Quantitative Changes in Tumor-Associated M2 Macrophages Characterize Cholangiocarcinoma and their Association with Metastasis

  • Thanee, Malinee (Department of Biochemistry, Faculty of Medicine, Khon Kaen University) ;
  • Loilome, Watcharin (Department of Biochemistry, Faculty of Medicine, Khon Kaen University) ;
  • Techasen, Anchalee (Center for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University) ;
  • Namwat, Nisana (Department of Biochemistry, Faculty of Medicine, Khon Kaen University) ;
  • Boonmars, Thidarut (Department of Parasitology, Faculty of Medicine, Khon Kaen University) ;
  • Pairojkul, Chawalit (Department of Pathology, Faculty of Medicine, Khon Kaen University) ;
  • Yongvanit, Puangrat (Department of Biochemistry, Faculty of Medicine, Khon Kaen University)
  • Published : 2015.04.14


The tumor microenvironment (TME) includes numerous non-neoplastic cells such as leukocytes and fibroblasts that surround the neoplasm and influence its growth. Tumor-associated macrophages (TAMs) and cancerassociated fibroblasts (CAFs) are documented as key players in facilitating cancer appearance and progression. Alteration of the macrophage (CD68, CD163) and fibroblast (${\alpha}-SMA$, FSP-1) cells in Opisthorchis viverrini (Ov) -induced cholangiocarcinoma (CCA) was here assessed using liver tissues from an established hamster model and from 43 human cases using immunohistochemistry. We further investigated whether M2-activated TAMs influence CCA cell migration ability by wound healing assay and Western blot analysis. Macrophages and fibroblasts change their phenotypes to M2-TAMs (CD68+, CD163+) and CAFs (${\alpha}-SMA+$, FSP-1+), respectively in the early stages of carcinogenesis. Interestingly, a high density of the M2-TAMs CCA in patients is significantly associated with the presence of extrahepatic metastases (p=0.021). Similarly, CD163+ CCA cells are correlated with metastases (p=0.002), and they may be representative of an epithelial-to-mesenchymal transition (EMT) with increased metastatic activity. We further showed that M2-TAM conditioned medium can induce CCA cell migration as well as increase N-cadherin expression (mesenchymal marker). The present work revealed that significant TME changes occur at an early stage of Ov-induced carcinogenesis and that M2-TAMs are key factors contributing to CCA metastasis, possibly via EMT processes.


Tumor-associated macrophages;cancer-associated fibroblasts;cholangiocarcinoma;metastasis


Supported by : Khon Kaen University


  1. Adjei IM, Blanka S (2015). Modulation of the tumor microenvironment for cancer treatment: a biomaterials approach. J Funct Biomater, 6, 81-103.
  2. Albini A, Sporn MB (2007). The tumour microenvironment as a target for chemoprevention. Nat Rev Cancer, 7, 139-47.
  3. Bhamarapravati N, Thammavit W, Vajrasthira S (1978). Liver changes in hamsters infected with a liver fluke of man, Opisthorchis viverrini. Am J Trop Med Hyg, 27, 787-94.
  4. Campbell DJ, Dumur CI, Lamour NF, et al (2012). Novel organotypic culture model of cholangiocarcinoma progression. Hepatol Res, 42, 1119-30.
  5. Chen SJ, Zhang QB, Zeng LJ, et al (2015). Distribution and clinical significance of tumour-associated macrophages in pancreatic ductal adenocarcinoma: a retrospective analysis in China. Curr Oncol, 22, 11-9.
  6. Chuaysri C, Thuwajit P, Paupairoj A, et al (2009). Alphasmooth muscle actin-positive fibroblasts promote biliary cell proliferation and correlate with poor survival in cholangiocarcinoma. Oncol Rep, 21, 957-69.
  7. Condeelis J, Pollard JW (2006). Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell, 124, 263-6.
  8. De Wever O, Demetter P, Mareel M, et al (2008). Stromal myofibroblasts are drivers of invasive cancer growth. Int J Cancer, 123, 2229-38.
  9. Diaz R, Kim JW, Hui JJ, et al (2008). Evidence for the epithelial to mesenchymal transition in biliary atresia fibrosis. Hum Pathol, 39, 102-15.
  10. Ebralidze A, Tulchinsky E, Grigorian M, et al (1989). Isolation and characterization of a gene specifically expressed in different metastatic cells and whose deduced gene product has a high degree of homology to a Ca2+-binding protein family. Genes Dev, 3, 1086-93.
  11. Erez N, Truitt M, Olson P, et al (2010). Cancer-Associated Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting Inflammation in an NF-kappaB-Dependent Manner. Cancer Cell, 17, 135-47.
  12. Forssell J, Oberg A, Henriksson ML, et al (2007). High macrophage infiltration along the tumor front correlates with improved survival in colon cancer. Clin Cancer Res, 13, 1472-9.
  13. Hasita H, Komohara Y, Okabe H, et al (2010). Significance of alternatively activated macrophages in patients with intrahepatic cholangiocarcinoma. Cancer Sci, 101, 1913-9.
  14. Hazan RB, Phillips GR, Qiao RF, et al (2000). Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis. J Cell Biol, 148, 779-90.
  15. Hsu SM, Raine L (1981). Protein A, avidin, and biotin in immunohistochemistry. J Histochem Cytochem, 29, 1349-53.
  16. Huang M, Li Y, Zhang H, et al (2010). Breast cancer stromal fibroblasts promote the generation of CD44+CD24- cells through SDF-1/CXCR4 interaction. J Exp Clin Cancer Res, 29, 80.
  17. IARC (1994). Infection with liver flukes (Opisthorchis viverrini, Opisthorchis felineus and Clonorchis sinensis). IARC Monogr Eval Carcinog Risks Hum, 61, 121-75.
  18. Ishiguro K, Yoshida T, Yagishita H, et al (2006). Epithelial and stromal genetic instability contributes to genesis of colorectal adenomas. Gut, 55, 695-702.
  19. Iwano M, Plieth D, Danoff TM, et al (2002). Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest, 110, 341-50.
  20. Jezequel AM, Mancini R, Rinaldesi ML, et al (1989). Dimethylnitrosamine-induced cirrhosis. Evidence for an immunological mechanism. J Hepatol, 8, 42-52.
  21. Joyce JA, Pollard JW (2009). Microenvironmental regulation of metastasis. Nat Rev Cancer, 9, 239-52.
  22. Kalluri R, Zeisberg M (2006). Fibroblasts in cancer. Nat Rev Cancer, 6, 392-401.
  23. Kang JC, Chen JS, Lee CH, et al (2010). Intratumoral macrophage counts correlate with tumor progression in colorectal cancer. J Surg Oncol, 102, 242-8.
  24. Kim S, Cho SW, Min HS, et al (2013). The expression of tumorassociated macrophages in papillary thyroid carcinoma. Endocrinol Metab (Seoul), 28, 192-8.
  25. Liu CY, Xu JY, Shi XY, et al (2013). M2-polarized tumorassociated macrophages promoted epithelial-mesenchymal transition in pancreatic cancer cells, partially through TLR4/IL-10 signaling pathway. Lab Invest, 93, 844-54.
  26. Loilome W, Yongvanit P, Wongkham C, et al (2006). Altered gene expression in Opisthorchis viverrini-associated cholangiocarcinoma in hamster model. Mol Carcinog, 45, 279-87.
  27. Ma J, Liu L, Che G, et al (2010). The M1 form of tumorassociated macrophages in non-small cell lung cancer is positively associated with survival time. BMC Cancer, 10, 112.
  28. Maniecki MB, Etzerodt A, Ulhoi BP, et al (2012). Tumorpromoting macrophages induce the expression of the macrophage-specific receptor CD163 in malignant cells. Int J Cancer, 131, 2320-31.
  29. Mantovani A, Sica A, Sozzani S, et al (2004). The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol, 25, 677-86.
  30. Martinez FO, Helming L, Gordon S (2009). Alternative activation of macrophages: an immunologic functional perspective. Annu Rev Immunol, 27, 451-83.
  31. McAnulty RJ (2007). Fibroblasts and myofibroblasts: their source, function and role in disease. Int J Biochem Cell Biol, 39, 666-71.
  32. McLean MH, Murray GI, Stewart KN, et al (2011). The inflammatory microenvironment in colorectal neoplasia. PLoS One, 6, 15366.
  33. Medrek C, Ponten F, Jirstrom K, et al (2012). The presence of tumor associated macrophages in tumor stroma as a prognostic marker for breast cancer patients. BMC Cancer, 12, 306.
  34. Mertens JC, Fingas CD, Christensen JD, et al (2013). Therapeutic effects of deleting cancer-associated fibroblasts in cholangiocarcinoma. Cancer Res, 73, 897-907.
  35. Moghaddam SJ, Li H, Cho SN, et al (2009). Promotion of lung carcinogenesis by chronic obstructive pulmonary diseaselike airway inflammation in a K-ras-induced mouse model. Am J Respir Cell Mol Biol, 40, 443-53.
  36. Nabeshima A, Matsumoto Y, Fukushi J, et al (2015). Tumourassociated macrophages correlate with poor prognosis in myxoid liposarcoma and promote cell motility and invasion via the HB-EGF-EGFR-PI3K/Akt pathways. Br J Cancer, 112, 547-55.
  37. Ni YH, Ding L, Huang XF, et al (2015). Microlocalization of CD68 tumor-associated macrophages in tumor stroma correlated with poor clinical outcomes in oral squamous cell carcinoma patients. Tumour Biol.
  38. Ohno S, Inagawa H, Dhar DK, et al (2003). The degree of macrophage infiltration into the cancer cell nest is a significant predictor of survival in gastric cancer patients. Anticancer Res, 23, 5015-22.
  39. Osterreicher CH, Penz-Osterreicher M, Grivennikov SI, et al (2011). Fibroblast-specific protein 1 identifies an inflammatory subpopulation of macrophages in the liver. Proc Natl Acad Sci U S A, 108, 308-13.
  40. Pinlaor S, Tada-Oikawa S, Hiraku Y, et al (2005). Opisthorchis viverrini antigen induces the expression of Toll-like receptor 2 in macrophage RAW cell line. Int J Parasitol, 35, 591-6.
  41. Prakobwong S, Pinlaor S, Yongvanit P, et al (2009). Time profiles of the expression of metalloproteinases, tissue inhibitors of metalloproteases, cytokines and collagens in hamsters infected with Opisthorchis viverrini with special reference to peribiliary fibrosis and liver injury. Int J Parasitol, 39, 825-35.
  42. Prakobwong S, Yongvanit P, Hiraku Y, et al (2010). Involvement of MMP-9 in peribiliary fibrosis and cholangiocarcinogenesis via Rac1-dependent DNA damage in a hamster model. Int J Cancer, 127, 2576-87.
  43. Qian BZ, Pollard JW (2010). Macrophage diversity enhances tumor progression and metastasis. Cell, 141, 39-51.
  44. Rasanen K, Vaheri A (2010). Activation of fibroblasts in cancer stroma. Exp Cell Res, 316, 2713-22.
  45. Sack U, Walther W, Scudiero D, et al (2011). S100A4-induced cell motility and metastasis is restricted by the Wnt/betacatenin pathway inhibitor calcimycin in colon cancer cells. Mol Biol Cell, 22, 3344-54.
  46. Shabo I, Stal O, Olsson H, et al (2008). Breast cancer expression of CD163, a macrophage scavenger receptor, is related to early distant recurrence and reduced patient survival. Int J Cancer, 123, 780-6.
  47. Shaykhiev R, Bals R (2007). Interactions between epithelial cells and leukocytes in immunity and tissue homeostasis. J Leukoc Biol, 82, 1-15.
  48. Sithithaworn P, Yongvanit P, Duenngai K, et al (2014). Roles of liver fluke infection as risk factor for cholangiocarcinoma. J Hepatobiliary Pancreat Sci, 21, 301-8.
  49. Solinas G, Germano G, Mantovani A, et al (2009). Tumorassociated macrophages (TAM) as major players of the cancer-related inflammation. J Leukoc Biol, 86, 1065-73.
  50. Sripa B, Kaewkes S, Sithithaworn P, et al (2007). Liver fluke induces cholangiocarcinoma. PLoS Med, 4, 201.
  51. Subimerb C, Pinlaor S, Khuntikeo N, et al (2010a). Tissue invasive macrophage density is correlated with prognosis in cholangiocarcinoma. Mol Med Report, 3, 597-605.
  52. Subimerb C, Pinlaor S, Lulitanond V, et al (2010b). Circulating CD14(+) CD16(+) monocyte levels predict tissue invasive character of cholangiocarcinoma. Clin Exp Immunol, 161, 471-9.
  53. Sugimoto H, Mundel TM, Kieran MW, et al (2006). Identification of fibroblast heterogeneity in the tumor microenvironment. Cancer Biol Ther, 5, 1640-6.
  54. Techasen A, Namwat N, Loilome W, et al (2014). Tumor necrosis factor-alpha modulates epithelial mesenchymal transition mediators ZEB2 and S100A4 to promote cholangiocarcinoma progression. J Hepatobiliary Pancreat Sci, 21, 703-11.
  55. Thamavit W, Bhamarapravati N, Sahaphong S, et al (1978). Effects of dimethylnitrosamine on induction of cholangiocarcinoma in Opisthorchis viverrini-infected Syrian golden hamsters. Cancer Res, 38, 4634-9.
  56. Trimboli AJ, Cantemir-Stone CZ, Li F, et al (2009). Pten in stromal fibroblasts suppresses mammary epithelial tumours. Nature, 461, 1084-91.
  57. Tsutsui S, Yasuda K, Suzuki K, et al (2005). Macrophage infiltration and its prognostic implications in breast cancer: the relationship with VEGF expression and microvessel density. Oncol Rep, 14, 425-31.
  58. Wang X, Wang H, Li G, et al (2014). Activated macrophages down-regulate expression of E-cadherin in hepatocellular carcinoma cells via NF-kappaB/Slug pathway. Tumour Biol, 35, 8893-901.
  59. Weber F, Shen L, Fukino K, et al (2006). Total-genome analysis of BRCA1/2-related invasive carcinomas of the breast identifies tumor stroma as potential landscaper for neoplastic initiation. Am J Hum Genet, 78, 961-72.
  60. Yongvanit P, Pinlaor S, Bartsch H (2012). Oxidative and nitrative DNA damage: key events in opisthorchiasis-induced carcinogenesis. Parasitol Int, 61, 130-5.
  61. Yongvanit P, Pinlaor S, Loilome W (2014). Risk biomarkers for assessment and chemoprevention of liver fluke-associated cholangiocarcinoma. J Hepatobiliary Pancreat Sci, 21, 309-15.
  62. Zeisberg M, Yang C, Martino M, et al (2007). Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition. J Biol Chem, 282, 23337-47.
  63. Zhang BC, Gao J, Wang J, et al (2011). Tumor-associated macrophages infiltration is associated with peritumoral lymphangiogenesis and poor prognosis in lung adenocarcinoma. Med Oncol, 28, 1447-52.
  64. Zhi K, Shen X, Zhang H, et al (2010). Cancer-associated fibroblasts are positively correlated with metastatic potential of human gastric cancers. J Exp Clin Cancer Res, 29, 66.

Cited by

  1. CD44 variant-dependent redox status regulation in liver fluke-associated cholangiocarcinoma: A target for cholangiocarcinoma treatment vol.107, pp.7, 2016,
  2. Serum Antibody Ig G and Ig M Titers for Opisthorchis felineus Correlate with Eggs in Faeces - a Comprehensive Study in Chuvash Republic, Russia vol.17, pp.1, 2016,
  3. Role of tumor-associated macrophages in human malignancies: friend or foe? vol.66, pp.9, 2016,
  4. LncRNA-MALAT1 Promotes Angiogenesis of Thyroid Cancer by Modulating Tumor-Associated Macrophage FGF2 Protein Secretion vol.118, pp.12, 2017,
  5. M2 macrophage is the predominant phenotype in airways inflammatory lesions in patients with granulomatosis with polyangiitis vol.19, pp.1, 2017,
  6. Establishment of cholangiocarcinoma cell lines from patients in the endemic area of liver fluke infection in Thailand vol.39, pp.11, 2017,
  7. Tumor reactive stroma in cholangiocarcinoma: The fuel behind cancer aggressiveness vol.9, pp.9, 2017,
  8. Epithelial-to-Mesenchymal Transition and Cancer Invasiveness: What Can We Learn from Cholangiocarcinoma? vol.4, pp.12, 2015,
  9. The microRNA-15a-PAI-2 axis in cholangiocarcinoma-associated fibroblasts promotes migration of cancer cells vol.17, pp.1, 2018,
  10. The role of tumour microenvironment: a new vision for cholangiocarcinoma pp.15821838, 2018,
  11. Fucosyl-Agalactosyl IgG1 Induces Cholangiocarcinoma Metastasis and Early Recurrence by Activating Tumor-Associated Macrophage vol.10, pp.11, 2018,
  12. Prognostic biomarkers for cholangiocarcinoma and their clinical implications vol.18, pp.6, 2018,