| BACKGROUND & OBJECTIVEColorectal cancer (CRC) is the primary cause of death in patients with liver metastasis, about one third patients was diagnosed with liver metastasis within three years [1].Only 25% CRC patients accompanied with isolated liver metastatic lesions can be effectively removed surgically treated, and only 21%-21% of patients with postoperative survival more than 5 years [2; 3].Treatment failure and death in patients with colorectal cancer is the major reason for the shift and form the metastatic tumor[4].In 1889 Paget has been proposed the hypothesis of "seed and soil" which provided the guidance for researching colorectal cancer metastasis, the tumor cells and the stromal cells in microenvironment and their released cytokines jointly participated in the invasion and metastasis of tumor[5;6; 13]. In 2003 Fidler the revised hypothesis of "seed and soil" explicitly presented the formation of metastases is the factor of target organ microenvironment correlation with the metastasizing cell, therapy of anti-metastasis should not only target the high metastatic characteristic of tumor cells, but also the cell and cytokines in the microenvironment which adapted tumor cell growth, angiogenesis, invasion and metastasis, and prevented and fighted against malignant tumor metastasis. Liver is commonly regarded as the target organ of colorectal cancer metastases, its environment is very suitable for tumor cell growth and metastasis. Therefore, it is particularly important to explore the correlation between microenvironment and colorectal cancer growth and metastasis.This study compared by the chemotaxis between colorectal cancer cells and parenchyma or mesenchymal cells derive from metastatic target organs (liver) or non-target organ, screening and identifying the key cell and cytokines of impacting the tumor cells, clarifying the molecular mechanism of adjusting the growth and metastasis of tumor cells, playing a therapy to tumor cells and the cytokines of target organ microenvironment, and providing a target treatment of antirumor for anti-soil and anti-seed.METHODS1. Screening the key chemotaxis cell between target organs (liver) of colorectal cancer metastasis and tumor cells.We first assessed whether normal cells originating from the liver and non-specific target organs exerted differential effects on the migration of CRC cells. A transwell assay was applied to compare the attractant ability of CRC cell migration, where human normal cells were placed in the bottom chamber, and the CRC cells (SW480, HCT116 and LS174T) were placed in the upper chamber, respectively. The normal cells of the liver include HHSECs, HL7702 (human hepatocyte) and LX-2 (human hepatic stellate cell), and corresponding cells such as HUVECs (human umbilical vein endothelial cells),293A (human embryonic kidney cell) and BJ (human foreskin fibroblast cell) were compared as analog-control cells originating from non-specific target organs of CRC metastasis. Subsequently, when the cells’ position was reversed in the transwell chamber, HHSECs, HUVECs, HL7702 and LX-2 at the upper chamber and the CRC cells at the bottom chamber, observing whether tumor cells have the ability of chemotracting the normal cells to migrate.2. Screening the key cytokines chemotracted tumor cell migration from hepatic sinus endothelial cells (HHSECs).To ascertain which mediator(s) might be released from HHSECs to induce CRC cell migration, we compared the culture supernatants (conditioned media) that were collected from upper and bottom chamber of the transwell by using human cytokine arrays containing antibodies against 1000 cytokines. Human cytokine arrays revealed that macrophage migration inhibitory factor (MIF) released by HHSECs was a key role of chemotracting CRC cells. To assess the chemotactic significance of MIF in HHSECs, MIF expression was inhibited by lentiviral vector-mediated small hairpin (shRNA) in HHSECs. Quantitative real-time PCR (qPCR), western blots (WB) and enzyme-linked immune sorbent assay (ELISA) verified that MIF knockdown and MIF secretion blockage were effective. To explore whether MIF is released by HHSECs or by CRC cells themselves act as a main factor in migration, we use different conditioned medium of mock/HHSECs or shMIF/HHSECs,or plus p425 in conditioned medium of mock/HHSECs or rhMIF in conditioned medium of shMIF/HHSECs to chemotracted CRC cells or shMIF/CRC cells.3. MIF paracrined from HHSECs influence the biological functions of the colorectal cancer cells.(1) Observe the CRC cells morphological changes after cultured with Conditioned medium of HHSECs.(2) Transwell migration model was used to determine whether the EMT that occurred during the CRC cells exhibited chemotaxis towards HHSECs. Using immunofluorescence technique to detect the migrated and non-migrated CRC cells.(3) Western blots to validate MIF secreted by HHSECs inducing EMT of colorectal cancer cells.(4) The proliferative effect of paracrined MIF on CRC cells was assessed with CCK.8 assay and EdU (5-ethynyl-2’-deoxyuridine) assay.(5) Using flow cytometry to measure MIF paracrined from HHSECs influence on cell cycle and apoptosis of CRC cells.4. Validate paracrined MIF from HHSECs effect the growth and metastasis of the CRC cells in vivo.(1) An orthotopic transplantation of nude mice for experimental metastasis was used to ascertain whether the MIF paracrined from HHSECs affects the tumor growth, invasiveness and liver metastases of CRC cells. CRC cells, or CRC cells mixed Mock/HHSECs, CRC cells mixed shMIF/HHSECs, HHSECs, and were implanted into the cecal wall of nude mice(2) To exclude the effect of MIF from CRC cells themselves in tumorigenesis and tumor growth, shMIF/CRC with Mock/HHSECs cells or shMIF/HHSECs were subcutaneously co-transplanted into nude mice subcutaneously.5. The correlation of paracrined MIF from liver metastases of colorectal cancer.Using immunohistochemistry staining to examine the MIF protein expressed in primary CRC samples of 229 patients collected from the Pathology Department of Nanfang Hospital, Southern Medical University, and combined with the clinical pathological characteristics, discussed the relationship between secreted MIF and liver metastases of colorectal cancer.6. Find out the mechanism of pacrined MIF from HHSECs inducing migration of CRC cells.Using western blot and immunofluorescence to detect the change of skeleton protein and the related regulatory factor in CRC cells which cultured with conditioned media.RESULTS1. HHSECs induce chemotaxis during CRC cell migration.We first assessed whether normal cells originating from the liver and non-specific target organs exerted differential effects on the migration of CRC cells. A Transwell assay was utilized to compare the attractant ability toward CRC cell migration, wherein human normal cells were placed in the bottom chamber, and CRC cells (SW480, HCT116, or LS174T) were placed in the upper chamber. The normal cells of the liver included HHSECs, HL7702s (human hepatocytes), and LX-2s (human hepatic stellate cells), and corresponding cells including HUVECs (human umbilical vein endothelial cells),293As (human embryonic kidney cells), and BJs (human foreskin fibroblast cells) were compared as analog-control cells originating from non-specific target organs of CRC metastasis. This model simulates the prometastatic cancer cells in the liver sinusoids chemotracted by the adjacent cells.The results showed that HHSECs were 3 to 14 times more active than HUVECs in stimulation of CRC cells migration. HL7702,293A, LX-2, and BJ cells induced the migration of CRC cells in a way that was not obviously different from that of the controls, and the cells that originated from the target organ (liver), such as HL7702 and LX-2, did not show any positive differential roles in promoting migration of CRC cells, but had similar effects to those of the non-target organ cells, such as 293A and BJ.Subsequently, when the cell positions were reversed in the Transwell chamber, the HHSECs, HUVECs, HL7702, and LX-2 in the upper chamber were not chemotracted by CRC cells in the bottom chamber. Furthermore, when HHSECs, and HL7702 and LX-2 cells were mixed in a co-cultured system to induce CRC cell migration, the chemoattractant effect of the mixed cells was not much greater than that of HHSECs alone. In addition, we also tried to demonstrate whether another tumor cell that metastasizes to the liver as a specific target organ, HCC1937s (human breast cancer cells), used as a positive control, was attracted by HHSECs or HL7702 or LX-2 cells. We used RL95s (endometrial cancer cells) as the negative control, as it rarely metastasizes to the liver. Interestingly, HHSECs induced HCC1937 migration more markedly than that of RL95, but neither the breast nor endometrial cancer cell lines chemotracted HHSECs or HUVECs to migrate. Thus, the Transwell assays demonstrated that HHSECs were the dominant cells for chemotracting CRC cells to metastasize to the liver.2. MIF is a critical factor released by HHSECs and contributes to the chemotaxis of CRC cell migration.To ascertain which mediator(s) might be released from HHSECs to induce CRC cell migration, we compared the culture supernatants (conditioned media) that were collected from the upper and lower chambers of the Transwell dish by using human cytokine arrays containing antibodies against 1000 cytokines. Analysis of the antibody array demonstrated that MIF, IGFBP-7, Smad 4, SPARC, thrombospondin (TSP), and Ras are mediators whose expression levels are significantly higher in HHSECs than in HUVECs and CRC cells. Among these proteins, MIF showed the greatest expression in the conditioned media from HHSECs, particularly in SW480/HHSECs and HCT116/HHSECs, in comparison with that from HUVECs, and SW480 and HCT116 cells. Application of either of two specific MIF inhibitors (ISO-1 and P425) resulted in the inhibition of HHSEC-induced migration of CRC cells. As a positive control, rhMIF (human recombinant MIF) was confirmed to promote CRC cell migration. To assess the chemotactic significance of MIF from HHSECs, MIF expression was inhibited by lentiviral vector-mediated small hairpin (shRNA) expression in HHSECs. Quantitative real-time PCR (qPCR), western blot (WB), and enzyme-linked immune sorbent assay (ELISA) verified the efficacies of MIF knockdown and MIF secretion blockage.We found that Mock/HHSECs treated by the MIF inhibitor p425(100 nM) or shMIF/HHSECs resulted in the inhibition of HHSEC-induced migration, but that the inhibitory effect could be recovered by supplementation with rhMIF (50 nM). To explore whether MIF was released by HHSECs or whether MIF in the CRC cells themselves could act as a main factor in migration, we knocked down MIF in SW480 and HCT116 cells.Mock/HHSECs chemotracted shMIF/SW480 or shMIF/HCT1 16 to migrate was markedly increased, in comparison with shMIF/HHSECs or Mock/HHSECs plus p425 chemotracted. We also utilized WB and ELISA to detect whether the MIF was expressed or secreted by other metastatic microenvironmental cells including HL7702 and LX-2 cells and HUVECs. HL7702s, LX-2s, and HUVECs also expressed intracellular MIF, and hardly excreted MIF. The mRNA coding sequence of MIF in HHSECs was the same as that in SW480, HCT116, and HUVECs as assessed by reverse transcription (RT)-PCR amplification. Thus, these results suggested that the MIF released from HHSECs is a major mediator contributing to the HHSEC-induced migration of CRC cells.3. MIF released by HHSECs promotes the epithelial-mesenchymal transition (EMT), proliferation, and apoptotic resistance of CRC cells.When CRC cells were cultured with conditioned media from HHSECs, the CRC cells appeared starfish shaped, which were composed of cytoplasmic protuberances. The Transwell migration model was used to determine whether the EMT that occurred within the CRC cells exhibited chemotaxis towards HHSECs. We found that the migrated CRC cells induced by HHSECs strongly expressed mesenchymal products such as N-cadherin (N-ca) and vimentin (VIM), and underexpressed epithelial products such as E-cadherin (E-ca) in comparison with non-migrated CRC cells. To confirm whether EMT was stimulated by MIF released from HHSECs, the CRC cells were cultured with different conditioned media for 24 hours. WB analysis showed that the CRC cells exhibited elevated N-ca and VIM expression but lost E-ca when grown in conditioned media containing higher levels of soluble MIF.The proliferative effect of paracrined MIF on CRC cells was assessed with a CCK8 assay, following treatment of SW480 and HCT116 cells with different conditioned media. The CCK8 assay results indicated that MIF released at high concentrations from HHSECs was favorable to CRC cell proliferation, which was similar to the result of the EdU (5-ethynyl-2’-deoxyuridine) assay.To explore the mechanism by which CRC cell proliferation was promoted by exogenous MIF, we studied the effect of MIF on the cell-cycle phases using flow cytometer. The percentage of cells in G2 phase was significantly increased by the presence of MIF in the conditioned media, while no significant effect on the percentage of cells in G1 or S phase was observed. Furthermore, Annexin V staining as an indicator of apoptosis was used to measure the apoptotic rates in association with the proliferative effect. This analysis demonstrated that soluble MIF inhibited the apoptosis of CRC cells induced by 5-fluorouracil (5-FU). Collectively, these data implied that MIF released by HHSECs activated EMT, proliferation, and apoptotic resistance during CRC cell migration.4. MIF released by HHSECs facilitates CRC growth and migration in vivo.Orthotopic transplantation of nude mice to generate experimental metastasis was utilized to ascertain whether the MIF released from HHSECs increases the growth,invasiveness, and liver metastases of CRC cell-derived tumors. CRC cells, or CRC cells mixed Mock/HHSECs, CRC cells mixed with shMIF/HHSECs, and HHSECs alone, were implanted into the cecal wall of nude mice for 8 weeks. The mice were euthanized and subjected to gross and microscopic examination. Gross and microscopic examination of hepatic and pulmonary metastases revealed that the tumor growth of the CRC and Mock/HHSEC cell mixture at the primary site was dramatically increased (Figure 4A) and that the tumor growth rate, volume, weight, and foci were markedly higher than those of the tumors of CRC cells alone or of CRC cells mixed with shMIF/HHSECs (Figure 4B-D). However, HHSECs injected alone did not generate any masses.Additionally, shMIF/SW480 cells, shMIF/SW480 cells mixed with Mock/HHSECs, or shMIF/SW480 cells mixed with shMIF/HHSECs were also subcutaneously implanted into nude mice. This revealed that Mock/HHSEC cells with MIF secretion activated tumorigenesis and tumor growth (Figure 4E). In summary, these results suggest that MIF secreted from HHSECs promotes tumorigenesis and the development of CRC cell metastases in vivo.5. MIF is associated with the metastatic outgrowth of CRC.We used immunohistochemistry staining to examine the MIF protein expression in primary CRC samples from 229 patients collected from the Pathology Department of Nanfang Hospital, Southern Medical University, Guangzhou, China. There was no statistically significant correlation between MIF expression levels and cancer invasion, histological grades, survival time, or lymph node or distant metastases. Therefore, the MIF expression levels that originated from CRC cells or from other undefined cells in the primary tumor microenvironment did not appear to have an important prometastatic effect. We also used immunohistochemistry to examine the MIF expression in paired samples of tubular adenocarcinoma from 29 patients with CRC in primary tumors and liver metastases. According to the distinct expression of MIF in primary tumors from metastatic tumors, we classified all samples into two groups. Group A included the samples that expressed MIF in the primary cancer tissues at levels less than in the liver metastases, while Group B included the samples that expressed MIF in primary cancer tissues at levels greater than or equal to that of the liver metastases. Of the 29 paired samples that were analyzed,0 (0%) patients in Group A and 5 (45%) in Group B produced liver metastases with a maximum size less than 3 cm. Approximately 18 (100%) patients in Group A and 6 (55%) in Group B produced liver metastases with a maximum size greater than or equal to 3 cm (P<0.05). There were no statistically significant differences in age or gender between Groups A and B (P>0.05). These results suggested that the sizes of the liver metastases were highly positively correlated with the expression of MIF, and that HHSECs had a promoting effect.6. MIF paracrined from HHSECs induces CRC cell migration through p-cofilin to increase F-actin polymerization.To determine whether the signaling pathways involved in paracrine MIF also induce the migration of CRC cells, cells were cultured with conditioned media as previously described. We identified that p-cofilin expression in the CRC cells was more marked when cells were cultured in the conditioned media from Mock/HHSECs than in other media (conditioned media from shMIF/HHSECs, Mock/HHSECs supplementing p425, and fundamental medium). However, p-cofilin expression was restored following cultivation in the conditioned media of shMIF/HHSECs supplemented with rhMIF (Figure 5A and Supplementary Figure S5A). Phosphorylation inactivates cofilin, leading to the accumulation of actin filaments [12]. As shown by WB and immunofluorescence, intracellular F-actin was more prominent when the cells were cultured by the conditioned media of Mock/HHSECs, but the enhanced expression was offset following cultured in the conditioned media of shMIF/HHSECs or Mock/HHSECs plus p425 (Figure 5B). To confirm whether signaling of CRC cells by secreted MIF leads to cofilin phosphorylation and is involved in F-action regulation, we treated CRC cells with rhMIF at the indicated concentrations. As shown in Figure 5C, addition of rhMIF at 50 nM led to increased p-cofilin and F-actin expression, whereas addition of rhMIF at concentrations greater than 50 nM did not promote further expression. The characteristic effects of MIF derived from HHSECs on the cofilin/F-actin cytoskeleton suggested that it might be specifically involved in the cytoskeletal remodeling that promotes CRC migration.CONCLUSION(1) HHSECs was the dominant cells for CRC prometastatic chemotaxis in the liver.(2) MIF paracrined from HHSECs was a key molecule contributing to the chemotaxis of CRC cell migration.(3) MIF paracrined from HHSECs promotes chemotaxis and outgrowth of colorectal cancer during liver prometastasis.(4) MIF stimulated to increase the p-cofilin of the CRC cells was associated with inhibiting the F-actin depolymerized.(5) There was no statistically significant correlation of MIF expression levels in primary colorectal cancer to the prognosis of patients, but MIF could promote the growth of intrahepatic metastatic carcinoma.INNOVATION IN THE STUDY(1) The data show the organ-specific metastasis of stromal cells participate in paracrine signaling to tumor cells.(2) Determine the HHSECs chemotracted the colorectal cancer cell to migrate was stronger than the endothelial cells of non-target organs.(3) Prove the MIF paracrined from HHSECs rather than autocrined from tumor cells per se play a key role in promoting tumorigenesis, tumor growth and metastasis.(4) HHSECs and their paracrined MIF might be an ideal target for anti-soil and anti-seed therapy, and provided antagonists of concept for migration and proliferation of CRC cells during prometastasis. |
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