derived hypoxic exosomes (HCT116-H-EXO, LoVo-H-EXO) for 48h. Consistent with our previous mRNA sequencing results, the expression of M2 markers (CD163 and TGF-β) in HMDMs was dramatically upregulated compared to that in the PBS group (Figure 6A). In addition, to investigate the direct effect of KRAS G13D mutated protein, we further exogenously expressed KRAS G13D mutation protein in HMDMs. Compared to GFP coding plasmid, KRAS G13D mutation protein overexpression significantly induced the M2-like polarization of HMDMs (Figure 6B and C). This finding suggests that the KRAS G13D mutation directly promotes macrophage M2-like polarization. 3.8. Inhibition of AKT signaling reduces KCs M2-like polarization and alleviates CRC liver metastasis In our previous study, we found that phagocytosis-induced Kupffer cell M2 polarization involves AKT signaling dependent 25 , and AKT has been acknowledged as a crucial mediator of macrophage survival and polarization 26 . The Protein-Protein Interaction Database string revealed several interactions between KRAS proteins and the AKT pathway. To probe the involvement of AKT signaling, we examined AKT phosphorylation using phosphoprotein flow cytometry, as shown Figure 6B, the phosphorylation of AKT was significantly increased in HMDMs incubated with hypoxic exosomes for 24h, compared with the PBS group. As shown in Figure 6A, after inhibiting AKT phosphorylation with 1μM GSK690693, the expression of CD163 in HMDMs and TGF-β secretion was significantly decreased compared with co-culture with hypoxic CRC-derived exosomes alone. This finding suggests that the administration of AKT inhibitors may be a prophylactic approach to prevent liver metastasis. To demonstrate the viability of this approach, we performed in vivo experiments, as shown in Figure 7A. To avoid the direct tumor suppressive effect of the AKT inhibitor, GSK690693 was only used in the exosome treatment stage (21d), followed by spleen injection of 10 6 CT26 cells to evaluate liver metastatic burden. As shown in Figure 7B and 7D, the inhibition of AKT by GSK690693 significantly depleted the expression of both F4/80 and CD163, suggesting a decrease in the number of KC cells and their M2-like polarization. By weighing the liver, we found that the administration of GSK690693 reduced the liver metastatic burden, especially the macrometastatic tumor nodules (Figure 7C). Taken together, our results demonstrated that hypoxic CRC-derived exosome KRAS mutation protein facilitates Kupffer cell M2-like polarization via AKT signaling. Discussion Tumor metastasis is a complex multifactorial process, in which tumor cells leave the primary site, enter the circulatory system, and survive and proliferate in distant organs. The survival and proliferation of tumor cells at the metastasis site is undoubtedly the core aspect that determines whether tumor macrometastasis eventually occurs 27 . According to the seed and soil hypothesis, as first proposed by Paget, the relationship between tumor cells and organ stromal cells is similar to that between seed and soil, and metastasis only develops if both seed and soil are compatible. The liver is one of the organs most liable for tumor metastasis 10 , 28 , and some studies have suggested that the pre-metastatic microenvironment may promote liver metastasis in numerous types of tumors 10 . Pre-metastatic niche is a tumor favorable microenvironment in distant organs, created by primary tumors for subsequent metastases In recent years, an increasing number of studies demonstrated that tumor-derived exosomes are involved in the development of tumor metastasis, by promoting carcinogenesis of normal cells, enhancing the ability of migration and invasion in tumor cells 29 , enhancing stemness and promoting cell proliferation 30 . In addition, it fosters a microenvironment suitable for tumors by increasing vascular angiogenesis and permeability 31 , 32 , inhibiting immune surveillance 33 , 34 , and remodeling the extracellular matrix 16 , 35 , 36 . It is well known that macrophages are the predominant stromal cells in primary tumor environments 37 . Depending on the signaling molecules and environmental stresses received, macrophages are activated in two different ways: classical activation (M1-type polarization) and alternative activation (M2-type polarization). M1-type macrophages induce inflammatory and anti-tumor responses by secreting pro-inflammatory cytokines 38 , while alternative-type activation secretes anti-inflammatory cytokines to inhibit excessive inflammatory responses and promote tissue repair 39 . Among them, tumor-associated macrophages (TAM) exhibit an M2-like polarization pattern that promotes tumor cell migration and metastasis 40 , and the liver has the highest abundance of macrophages 41 . Our exosome tracing experiments also demonstrated the organophilicity of colorectal cancer-derived exosomes to the liver, and Zhang et al. also reported that the hepatic synthesis of complement C1q promotes phagocytosis in KCs, which might explain why the liver is the main organ in exosomes 42 . In contrast, a study by Shao et al. found that exosomes of breast cancer have a higher affinity to the lung than colorectal cancer-derived exosomes 14 , and a study by Moller et al. also demonstrated that the liver is the major uptake organ of exosomes in pancreatic ductal carcinoma 43 . The specific tropism of these tumor exosomes coincides with tumor-prone metastatic organs, which is further evidence suggesting that tumor exosomes may play a key role in tumor metastasis. In the present study, we found an increase in M2-like polarized KCs after treatment with hypoxic CRC-derived exosomes. In contrast, Shao et al. developed a xenogeneous model by treating mice with human CRC exosomes, and their experimental results demonstrated that human colorectal carcinoma cell-derived exosomes have pro-inflammatory effects on the mouse macrophage cell line RAW264.7, while human-derived colorectal carcinoma cell-derived exosomes ultimately increased liver metastatic burden by promoting IL6 pro-inflammatory factor IL6 secretion in mouse KCs 22 . Hao et al. utilized a patient-derived xenograft model to reveal that hypoxic exosomes containing miR-135-5p promote CRC liver metastasis via P65 immunosuppression signaling 44 . Cooks et al. demonstrated that P53 mutant colorectal carcinoma cell-derived miR-1246 is transferred via exosomes to macrophages in the CRC primary tumor microenvironment, promoting their M2-like polarization and liver and lung metastasis 45 . Recently, it was reported that macrophages are not the only target cell components. Zeng et al. found that exosomes secreted by human colorectal carcinoma cell lines carrying miR-25-3p were taken up by the vascular endothelium in the liver and lung to increase vascular permeability by affecting the tight junctions between endothelial cells and enhancing angiogenesis to support tumor cells for colonization and proliferation 31 . Tian et al. found that liver carcinoma-derived exosomes transfer miR-1247-3P to lung fibroblasts, induce pro-inflammatory cytokine release, and further promote cancer lung metastasis 46 . KRAS is the most frequently mutated proto-oncogene in human solid tumors, and KRAS mutations have been detected in up to 85% of metastatic colorectal carcinoma 47 . Furthermore, G13D mutations are mainly found in colorectal cancer 48 . This mutated protein enhances the malignant biological behavior of tumor cells and contributes to tumor progression by promoting tumor cell proliferation, migration, and invasion, and anti-apoptosis effects via AKT and MAPK signaling 49 , 50 . Recently, ISCHENKO et al. demonstrated by single-cell sequencing that KRAS mutation activates the RAF pathway to promote the secretion of multiple cytokines to establish an immunosuppressive microenvironment in pancreatic cancer, while knockdown of KRAS mutations resulted in enhanced T-cell anti-tumor immunity 51 . Beckler et al. found by mass spectrometry that mutated KRAS proteins could be detected in the exosomes of the HCT116 cell line, and KRAS protein can be transferred to non-cancerous normal cells 52 . Dai et al. found that oxidative stress-induced secretion of pancreatic adenocarcinoma-derived exosomes is enriched with KRAS-mutated proteins, which are taken in by tumor-associated macrophages in the primary tumor microenvironment, supporting the M2-like polarization of tumor cells via fatty acid oxidation metabolism 24 . Due to the pro-cancer activity of mutant KRAS proteins, a small molecule inhibitor of KRAS G12C-type mutations, entering phase I clinical trials in recent years, this genotype-specific inhibitor is not effective in most CRC patients 50 . In the present study, we established two vivo models to validate the participation of CRC derived exosomes in liver metastasis we confirmed hypoxia exosomes has greater pro-metastasis ability compared with normoxia exosomes. Exosome tracing experiments and mRNA sequencing revealed that the key regulator in the treatment of hypoxic CRC-derived exosomes is liver macrophages (KCs), and that treatment with hypoxic CRC exosomes upregulates the KCs population and M2-like polarization. Moreover, bioinformatics analysis indicated that KRAS-mutated proteins and AKT cell pathways mediate macrophage M2 polarization. Since KRAS protein is a nearly non-druggable target, we successfully reversed the pro-tumor metastatic effect of KRAS mutant CRC-derived exosomes by administering AKT inhibitors. Overall, our study determined the role of exosome mutated KRAS-induced AKT signaling in the process of KCs M2-like polarization, AKT inhibitors, and might potentially be used as a therapeutic approach to prevent liver metastases in colorectal carcinoma. Declarations Acknowledgments This project was supported by the National Science Foundation of China (No. 81170442, 81470899, 81702357, 82070678), Chen Xiao-ping foundation for the development of science and technology (No. CXPJJH12000001-2020330), Kuanren Talents Program of the second affiliated hospital of Chongqing Medical University (No. kryc-yq-2208), Chongqing Natural Science Foundation (CSTB2023NSCQ-MSX0150) and Chongqing Municipal Science and Health Joint Medical Research Project (2024QNXM003). Author Contributions Conceived and designed the study: Zuojin LIU Performed the experimental procedures: Zhihao FENG, Jiao LU, Hua SONG, Fuyao LIU Analyzed the data: Jie XU, Ke YOU, Tianzhu WU Drafted the manuscript: Zhihao FENG, You YU Conflicts of interest None. Funding This project was supported by the National Science Foundation of China (No. 81170442, 81470899, 81702357, 82070678), Chen Xiao-ping foundation for the development of science and technology (No. CXPJJH12000001-2020330), Kuanren Talents Program of the second affiliated hospital of Chongqing Medical University (No. kryc-yq-2208), Chongqing Natural Science Foundation (CSTB2023NSCQ-MSX0150) and Chongqing Municipal Science and Health Joint Medical Research Project (2024QNXM003). Data Availability Statement: The datasets generated and analysed during the current study are not publicly available due ethical, privacy or security reasons, but are available from the corresponding author on reasonable request. References Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA: A Cancer Journal for Clinicians. 2020;70(1):7-30. GY L, S H, J H, et al. ASCO 2006 update of recommendations for the use of tumor markers in gastrointestinal cancer. 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H W, X X, J L, J G, M L. TIM‑4 blockade of KCs combined with exogenous TGF‑β injection helps to reverse acute rejection and prolong the survival rate of mice receiving liver allografts. International journal of molecular medicine. 2018;42(1):346-358. Zhang G, Huang X, Xiu H, et al. Extracellular vesicles: Natural liver-accumulating drug delivery vehicles for the treatment of liver diseases. Journal of extracellular vesicles. 2020;10(2):e12030. Liu Y, Gu Y, Han Y, et al. Tumor Exosomal RNAs Promote Lung Pre-metastatic Niche Formation by Activating Alveolar Epithelial TLR3 to Recruit Neutrophils. Cancer cell. 2016;30(2):243-256. Sun H, Meng Q, Shi C, et al. Hypoxia-inducible exosomes facilitate liver-tropic pre-metastatic niche in colorectal cancer. Hepatology (Baltimore, Md). 2021. T C, IS P, LM J, et al. Mutant p53 cancers reprogram macrophages to tumor supporting macrophages via exosomal miR-1246. Nature communications. 2018;9(1):771. Fang T, Lv H, Lv G, et al. Tumor-derived exosomal miR-1247-3p induces cancer-associated fibroblast activation to foster lung metastasis of liver cancer. Nature communications. 2018;9(1):191. Lee H, Son E, Lee K, et al. Promising Therapeutic Efficacy of GC1118, an Anti-EGFR Antibody, against KRAS Mutation-Driven Colorectal Cancer Patient-Derived Xenografts. International journal of molecular sciences. 2019;20(23). Peng N, Zhao X. K-rasComparison of mutations in lung, colorectal and gastric cancer. Oncology letters. 2014;8(2):561-565. Mustachio L, Chelariu-Raicu A, Szekvolgyi L, Roszik J. KRASTargeting in Cancer: Promising Therapeutic Strategies. Cancers. 2021;13(6). Hong D, Fakih M, Strickler J, et al. KRAS Inhibition with Sotorasib in Advanced Solid Tumors. The New England journal of medicine. 2020;383(13):1207-1217. Ischenko I, D'Amico S, Rao M, et al. KRAS drives immune evasion in a genetic model of pancreatic cancer. Nat Commun. 2021;12(1):1482. M DB, JN H, JL F, et al. Proteomic analysis of exosomes from mutant KRAS colon cancer cells identifies intercellular transfer of mutant KRAS. Molecular & cellular proteomics : MCP. 2013;12(2):343-355. Supplementary Table 1 Supplementary table 1 is not available with this version. Additional Declarations No competing interests reported. Supplementary Files WB.doc SupplementoryFigures.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6300438","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":434368281,"identity":"617e5f9e-01c1-49ff-ae65-a49412ac35a0","order_by":0,"name":"Yu You","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"You","suffix":""},{"id":434368282,"identity":"48cfa46d-56fd-447d-928e-3ec948b7351e","order_by":1,"name":"Zhihao FENG","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhihao","middleName":"","lastName":"FENG","suffix":""},{"id":434368283,"identity":"32fbce06-4abb-4082-88a7-b462a4975b79","order_by":2,"name":"Jiao LU","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiao","middleName":"","lastName":"LU","suffix":""},{"id":434368284,"identity":"9d812e49-b51f-4258-9d38-4b11f91310e8","order_by":3,"name":"Jie XU","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"XU","suffix":""},{"id":434368285,"identity":"974322e4-3717-4c1e-9ba3-872520be8f98","order_by":4,"name":"Ke YOU","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"YOU","suffix":""},{"id":434368286,"identity":"9ad79e24-5c8e-4091-8f69-30a943a871ac","order_by":5,"name":"Fuyao LIU","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fuyao","middleName":"","lastName":"LIU","suffix":""},{"id":434368287,"identity":"c930c405-09f6-4d12-8346-ae4e76a84542","order_by":6,"name":"Tianzhu WU","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tianzhu","middleName":"","lastName":"WU","suffix":""},{"id":434368288,"identity":"0909bac0-cdee-4528-a5d8-81c9ea0997f3","order_by":7,"name":"Hua SONG","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"SONG","suffix":""},{"id":434368289,"identity":"73ee1111-fd9e-46f2-867c-91ddc949200e","order_by":8,"name":"Zuojin LIU","email":"data:image/png;base64,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","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Zuojin","middleName":"","lastName":"LIU","suffix":""}],"badges":[],"createdAt":"2025-03-25 05:53:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6300438/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6300438/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80014968,"identity":"05ccdcc1-620f-451c-98f7-64124c3d6111","added_by":"auto","created_at":"2025-04-07 03:04:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":108326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eRab27a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e knockdown decrease liver metastatic burden in the spontaneous liver metastasis model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Expression of \u003cem\u003eRab27a\u003c/em\u003e in CT26-NC and CT26-RAB27a KD were detected by Western Blot. (\u003cstrong\u003eB\u003c/strong\u003e) Relative exosomes concentration of CT26 NC and CT26 were determined by ELISA. n=4 (\u003cstrong\u003eC\u003c/strong\u003e) Particles concentration of CT26-NC and CT26-RAB27a KD culture supernatant were tested by NTA. (\u003cstrong\u003eD\u003c/strong\u003e) Representative VIVO image in spontaneous metastasis model. (\u003cstrong\u003eE\u003c/strong\u003e) Bar chart of luminescence intensity of CT26-NC and CT26-RAB27a KD in spontaneous liver metastasis model (A.U). n=5 (\u003cstrong\u003eF\u003c/strong\u003e) Bar chart of liver weight in spontaneous metastasis model at 28d. n=5 **p＜0.01, ***p＜0.001, ****p＜0.0001\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6300438/v1/7ca05aaef7ed18049f5b24ec.png"},{"id":80014971,"identity":"873e948a-3ed2-404a-827e-3939c72fd558","added_by":"auto","created_at":"2025-04-07 03:04:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":144973,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHypoxia CT26 exosomes increase liver metastatic burden in experimental liver metastasis model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Bioluminescence image of experimental liver metastasis model at 14d, established by spleen injection of CT26-luciferase cells after 21d CT26 exosome or PBS treatment. CT26-N-EXO: CT26-derived exosomes; CT26-H-EXO: hypoxia CT26-derived exosomes (\u003cstrong\u003eB\u003c/strong\u003e) Bar chart of bioluminescence intensity of liver region in experimental liver metastasis model. n=4-5 (\u003cstrong\u003eC\u003c/strong\u003e) Bar chart of liver weight in experimental liver metastasis model. n=5 *P＜0.05,***p＜0.001, ns: no significance\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6300438/v1/03b3265ba155860c1b74016a.png"},{"id":80015669,"identity":"0a65eb56-ad3c-4fde-a2f5-c13a1b3f7d2c","added_by":"auto","created_at":"2025-04-07 03:12:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":253066,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKupffer cells are the dominant cells that take up CT26 hypoxia exosomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Distribution of CT26 hypoxia exosomes was visualized by vivo fluorescence imaging, DIR was used to stain CT26 hypoxia exosomes. (\u003cstrong\u003eB\u003c/strong\u003e) Different injection manners do not affect the distribution of CT26 hypoxia exosomes in vivo, determined by DIR Vivo fluorescence imaging. (\u003cstrong\u003eC\u003c/strong\u003e) Representative flow cytometry plots (left) and quantitative analysis (right) of the subsets of cells taken up by CT26 hypoxic exosomes, and PKH26 labeled CT26 hypoxic exosomes. The proportion of exosomes uptake by F4/80\u003csup\u003e+\u003c/sup\u003e cells, n=9, was calculated from the summary of three experiments. The proportion of CT26 hypoxic exosomes uptake by F4/80\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e-\u003c/sup\u003e cells n=9 (\u003cstrong\u003eD\u003c/strong\u003e) Represented immunofluorescence results of mouse KCs cells uptake of CT26 hypoxia exosomes and human monocyte derived macrophages uptake of hypoxia exosomes from human colon cancer cell lines, CD63-GFP was to label exosomes. \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6300438/v1/1f3840dcca3758bfa3f3738e.png"},{"id":80015668,"identity":"7b807711-c148-42e7-90b4-f07882393edd","added_by":"auto","created_at":"2025-04-07 03:12:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":306741,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCT26-H-EXO treatment increases Kupffer cells numbers and activated towards M2-like phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic timeline of CT26-H-EXO treated mice model. (\u003cstrong\u003eB\u003c/strong\u003e) Bar chart of overall differential mRNA expression in mouse liver, PBS treated mice were used as control. n=3 or 8, pooled from two experiments. Results of GO pathway enrichment on differential expression genes (top5 were shown). Heatmap of cytokines mRNA expression in mice liver. n=3 or 8 (\u003cstrong\u003eC\u003c/strong\u003e) Representative Immunofluorescence in frozen sections of CT26-H-EXO treated livers, PBS group livers were used as control. (\u003cstrong\u003eD\u003c/strong\u003e) Representative IHC staining of Paraffin section of CT26-H-EXO treated livers, and PBS treated mice were used as control. Immunofluorescence quantification in arbitrary units (A.U.) of F4/80 expression in mouse livers. n=7 pooled from two experiments. Immunofluorescence quantification in arbitrary units (A.U.) of CD163 expression in mouse livers. n=7 pooled from two experiments. The data are represented as Mean±SD. *P \u0026lt; 0.05, ****P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6300438/v1/9d8c6490173db767dbefa828.png"},{"id":80015884,"identity":"e91e1ea2-19c7-4294-9ed1-ebf1001c183d","added_by":"auto","created_at":"2025-04-07 03:20:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":187154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHypoxia condition increased KRAS abundance in CRC derived exosomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Overview of conventional signaling pathway enrichment in KEGG analysis of differential expressed genes from mRNA sequencing. (\u003cstrong\u003eB\u003c/strong\u003e) Waterfall plot of CRC mutation generated by Dr. bioRight website from TCGA database. (\u003cstrong\u003eC\u003c/strong\u003e) The effects of hypoxia condition on the expression of KRAS G13D in LoVo derived exosomes and HCT116 derived exosomes were tested using western blotting. H2O2 treatment group was used as positive control, and COLO 205 (KRAS WT) derived exosomes were used as negative control. PDCD61P was used as loading control of exosomes. (\u003cstrong\u003eD\u003c/strong\u003e) Representative fluorescence image of Raw264.7 cells take in exosomal KRAS G13D protein.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6300438/v1/bef5407d85c01bd2f471ee33.png"},{"id":80014973,"identity":"1ff14286-c761-441e-97ef-db80bb5d7174","added_by":"auto","created_at":"2025-04-07 03:04:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":200849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHypoxia CRC exosomes promote M2-like phenotype via AKT signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Flow cytometry of the expressing of CD163 in HMDMs after co-culture with human CRC cell-derived exosomes, with or without GSK690693. HMDMs were co-cultured with HCT116-H-EXO or LoVo-H-EXO, with or without GSK690693 for 48h, concentrate of TGF-β1 was determined by ELISA. n=3-5, pooled from two experiments. (\u003cstrong\u003eB\u003c/strong\u003e) Phosphorylation of AKT in HMDMs after co-culture with human CRC cell-derived exosomes was determined by flow cytometry (left). Bar chart (right) of the median fluorescence intensity (MFI) of P-AKT expression after 10μg LoVo-H-EXO or HCT116-H-EXO treatment, PBS group were used as control. (\u003cstrong\u003eC\u003c/strong\u003e) Expressing of CD163 and GFP in HMDMs after overexpression of KRAS G13D, GFP is the marker of transfected cells, determined by intracellular flow cytometry. GFP coding plasmid was used as control. The concentration of TGF-β1 of HMDMs supernatant after transfection of KRAS G13D coding plasmid or GFP coding plasmid, determined by ELISA. n=5, pooled from three experiments. **p＜0.01,***p＜0.001,****p＜0.0001\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6300438/v1/709d28c4c0c274581e040324.png"},{"id":80015670,"identity":"cab372a4-f5d3-4101-8423-676cb3a08612","added_by":"auto","created_at":"2025-04-07 03:12:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":379690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAKT inhibiting attenuates the tumor burden caused by hypoxia exosome treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A\u003c/strong\u003e) Schematic of GSK690693 administration in CT26-H-EXO treated mice model. (\u003cstrong\u003eB\u003c/strong\u003e) Representative IHC staining of Paraffin section of AKT inhibited CT26-H-EXO treated livers, and CT26-H-EXO treated mouse livers. Nuclei were stained with hematoxylin. M2 polarization marker CD163 was stained with CD163 antibody. Scale bars: 100 μm. (\u003cstrong\u003eC\u003c/strong\u003e) Liver weight of AKT inhibited CT26-H-EXO treated mouse livers and CT26-H-EXO treated mouse livers in experimental liver metastasis model. n=3-4 pooled from two independent experiments. (\u003cstrong\u003eD\u003c/strong\u003e) Representative Immunofluorescence of AKT inhibited CT26-H-EXO treated mouse livers and CT26-H-EXO treated mouse livers, PBS group livers were used as control. Nuclei were stained with DAPI. M2 polarization marker CD163 was stained with CD163 antibody (red). Macrophages were stained with F4/80 antibody (green). Scale bars: 100 μm.**p＜0.01.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6300438/v1/da93023a28185bd564df6f84.png"},{"id":80014976,"identity":"4a818a54-b9f8-46fd-b424-995cc6257eaf","added_by":"auto","created_at":"2025-04-07 03:04:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":220300,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not available with this version.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6300438/v1/d1e497a48ae0a4044f6d57e9.png"},{"id":80014969,"identity":"d56b9722-a0b2-4d89-bf6b-de50762c8c72","added_by":"auto","created_at":"2025-04-07 03:04:01","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2129242,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not available with this version.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-6300438/v1/53c876b6493cf1d9af560221.png"},{"id":90879058,"identity":"33912d3e-8edf-48ac-8692-24ad52f6e447","added_by":"auto","created_at":"2025-09-09 09:24:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4938596,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6300438/v1/2a3affa3-6dfb-4e27-a5ef-6393462f8327.pdf"},{"id":80015675,"identity":"8db4ec9b-5f96-4512-b6fa-a3fdea06c72e","added_by":"auto","created_at":"2025-04-07 03:12:02","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3503616,"visible":true,"origin":"","legend":"","description":"","filename":"WB.doc","url":"https://assets-eu.researchsquare.com/files/rs-6300438/v1/d9e4330fda54bf2aeb25d06f.doc"},{"id":80015883,"identity":"a1f18b96-dcff-4dac-94d9-bf2db4ca6939","added_by":"auto","created_at":"2025-04-07 03:20:01","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":748651,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementoryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6300438/v1/a37af6a8af5ab26d65a34216.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Kupffer cells M2-like polarization increases liver metastatic burden via uptake of exosomal KRAS mutant protein from hypoxia colorectal carcinoma cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is the world\u0026rsquo;s second-deadliest cancer after lung ccancer\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. More than 90% of CRC-related deaths can be attributed to metastatic outgrowth in distant organs\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The liver is the most common organ for CRC metastasis, and patients with liver metastasis have a much poorer prognosis; the 5-year survival rate of untreated CRC patients with liver metastasis is 7\u0026ndash;12%\u003csup\u003e3,4\u003c/sup\u003e. And approximately 60\u0026ndash;80% of CRC patients eventually develop liver metastases\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Even in patients with early stage disease, intrahepatic metastases frequently occur after curative resection, and the underlying mechanisms are not well understood. Although colorectal cancer metastasis is influenced by multiple factors, the significance of hypoxia is well established\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Hypoxia is one of the hallmarks of the tumor microenvironment due to rapid tumor cell proliferation, unique tumor metabolism, and abnormal blood vessel \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Recent studies have revealed that, before the arrival of tumor cells, the formation of tumor-favoring microenvironments in distant organs can be forged\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. These specific microenvironments are termed \u0026ldquo;pre-metastatic niches\u0026rdquo; (PMNs). PMNs are characterized by abnormal angiogenesis, extracellular matrix remodeling, and immunosuppression, which allows tumor cells to colonize and rapidly proliferate in distant organ\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. A growing number of reports indicate that tumor-derived exosomes contribute to tumor metastasis by directly enhancing the tumor malignant phenotype and the formation of the tumor pre-metastatic niche\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eExosomes are small extracellular vesicles that are mainly distributed in size between 30\u0026ndash;150 nm, secreted by all kinds of living cells. The phospholipid bilayer of exosomes protects their cargo from enzymatic degradation. Through direct fusion, phagocytosis, micropinocytosis, and receptor-mediated uptake, exosomes enter the recipient cell and transfer multiple nucleic acids, proteins, and lipids contained in the recipient cells to exercise long-distance cell-cell communication\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Our previous study revealed that hypoxia promotes exosome secretion and cancer metastasis. In this study, we found that colorectal carcinoma cells under hypoxia secrete exosomes containing mutated KRAS proteins, which are taken up by Kupffer cells and facilitate their M2-like polarization to modify the liver microenvironment and accelerate tumor metastasis via AKT signaling.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Cell lines and cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse macrophage cell line RAW264.7, human colorectal cancer cell lines LoVo, HCT116, and COLO205 were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). Mouse colorectal cancer CT26 cells were purchased from Fenghui (Changsha, China). LoVo，HCT116，COLO205 and CT26 were maintained in RPMI1640 complete medium (Gibco, USA), RAW 264.7 were maintained in DMEM complete medium (Gibco, USA). The complete culture medium contained 10% fetal bovine serum (PAN, German) 4mM L-glutamine (Beyotime, China), penicillin(100u/ml) and streptomycin (100μg/ml). Before exosome isolation, regular culture media were removed and replaced with a complete culture medium supplemented with 10% exosome-depleted fetal bovine serum. Exosome-depleted FBS was prepared by 16h, 120,000g ultra-centrifugation at 4℃. CT26 cells were cultured in RPMI1640 containing 10% FBS to 70% confluence, washed twice with PBS, replaced with RPMI1640 exosome-depleted complete medium, transferred to a triple-gas incubator, and incubated for 48 h at 1% oxygen concentration and 5% CO\u003csub\u003e2\u003c/sub\u003e, and the cell culture supernatant was harvested. The RAB27 stable knockdown cell line was constructed using lentiviral transfection (Genchem Company Ltd., China). The antibodies and auxiliary reagents used are listed in Supplementary Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2\u003c/strong\u003e \u003cstrong\u003eIsolation of exosomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUltracentrifuge tubes were treated overnight with sodium hypochlorite solution prior to use to remove potential LPS contamination. The cell culture supernatant was centrifuged 3 times with different parameters to remove dead cells, cell debris, and large extracellular vesicles. First, the supernatant was centrifuged at 350 × g for 5 min and the precipitate was discarded. Secondly the supernatant was then centrifuged at 2000 × g for 10 min, and the precipitate was discarded. Thirdly the supernatant was centrifuged at 10000 g for 1 h and the precipitate was discarded. All centrifugations were performed at 4℃. At this point, the pretreated supernatant can be stored at -80℃ for 1 month. Exosomes were isolated by ultracentrifugation at 100,000g for 1h. The supernatant was carefully removed and the exosomes were washed by adding at least 15 mL of sterile PBS to resuspend the exosomes. After ultracentrifugation at 100,000g for 1h, the exosomes were resuspended in 50-200 μL sterile PBS for further experiments. Nanoparticle tracking analysis was performed using a Viva Cell (Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3\u003c/strong\u003e \u003cstrong\u003eExosome fluorescent labeling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDIR fluorescent labeling is suitable for in vivo imaging. DIR labeling was performed according to the manufacturer's instructions. Briefly, DIR dye was added to the exosome suspension to a final concentration of 25 μg/mL and incubated at 37°C for 20 min away from light. Sterile PBS (20 ml of sterile PBS was added to terminate the reaction and the exosomes were centrifuged and set aside. PKH26 provides a highly specific and long-lasting fluorescent signal. PKH26 labeling was performed according to the manufacturer's instructions by mixing equal volumes of exosome suspension and PKH working solution for 10 min and then adding 10 ml of sterile complete medium to terminate the reaction. The labeled exosomes were separated by ultracentrifugation. GFP-labeled exosomes have a fine microscopic morphology but are vulnerable to fluorescence bursts and are not fixable. After the overexpression of the CD63-GFP fusion protein in tumor cells, the corresponding exosomes were labeled with GFP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Flow Cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo digest tissue or in vitro cultured cells into a single cell suspension, the cell density was adjusted to 1x10\u003csup\u003e6\u003c/sup\u003e/100μL, antibody titrated to optimize the amount of fluorescent antibody, allowed to stand on ice for 15-20 min away from light, washed three times with PBS to resuspend, and then detected by flow cytometry. When the target protein was phosphorylated, the cell samples were fixed with 4% paraformaldehyde at room temperature for 20 min and then permeabilized at -20°C for more than 2 h using a final concentration of 90% pre-cooled methanol. The subsequent steps are identical.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Western blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were lysed in RIPA buffer (Beyotime, China) and subjected to ultrasound lysis for 30s to obtain whole-cell lysis. After 12000 g centrifugation for 15 min, the precipitate was discarded, and the protein concentration was determined by BCA assay. The protein sample was complete with 5X loading buffer (Beyotime, China) and boiled at 100℃or 10min. The protein samples were run on a 7.5-12.5% gel and transfer to a 0.45μM PVDF membrane (Millipore, USA). The membrane was blocked with 5% skimmed milk for 1h at room temperature. And the membrane was incubated with primary antibody at 4℃or overnight. The membrane was washed 3 times with TBST and incubated with a secondary antibody at room temperature for 1h. The results were visualized using a Bio-Rad ChemiDot Imagine system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Enzyme-linked immunosorbent assay (Elisa)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eELISA was used to detect exosomes (299-77603, WAKO, Japan) and TGF-beta1 (CHE0029,4A Biotech, China) in the cell culture supernatants. Briefly, cell culture supernatant was added to the ELISA plate, incubated for 2h, washed with wash buffer, and biotin-labeled primary antibody was added, followed by three washes and incubation with secondary antibody, followed by a 10 min reaction with color development solution. The reaction was terminated with a stop solution, and absorbance was measured immediately. The concentration of cytokines was calculated according to the standard curve and the dilution of the supernatant. Before using the kit for exosome concentration, dead cells, cell debris, and large extracellular vesicles were removed from the sample according to the method described above. Before testing for TGF-beta1, the cell supernatant was activated with HCl, as described in the kit instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Animal experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale BALB/c and BALB/c nude mice (male, 4-5 weeks old, each weighing 23-28 g) were provided by the Experimental Animal Center of Chongqing Medical University (Chongqing, China). Humane care guided by the National Institutes of Health was provided to all animals. The protocols used in this study were evaluated and approved by the Animal Use and Ethics Committee of the 2nd Affiliated Hospital of Chongqing Medical University (2018–2021). \u003c/p\u003e\n\u003cp\u003e(1) The spontaneous metastasis model simulates colorectal cancer metastasis under spontaneous conditions. CT26 cell masses were first prepared by subcutaneous injection of 10 6 CT26-NC or CT26 Rab27a KD cells into male nude mice. Approximately one week later, mice were euthanized, and the masses were dissected sterilely and divided to 0.1 cm\u003csup\u003e3\u003c/sup\u003e. After anesthetizing the nude mice, an incision was made in the abdomen, and the tumor mass was adhered to the sigmoid wall using biologic glue (3M, USA). Mice were stitched together and observed continuously.\u003c/p\u003e\n\u003cp\u003e(2) An experimental liver metastasis model was used to assess the suitability of the liver environment for tumor growth. In brief, after anesthetizing the nude mice, an incision was made under the left rib, the spleen was dragged out, and 2 × 10\u003csup\u003e6\u003c/sup\u003e CT26-luc cells were injected along the long axis of the spleen using a 22G syringe for 5 min. The mice were stitched up, and their status was monitored continuously.\u003c/p\u003e\n\u003cp\u003e(3) Vivo imaging: Preparation of luciferase substrate working solution: Dissolve D-luciferin potassium salt in sterile PBS at a final concentration of 15 mg/mL, mix well, and store immediately at -80°C for no longer than 6 min; or -20°C for no longer than 1 min. Intraperitoneal injection of luciferase substrate working solution in mice at a dose of 10 μL/g; wait 10 min. After 5 min of isoflurane gas anesthesia, the mice were placed in the prone position, exposed in bioluminescence mode on a small animal imager, and images were recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 mRNA sequncing and bioinformation analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003emRNA sequencing was performed using Majorbio (Shanghai, China). To identify DEGs (differentially expressed genes) between two different samples, the expression level of each transcript was calculated based on transcripts per million reads (TPM). The expression level of each transcript was calculated based on the transcripts per million reads (TPM). RSEM (http://deweylab.biostat.wisc.edu/rsem/) was used to quantify the gene abundance. Basically, differential expression analysis was performed using DESeq2/DEGseq/EdgeR with Q-values ≤ 0.05, log2FC|\u0026gt;1 for DEGs and Q-values \u0026lt;=0.05 (DESeq2 or EdgeR)/Q-value \u0026lt;= 0.001 (DEGseq) were considered significantly different (expressed genes). Functional enrichment analyses, including GO and KEGG analyses, were also performed. Functional enrichment analysis, including GO and KEGG, was performed to determine the DEGs that were significantly enriched in GO terms and metabolic pathways. DEGs were significantly enriched in GO terms and metabolic pathways at Bonferroni-corrected P-values ≤0.05 compared to the whole transcriptome background. GO functional enrichment and KEGG pathway analyses were performed using Goatools (https://github.com/tanghaibao/Goatools) and KOBAS (http://kobas.cbi.pku.edu.cn/home.do), respectively. Methods: Bioinformatics analysis and mapping were performed using the Majorbio online platform.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Immunohistochemistry \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were euthanized by injecting an overdose of anesthetic. The right ear of the heart was immediately cut open and 20-30 mL of 4% paraformaldehyde was perfused into the apical part of the heart. The sections were then dewaxed three times in xylene, hydrated in gradient alcohol, placed in boiling sodium citrate buffer at pH 6.0, and maintained in a microwave oven at medium-low heat for 15 min in a subsoiling state for antigen retrieval. After the sections were cooled naturally in the buffer, they were washed three times with PBS, incubated in a 3% H2O2 solution for 25 min, and then washed three times with PBS. Subsequently, 5% normal goat serum was added dropwise for 1 h. After shaking off the goat serum, diluted primary antibody was added dropwise and incubated overnight at 4°C in a wet box. After incubation for at least 8 h, the secondary antibody was washed 3 times with PBS and incubated at room temperature for 1 h. After washing three times with PBS, freshly prepared DAB color development solution was added dropwise and observed under a microscope. The color development was terminated by timely rinsing with tap water. Hematoxylin was re-stained for 3 min and then rinsed with running water, and the blue color was returned by differentiation in hydrochloric acid ethanol differentiation solution. The sections were sequentially dehydrated in gradient alcohol, sealed with neutral resin after xylene transparency, and observed under a microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 Immunofluorescence Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue fixation, dehydration, embedding, sectioning, antigen retrieval, and sealing were performed in the same way as previously described for immunohistochemistry. Two primary antibodies of different species, CD163(1:100) and F4/80 (1:400), were simultaneously diluted in antibody diluent and incubated dropwise on the tissue overnight at 4°C. The secondary antibodies with different reactivities were diluted simultaneously in a secondary antibody diluent and incubated dropwise at room temperature for 90 min. After four washes with PBS, the sections were placed in 1% ethanolic solution of Sudan Black 3 B to remove tissue autofluorescence. The sections were then washed three times with PBS, sealed with an anti-fluorescent bursting agent containing DAPI, and observed under a fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11 Isolation of human monocytes and induction of HMDMs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeripheral blood collected from healthy donors was diluted 1:1 using the dilution solution in the kit, and cells were isolated according to the instructions in the Human Monocyte Isolation Kit (Boster, China). The cell layer on the lower side of the plasma was aspirated into serum-free 1640 basal medium in a CO2 incubator for 45 min. After washing thrice with cold PBS, the attached cells were human peripheral blood mononuclear cells. Human peripheral blood mononuclear cells were maintained in RPIM 1640+10% FBS with human recombinant M-CSF (NOVO protein, China) at a final concentration of 20ng/mL for 6-7days to differentiate into macrophages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data were analyzed using GraphPad Prism 8.5. A two-tailed t-test was performed for two sets of normally distributed data. Data are expressed as mean ± standard deviation, unless otherwise specified. Statistical significance was set at P \u0026lt; 0.05.\u003c/p\u003e"},{"header":"3. Results","content":"\u003ch2\u003e3.1 Identification of CRC derived exosomes\u003c/h2\u003e\n\u003cp\u003eDifferential centrifugation followed by ultracentrifugation is the most common method for isolating exosomes from a large volume of the culture medium supernatant. To verify the purity of the exosomes, we used transmission electron microscopy to obtain the morphology of CRC cell line-derived exosomes. A typical morphology of exosomes was observed (Figure S1A). And the Nanoparticle tracking analysis (NTA) also confirmed that the particle size of these vesicles was mainly concentrated around 130 nm (Figure S1B)，which is in accordance with previous reports\u003csup\u003e12\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMore importantly, we lysed these exosomes and examined their protein expression, while whole cell lysate (WCL) from CRC cell line served as a control, and our results confirmed that these vesicles expressed the exosome markers CD63, PDCD61P (ALIX), and TSG101, while the organelle marker protein calnexin ruled out organelle contamination\u003csup\u003e13\u003c/sup\u003e (Figure S1C).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.2 CT26 derived exosomes promote CRC liver metastasis\u003c/h2\u003e\n\u003cp\u003eTo confirm the involvement of exosomes in tumor metastasis in vivo, we utilized a mouse model of spontaneous colorectal cancer metastasis\u003csup\u003e14\u003c/sup\u003e. To regulate exosome production in CRC cells, we focused on RAB27a, which is a positive regulator of exosome biosynthesis\u003csup\u003e15\u003c/sup\u003e. A stable RAB27a knockdown CT26 cell line was established using lentivirus transduction. (Figure 1A). To minimize the inaccuracy associated with exosome isolation, we directly measured the concentration of exosomes in the culture supernatant of the CT26 cell line using NTA and ELISA. We found that RAB27a knockdown resulted in a 6-fold decrease in exosome secretion in CT26 cells. (Figure 1B and C). To determine the involvement of exosomes in liver metastasis and to avoid the rejection effect of xenotransplantation, we took advantage of a murine colorectal cancer cell line. CT26 and CT26 tumor masses were cultured and implanted into the sigmoid wall of BALB/c-nude mice. We found that CT26-Rab27a KD cells generated considerably less liver metastatic burden than CT26-NC cells, as demonstrated by vivo bioluminescence imaging and measurement of liver weight, the increased liver weight is due to the metastatic burden of tumors. (Figure 1D, E and F). This suggested that the effect of exosome-enhanced metastatic ability was partially abolished. Interestingly, Rab27-KD resulted in a larger primary tumor volume.\u003c/p\u003e\n\u003cp\u003eTo test whether exosomes promote metastasis by affecting the carcinoma cells themselves, we performed a series of classical tumor phenotype assays in vivo and in vitro and found that RAB27A knockdown affected the proliferation and apoptosis of CT26 cells (Figure S2A). The migration and invasion capacity in vitro were minimally affected. The tumorigenicity of CT26 cells also did not change significantly, as determined by the subcutaneous tumor assay (Figure S2B). Taken together, CT26-derived exosomes indeed contribute to colorectal cancer liver metastasis, possibly by modifying the hepatic microenvironment to make it more suitable for tumor cell growth.\u003c/p\u003e\n\u003ch2\u003e3.3 Hypoxia CT26 exosomes increase liver metastatic burden in experimental liver\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eTo investigate the direct effect of CRC-derived exosomes on liver metastasis, we utilized an experimental liver metastasis model\u003csup\u003e16\u003c/sup\u003e, which assesses the suitability of the hepatic microenvironment for tumor growth, which generates immediate liver metastasis. We isolated exosomes from the CT26 culture supernatant and treated BALB/c-nude mice by injecting 10 μg CT26 derived exosomes into the tail vein once every other day, and after 10 times (21 d), we injected 2x10\u003csup\u003e6\u003c/sup\u003e CT26-luciferase cells into the mouse spleen under direct vision. After 14d (35d after the first exosome injection), we assessed the liver metastatic burden by in vivo bioluminescence imaging and measurement of liver weight (Figure 2A). Interestingly, we found no statistically significant difference between the liver metastatic burden of the CT26-N-EXO-treated and control groups (Figure 2B). Owing to the rapid proliferation of tumor cells and abnormal blood vessel structure within tumors, hypoxia is one of the most common features of the tumor microenvironment\u003csup\u003e6,7\u003c/sup\u003e. We hypothesized that the hypoxic microenvironment within the primary tumor may play a crucial role in this process. We further cultured CT26 cells under hypoxic conditions, isolated hypoxic CT26-derived exosomes (CT26-H-EXO) and treated BALB/c-nude mice with equal amounts of exosomes. Notably, we found that CT26-H-EXO treatment significantly increased the biofluorescence intensity in the liver region and the liver weight in the model of experimental liver metastasis (Figure 2C), suggesting that hypoxic CRC-derived exosomes had a greater ability to promote liver microenvironment remodeling and cause higher metastatic burden.\u0026nbsp;Therefore, we used hypoxic exosomes in all subsequent experiments.\u003c/p\u003e\n\u003ch2\u003e3.4 Kupffer cells are the main cellular component of exosome ingestion\u003c/h2\u003e\n\u003cp\u003eTo further understand the biological function of hypoxic exosomes in colorectal cancer, we traced the distribution of CT26-H-EXO in vivo by optimizing multiple fluorescent labeling schemes. DIR is a far-IR lipophilic dye that offers low-background in vivo imaging\u003csup\u003e17\u003c/sup\u003e. To visualize the organ distribution of CRC-exosomes, we injected 50 μg of DIR-labeled CT26-H-EXO into the tail vein of BALB/c mice and performed in vivo imaging 24 h later. We found that the liver was the main organ uptake of CT26-H-EXO (Figure 3A). To exclude the influence of anatomical structures on exosome uptake, we injected equal amounts of DIR-labeled CT26-H-EXO into four different groups: tail vein injection (TV), retro-orbital injection (RO), and trans-spleen injection (SP) into three groups of BALB/c mice; the PBS group was used as a control. Among them, tail vein injection and retro-orbital injection allow exosomes to enter the body circulatory system, while trans-spleen injection causes exosomes to enter the portal vein first\u003csup\u003e18\u003c/sup\u003e, which is more similar to the process of in situ tumor release of exosomes. As shown in Figure 3B, irrespective of the injection method, the mouse livers in all groups exhibited the highest fluorescence intensity, demonstrating the tropism of CRC-derived exosomes in the liver. To determine which cellular component uptakes the CRC-derived exosomes, we took advantage of PKH26 labeling. Compared with DIR labeling, PKH26 labeling showed better specificity in previous reports\u003csup\u003e19\u003c/sup\u003e. After 24h of injection of 50 μg of PKH26-labeled CT26-H-EXO, we examined liver single-cell suspensions via flow cytometry, and F4/80 antibody was used to distinguish macrophages. As shown in Figure 3C, macrophages dominated the liver cellular components that engulf exosomes, consistent with F4/80+ and F4/80+ CD11b int cells\u003csup\u003e20,21\u003c/sup\u003e. Liver-resident macrophages, also known as Kupffer cells (KCs), are essential cellular components in liver homeostasis and have been proven to engulf tumor-derived exosomes and mediate the establishment of a pro-metastasis niche\u003csup\u003e16,22\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo obtain a direct morphology of KC uptake by CRC-derived exosomes, we overexpressed CD63-GFP recombinant protein in CT26 cells to obtain GFP-expressing CT26 exosomes\u003csup\u003e23\u003c/sup\u003e. After co-culture of GFP-expressing CT26 exosomes with primary mouse KCs for 24h, we confirmed to KC engulfment of CT26-H-exosomes by laser confocal imaging. Similarly, we found that human monocyte-derived macrophages also take up hypoxic exosomes derived from human CRC cell lines (Figure 3D).\u003c/p\u003e\n\u003ch2\u003e3.5 CT26-H-EXO treatment increased Kupffer cells numbers and activated towards M2-like phenotype\u003c/h2\u003e\n\u003cp\u003eTo investigate the underlying mechanism, mRNA sequencing was performed on livers derived from the PBS-treated group and CT26-H-EXO treated group derived livers (Figure 4A). Statistical analysis of differentially expressed genes showed that 21day of CT26-H-EXO treatment upregulated 1996 genes. GO analysis also indicated that macrophages were the most affected cellular component, and neutrophils and fibroblasts showed varying degrees of functional activation. Macrophages, including Kupffer cells, perform biological functions by secreting cytokines. Hence, we focused on the most obvious differential expression of various soluble cytokines associated with macrophage M2-like polarization (Figure 4B), such as IL-10, IL-8, TGFB3, and MIP-1, suggesting that the KCs phenotype may be skewed towards M2-like polarization. As shown by our IHC results (Figure 4C), the expression of the macrophage M2 polarization marker CD163 was significantly higher than that in the PBS-treated group. Consistently, as shown in Figure 4D, immunofluorescence confirmed that the number of F4/80-positive cells and expression of CD163 were elevated.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.6 Hypoxia treatment increased KRAS abundance in CRC derived exosomes\u003c/h2\u003e\n\u003cp\u003eNext, we performed KEGG pathway analysis for the 2396 differentially expressed genes. As shown in Figure 5A, the AKT and MAPK pathways showed the most significant enrichment. Previous data suggested that mutated KRAS protein constitutively mediates AKT and MAPK activation signaling in carcinoma cells to induce a malignant phenotype. KRAS is reported to be one of the most frequent mutations in CRC (Figure 5B). We selected three human CRC cell lines, HCT116 and LoVo, harboring the KRAS G13D mutation, while colo205 was used as the wild-type control. We further confirmed KRAS G13D expression using western blotting with a KRAS G13D-specific antibody (Figure 5C). Similar to oxidative stress\u003csup\u003e24\u003c/sup\u003e, we found that KRAS G13D expression was considerably higher in tumor exosomes under hypoxic culture conditions, whereas KRAS G13D was rarely detectable in normoxic CRC-derived exosomes. To prove that KRAS-mutated proteins can be transmitted to macrophages via exosomes, we used KRAS G13D-FLAG fusion protein-coding plasmids. KRAS G13D-FLAG and CD63-GFP coding plasmids were co-transfected into CT26 cells to generate CD63-GFP and KRAS G13D-FLAG expressing CT26-H-EXO. As shown in Figure 5D, when the murine macrophage cell line RAW264.7 was co-cultured with CD63-GFP and KRAS G13D-FLAG expressing CT26-H-EXO, the confocal-laser images showed exosomes and KRAS mutated proteins in the RAW cells, indicating that KRAS G13D mutated protein can be transmitted to macrophages.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.7 Human CRC hypoxia exosomes induce HMDMs M2-like polarization\u003c/h2\u003e\n\u003cp\u003eTo address the effects of human CRC exosomes on macrophages. We used an in vitro macrophage model, human monocyte-derived macrophages (HMDMs). Human monocytes were differentiated into macrophages by incubating with 20ng/mL M-CSF for 7d, confirmed by the morphology and ICC results of CD68 expression. HMDMs were co-cultured with equal doses of HCT116 or LoVo-derived hypoxic exosomes (HCT116-H-EXO, LoVo-H-EXO) for 48h. Consistent with our previous mRNA sequencing results, the expression of M2 markers (CD163 and TGF-β) in HMDMs was dramatically upregulated compared to that in the PBS group (Figure 6A).\u0026nbsp;In addition, to investigate the direct effect of KRAS G13D mutated protein, we further exogenously expressed KRAS G13D mutation protein in HMDMs. Compared to GFP coding plasmid, KRAS G13D mutation protein overexpression significantly induced the M2-like polarization of HMDMs (Figure 6B and C). This finding suggests that the KRAS G13D mutation directly promotes macrophage M2-like polarization.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.8. Inhibition of AKT signaling reduces KCs M2-like polarization and alleviates CRC liver metastasis\u003c/h2\u003e\n\u003cp\u003eIn our previous study, we found that phagocytosis-induced Kupffer cell M2 polarization involves AKT signaling dependent\u003csup\u003e25\u003c/sup\u003e, and AKT has been acknowledged as a crucial mediator of macrophage survival and polarization\u003csup\u003e26\u003c/sup\u003e. The Protein-Protein Interaction Database\u0026nbsp;string revealed several interactions between KRAS proteins and the AKT pathway. To probe the involvement of AKT signaling, we examined AKT phosphorylation using phosphoprotein flow cytometry, as shown Figure 6B, the phosphorylation of AKT was significantly increased in HMDMs incubated with hypoxic exosomes for 24h, compared with the PBS group. As shown in Figure 6A, after inhibiting AKT phosphorylation with 1μM GSK690693, the expression of CD163 in HMDMs and TGF-β secretion was significantly decreased compared with co-culture with hypoxic CRC-derived exosomes alone. This finding suggests that the administration of AKT inhibitors may be a prophylactic approach to prevent liver metastasis. To demonstrate the viability of this approach, we performed in vivo experiments, as shown in Figure 7A. To avoid the direct tumor suppressive effect of the AKT inhibitor, GSK690693 was only used in the exosome treatment stage (21d), followed by spleen injection of 10\u003csup\u003e6\u003c/sup\u003e CT26 cells to evaluate liver metastatic burden. As shown in Figure 7B and 7D, the inhibition of AKT by GSK690693 significantly depleted the expression of both F4/80 and CD163, suggesting a decrease in the number of KC cells and their M2-like polarization. \u0026nbsp;By weighing the liver, we found that the administration of GSK690693 reduced the liver metastatic burden, especially the macrometastatic tumor nodules (Figure 7C).\u003c/p\u003e\n\u003cp\u003eTaken together, our results demonstrated that hypoxic CRC-derived exosome KRAS mutation protein facilitates Kupffer cell M2-like polarization via AKT signaling.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTumor metastasis is a complex multifactorial process, in which tumor cells leave the primary site, enter the circulatory system, and survive and proliferate in distant organs. The survival and proliferation of tumor cells at the metastasis site is undoubtedly the core aspect that determines whether tumor macrometastasis eventually occurs\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e According to the seed and soil hypothesis, as first proposed by Paget, the relationship between tumor cells and organ stromal cells is similar to that between seed and soil, and metastasis only develops if both seed and soil are compatible. The liver is one of the organs most liable for tumor metastasis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, and some studies have suggested that the pre-metastatic microenvironment may promote liver metastasis in numerous types of tumors\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Pre-metastatic niche is a tumor favorable microenvironment in distant organs, created by primary tumors for subsequent metastases In recent years, an increasing number of studies demonstrated that tumor-derived exosomes are involved in the development of tumor metastasis, by promoting carcinogenesis of normal cells, enhancing the ability of migration and invasion in tumor cells\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, enhancing stemness and promoting cell proliferation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In addition, it fosters a microenvironment suitable for tumors by increasing vascular angiogenesis and permeability\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, inhibiting immune surveillance\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, and remodeling the extracellular matrix\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt is well known that macrophages are the predominant stromal cells in primary tumor environments\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Depending on the signaling molecules and environmental stresses received, macrophages are activated in two different ways: classical activation (M1-type polarization) and alternative activation (M2-type polarization). M1-type macrophages induce inflammatory and anti-tumor responses by secreting pro-inflammatory cytokines\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, while alternative-type activation secretes anti-inflammatory cytokines to inhibit excessive inflammatory responses and promote tissue repair\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Among them, tumor-associated macrophages (TAM) exhibit an M2-like polarization pattern that promotes tumor cell migration and metastasis\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, and the liver has the highest abundance of macrophages\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Our exosome tracing experiments also demonstrated the organophilicity of colorectal cancer-derived exosomes to the liver, and Zhang et al. also reported that the hepatic synthesis of complement C1q promotes phagocytosis in KCs, which might explain why the liver is the main organ in exosomes\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In contrast, a study by Shao et al. found that exosomes of breast cancer have a higher affinity to the lung than colorectal cancer-derived exosomes \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, and a study by Moller et al. also demonstrated that the liver is the major uptake organ of exosomes in pancreatic ductal carcinoma\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The specific tropism of these tumor exosomes coincides with tumor-prone metastatic organs, which is further evidence suggesting that tumor exosomes may play a key role in tumor metastasis.\u003c/p\u003e \u003cp\u003eIn the present study, we found an increase in M2-like polarized KCs after treatment with hypoxic CRC-derived exosomes. In contrast, Shao et al. developed a xenogeneous model by treating mice with human CRC exosomes, and their experimental results demonstrated that human colorectal carcinoma cell-derived exosomes have pro-inflammatory effects on the mouse macrophage cell line RAW264.7, while human-derived colorectal carcinoma cell-derived exosomes ultimately increased liver metastatic burden by promoting IL6 pro-inflammatory factor IL6 secretion in mouse KCs \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Hao et al. utilized a patient-derived xenograft model to reveal that hypoxic exosomes containing miR-135-5p promote CRC liver metastasis via P65 immunosuppression signaling\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Cooks et al. demonstrated that P53 mutant colorectal carcinoma cell-derived miR-1246 is transferred via exosomes to macrophages in the CRC primary tumor microenvironment, promoting their M2-like polarization and liver and lung metastasis \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecently, it was reported that macrophages are not the only target cell components. Zeng et al. found that exosomes secreted by human colorectal carcinoma cell lines carrying miR-25-3p were taken up by the vascular endothelium in the liver and lung to increase vascular permeability by affecting the tight junctions between endothelial cells and enhancing angiogenesis to support tumor cells for colonization and proliferation\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Tian et al. found that liver carcinoma-derived exosomes transfer miR-1247-3P to lung fibroblasts, induce pro-inflammatory cytokine release, and further promote cancer lung metastasis\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eKRAS is the most frequently mutated proto-oncogene in human solid tumors, and KRAS mutations have been detected in up to 85% of metastatic colorectal carcinoma\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Furthermore, G13D mutations are mainly found in colorectal cancer\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. This mutated protein enhances the malignant biological behavior of tumor cells and contributes to tumor progression by promoting tumor cell proliferation, migration, and invasion, and anti-apoptosis effects via AKT and MAPK signaling\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Recently, ISCHENKO et al. demonstrated by single-cell sequencing that KRAS mutation activates the RAF pathway to promote the secretion of multiple cytokines to establish an immunosuppressive microenvironment in pancreatic cancer, while knockdown of KRAS mutations resulted in enhanced T-cell anti-tumor immunity\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Beckler et al. found by mass spectrometry that mutated KRAS proteins could be detected in the exosomes of the HCT116 cell line, and KRAS protein can be transferred to non-cancerous normal cells\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Dai et al. found that oxidative stress-induced secretion of pancreatic adenocarcinoma-derived exosomes is enriched with KRAS-mutated proteins, which are taken in by tumor-associated macrophages in the primary tumor microenvironment, supporting the M2-like polarization of tumor cells via fatty acid oxidation metabolism\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Due to the pro-cancer activity of mutant KRAS proteins, a small molecule inhibitor of KRAS G12C-type mutations, entering phase I clinical trials in recent years, this genotype-specific inhibitor is not effective in most CRC patients \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the present study, we established two vivo models to validate the participation of CRC derived exosomes in liver metastasis we confirmed hypoxia exosomes has greater pro-metastasis ability compared with normoxia exosomes. Exosome tracing experiments and mRNA sequencing revealed that the key regulator in the treatment of hypoxic CRC-derived exosomes is liver macrophages (KCs), and that treatment with hypoxic CRC exosomes upregulates the KCs population and M2-like polarization. Moreover, bioinformatics analysis indicated that KRAS-mutated proteins and AKT cell pathways mediate macrophage M2 polarization. Since KRAS protein is a nearly non-druggable target, we successfully reversed the pro-tumor metastatic effect of KRAS mutant CRC-derived exosomes by administering AKT inhibitors.\u003c/p\u003e \u003cp\u003eOverall, our study determined the role of exosome mutated KRAS-induced AKT signaling in the process of KCs M2-like polarization, AKT inhibitors, and might potentially be used as a therapeutic approach to prevent liver metastases in colorectal carcinoma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported by the National Science Foundation of China (No. 81170442, 81470899, 81702357, 82070678), Chen Xiao-ping foundation for the development of science and technology (No. CXPJJH12000001-2020330), Kuanren Talents Program of the second affiliated hospital of Chongqing Medical University (No. kryc-yq-2208), Chongqing Natural Science Foundation (CSTB2023NSCQ-MSX0150) and Chongqing Municipal Science and Health Joint Medical Research Project (2024QNXM003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceived and designed the study: Zuojin LIU\u003c/p\u003e\n\u003cp\u003ePerformed the experimental procedures: Zhihao FENG,\u0026nbsp;Jiao LU, Hua SONG,\u0026nbsp;Fuyao LIU\u003c/p\u003e\n\u003cp\u003eAnalyzed the data:\u0026nbsp;Jie XU, Ke YOU, Tianzhu WU\u003c/p\u003e\n\u003cp\u003eDrafted the manuscript: Zhihao FENG, You YU\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported by the National Science Foundation of China (No. 81170442, 81470899, 81702357, 82070678), Chen Xiao-ping foundation for the development of science and technology (No. CXPJJH12000001-2020330), Kuanren Talents Program of the second affiliated hospital of Chongqing Medical University (No. kryc-yq-2208), Chongqing Natural Science Foundation (CSTB2023NSCQ-MSX0150) and Chongqing Municipal Science and Health Joint Medical Research Project (2024QNXM003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are not publicly available due ethical, privacy or security reasons, but are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSiegel RL, Miller KD, Jemal A. 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Proteomic analysis of exosomes from mutant KRAS colon cancer cells identifies intercellular transfer of mutant KRAS. \u003cem\u003eMolecular \u0026amp; cellular proteomics : MCP.\u0026nbsp;\u003c/em\u003e2013;12(2):343-355.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Table 1","content":"\u003cp\u003eSupplementary table 1 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"KRAS, exosome, Kupffer cell, M2 polarization","lastPublishedDoi":"10.21203/rs.3.rs-6300438/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6300438/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e: This study aimed to investigate the metastasis-promoting effect of colorectal carcinoma cell-derived exosomes on liver metastasis, M2-like polarization of Kupffer cells, and the underlying mechanism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Mouse liver metastasis models were established to testify the involvement of CRC-derived exosomes on liver metastasis, and DIR and PKH26 fluorescent labeling strategies were used to trace the distribution of CRC-derived exosomes in vivo. GO and KEGG analyses of differentially expressed genes revealed the key cellular regulators and KRAS-induced signaling in CRC liver metastasis. The phenotype of Kupffer cells was determined using IHC and IF. In vitro model HMDMs were used to explore the polarization phenotype and therapeutic effects of AKT inhibition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Exosome mutant KRAS induced AKT signaling in the process of kupffer cells (KCs) M2-like polarization, promoting CRC liver metastasis. AKT inhibitors may potentially be used as a therapeutic approach to prevent liver metastasis in CRC.\u003c/p\u003e","manuscriptTitle":"Kupffer cells M2-like polarization increases liver metastatic burden via uptake of exosomal KRAS mutant protein from hypoxia colorectal carcinoma cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-07 03:03:56","doi":"10.21203/rs.3.rs-6300438/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f03a3841-2f91-43bf-8147-ba07c4a98691","owner":[],"postedDate":"April 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-09T09:23:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-07 03:03:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6300438","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6300438","identity":"rs-6300438","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}