Role And Mechanism Of Tumor-Associated Macrophages In The Regulation Of Cancer Stem Cell-like Properties In Hepatocellular Carcinoma

Background and AimsHepatocellular carcinoma (HCC) is one of the most common cancers in the world.And it’s the third most common cause of cancer-related death in adults. What`s more, ourchina is the “disaster area” of HCC. Each year more than half of global new cases havebeen confirmed in our country. In recent years, with the development of medicaltechnology, both diagnosis and treatment have made great progress in HCC. Althoughhepatic resection, liver transplantation and several minimally invasive therapies are widelyused to improving outcomes of the patients of HCC. Due to recurrence and metastasis, thefive-year survival of patients undergoing therapies still remains disappointingly low.Currently, an increasing number of studies have supported that cancer stem cells(CSC), which have many features of stem cells, are responsible for the poor prognosis ofpatients by promoting tumor invasion and metastasis. CSCs or cancer-initiating cells aredefined as a small subpopulation of cancer cells with the ability of self-renewed andpluripoteney. With the deepening of research, recent dates suggest that the maintaince ofCSC homeostasis rely on a specialized tumor microenvironment (TME). However, theeffect of TME on CSC-like properties remains unclear in HCC.As a kind of inflammatory cells existing widely in the tumor tissue, macrophagesoccupy an important position in TME. Macrophages could polarize into two functionallyphenotypes, classically activated M1and alternatively activated M2, in response todifferent microenvironment. Macrophages infiltrated in TME are define astumor-associated macrophages (TAM), and express the similar molecular and functionalcharacteristic of M2phenotype. TAMs is a pivotal component of tumor-infiltratingimmune cells which play a critical role in regulating tumor growth and progression. Atpresent, some studies about TAMs have showed that an increased density of TAMs wascorrelate with worse prognosis in many cancers. And many evidences has revealed thatTAM promoted cancer cells to gain CSC-like properties, and Epithelial to mesenchymaltransition (EMT). However, the role of TAMs in the maintenance of CSC-like properties inHCC and the detailed mechanisms underlying this process were remained elusive.EMT is a process epithelial tumor cells lose epithelial features and gain mesenchymalphenotypes. It is considered as the key step by which tumor cells gain the higher ability of invasive and metastatic. Some reports implies that tumor cells take advantage of EMT asan intermediary phenotype to achieve self-renewal and adapt to microenvironments. Andthese tumor cells could be enhanced the ability of invasion and metastasis by acquiringsome properties of the mesenchymal cells. Transforming growth factor-β1(TGF-β1) is oneof the most important members of the transforming growth factor family. It is one of themost power potent inducers of epithelial plasticity leading to EMT in cancer cells.Increasing studies have pointed out the importance of TGF-β1in cancer progression andmetastasis. As reported, TGF-β1released from the TME is essential to regulatemaintenance, differentiation and function of CSC-like properties in many epithelial cancerssuch as skin cancer, gastrointestinal tract cancer, and bladder cancer.In the study, we aim to analyze the role of TAMs in HCC progression with focus onTGF-β1. We treated the hepatoma cells Hepa1-6cells with conditioned medium (CM)from cultural TAMs. And we proceeded from the following three aspects to analyze TAMin maintaince of CSC-like properties:(1) Observation of the effect of tumor-associatedmacrophages induced EMT on cancer stem cell-like properties in HCC;(2) Themechanism of tumor-associated macrophages-induced EMT of hepatoma cells in HCC;(3)The role of tumor-associated macrophages and cancer stem cells in clinical outcomesassessment in HCC.Methods and ResultsPart1The effect of tumor-associated macrophages induced EMT on cancer stem cell-likeproperties in HCCIn part1, we performed immunofuorescence staining on the paraffin section of HCCspecimens to assess the expression and positional relationship of TAM and CSCs in thetumor tissue. We found that presence of CD68+macrophages has a correlation withlocation of EpCAM+CSCs in the marginal area of tumor. Then IL-4was used to stimulatemouse macrophage-like line RAW264.7cells to prepare TAM-CM, and investigate the roleof TAMs in the regulation of CSC-like properties. The mouse hepatoma cell lineHepa1-6cells was cocultured with TAM-CM, and we tested the ability of Hepa1-6cells toform colonies in vitro by colony formation assay and the levels of two CSC transcriptionalfactors Bmi1and Klf4by RT-PCR. As shown, TAM could promote the acquisition ofCSC-like properties of Hepa1-6cells. Meanwhile, cell immunofluorescence, RT-PCR andWestern blot were taken to analyze expression of epithelial marker E-cadherin, mesenchymal markers N-cadherin, vimentin and EMT associated transcription factor Snailin cellular, protein and genetic level. These tests suggested that TAM promoted EMT inHepa1-6cells. Moreover, whether TAM-CM could promote the invasive and migratoryabilities of Hepa1-6were tested by transwell assay and wound-healing assay. Comparedwith control, TAM-CM-treated Hepa1-6cells had fast closure of the wound and the resultwas confirmed by transwell assay. Finally, we adopted tumorigenicity experiments of C57mice to evaluate whether TAM regulated hepatoma cells in vivo. As result, the tumors oftreatment group were larger and heavier than control. Accordingly, TAM had a significantpromotion on the growth of tumors in mice. In summary, TAM mediated EMT toupregulate cancer cells stemness characteristic and promote migratory and invasivebehaviors in Hepa1-6cells.Part2The mechanism of tumor-associated macrophages-induced EMT of hepatoma cells inHCCIn part2, we focus on the mechanism by which TAM-CM promoted CSC-like propertiesof hepatoma cells. At first, we performed immunofuorescence staining on the HCC tissueto assess the expression macrophage marker CD68and TGF-β1. A high expression ofTGF-β1was detected in the microenvironment surrounding CD68+TAMs in the tumor.TGF-β1secreted by different phenotypes of RAW264.7macrophages and its mRNA levelwas measured by ELISA and RT-PCR. LPS plus IFN-γ were used to generate classicallyactivated M1macrophages. In contrast, alternatively activated M2macrophagesdifferentiated in response to IL-4. RAW264.7cells treated with IL-4had a high productionof TGF-β1compared with TAM treated with LPS plus IFN-γ. These results suggested thatTAMs, derived from murine macrophage-like line RAW264.7cells, had alternativelyactivated M2phenotypes with a higher production of TGF-β1. Next, in order to determinewhether TGF-β1would have effects on TAM-mediated stem-like characteristic ofhepatoma cells, we used TGF-β1neutralizing antibody to deplete TGF-β1in TAM-CM.After applied TGF-β1neutralizing antibody in TAM-CM, the colony formation ability ofhepa1-6cells was showed by colony formation assay, CSC transcriptional factors Bmi1and Klf4levels were tested by RT-PCR. RT-PCR and Western blot were performed to theexpression of epithelial marker E-cadherin, mesenchymal markers N-cadherin, Vimentinand EMT associated transcription factor snail were decreased in Hepa1-6cells coculturedwith TGF-β1depleted TAM-CM. In addition, the invasive and migratory abilities ofHepa1-6cells were tested by transwell assay and wound-healing assay. Collectively, the results presumably suggested that depletion of TGF-β1blocked the effect of TAM-inducedEMT on promotion of the CSC-like properties in Hepa1-6cells.Part3The role of tumor-associated macrophages and cancer stem cells in clinical outcomesassessment in HCC.Combined with the results of the first two parts of the experiment, we performedimmunohistochemical staining on the tissue samples collected from90patients with HCC.Our dates showed that the positive expression of EpCAM was an independent prognosticfactor for both OS and DF. In EpCAM+samples the DFS and OS rates for patients in thelow-CD68expression group were significantly higher than the DFS and OS rates forpatients in the high-CD68expression group. These results demonstrated that high densityof TAM would be a risk factor in EpCAM+sample of HCC. The combination of CSCmarkers and TAM markers have a better power to predict patients’ outcomes inHepatocellular carcinoma.[Conclusion]In summary, the combined results of three parts, we can draw the following conclusions:1.Tumor-Associated Macrophages enhance cancer stem cell-like properties by EMT.2.Tumor-Associated Macrophages promote cancer stem cell-like properties viatransforming growth factor-beta1-induced epithelial-mesenchymal transition.3.The combination of CSC markers and TAM markers have a better power to predictpatients’ outcomes in HCC. In EpCAM+samples, the low-CD68expression group has abetter prognosis than the high-CD68expression group.