Tumor Biology

, Volume 36, Issue 6, pp 4467–4477 | Cite as

Interpretation of immunohistochemistry data of tumor should consider microenvironmental factors

Research Article


The influence of tumor surrounding microenvironment is often neglected when immunohistochemistry is performed to investigate tumor properties and search biomarkers of cancer. This study was designed to evaluate whether the influence of tumor microenvironment on biological features of tumor cells should be taken into account for interpretation of the immunohistochemistry data of tumor specimens. In this study, we showed an example by using three tumor cell lines (HeLa, WSU-HN6, and Tca83) to establish tumor-caused bone destruction models in nude mice and then to investigate the influence of bone marrow microenvironment (BMM) on biological features of tumor cells. Immunohistochemistry results showed that, compared with tumor cells located outside of BMM, tumor cells located inside of BMM presented huge differences in the expression of inflammation-related proteins including tumor necrosis factor-α (TNF-α), TNF receptor-associated factor protein-6 (TRAF-6), phosphorylated-NF-κB p65 (p-p65), interleukin (IL)-6 and IL-11, matrix metalloproteinases including MMP-1, MMP-2, MMP-9, and MMP-13; and osteogenesis-related proteins including runt-related transcription factor 2 (RUNX2), bone sialoprotein (BSP), and osteocalcin (OCN) in all the models. However, when we compared the cell line pair derived from different sites (outside and inside of BMM, respectively) of the same HeLa tumor sample by real-time PCR, Western blot, and immunocytochemistry, the differences aforementioned in tumor tissues were not found. In addition, we verified that normal human bone marrow could not cause the above changes detected in vivo. Our results suggested that tumor-modified microenvironment could give the new biological features of the invaded tumor cells. Therefore, we should consider the influence of the surrounding microenvironment on tumor cells when we analyze tumor properties using immunohistochemistry.


Cancer microenvironment Immunohistochemistry Interpretation Homologous cell lines 



The work was supported by the research grants from Special Fund for Development of Capital Health care (2011-4025-02), Nature Science Foundation of Heilongjiang Province, China (Grant No. QC2014C107), and the Ministry of Science and Technology of China under contract International Science & Technology Cooperation Program Foundation (Grant No. 1019).

Conflicts of interest



  1. 1.
    Ordonez-Moran P, Huelsken J. Complex metastatic niches: already a target for therapy? Curr Opin Cell Biol. 2014;31C:29–38.CrossRefGoogle Scholar
  2. 2.
    Erin N, Kale S, Tanriover G, Koksoy S, Duymus O, Korcum AF. Differential characteristics of heart, liver, and brain metastatic subsets of murine breast carcinoma. Breast Cancer Res Treat. 2013;139:677–89.CrossRefPubMedGoogle Scholar
  3. 3.
    Wood SL, Pernemalm M, Crosbie PA, Whetton AD. The role of the tumor-microenvironment in lung cancer-metastasis and its relationship to potential therapeutic targets. Cancer Treat Rev. 2014;40:558–66.CrossRefPubMedGoogle Scholar
  4. 4.
    Rahim F, Hajizamani S, Mortaz E, Ahmadzadeh A, Shahjahani M, Shahrabi S, et al. Molecular regulation of bone marrow metastasis in prostate and breast cancer. Bone Marrow Res. 2014;2014:405920.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Rondeau G, Abedinpour P, Desai P, Baron VT, Borgstrom P, Welsh J. Effects of different tissue microenvironments on gene expression in breast cancer cells. PLoS One. 2014;9:e101160.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Shi H, Hayes M, Kirana C, Miller R, Keating J, Macartney-Coxson D, et al. TUFM is a potential new prognostic indicator for colorectal carcinoma. Pathology. 2012;44:506–12.CrossRefPubMedGoogle Scholar
  7. 7.
    Pei J, Fu W, Yang L, Zhang Z, Liu Y. Oxidative stress is involved in the pathogenesis of Keshan disease (an endemic dilated cardiomyopathy) in China. Oxid Med Cell Longev. 2013;2013:474203.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Cong L, Pu CQ, Shi Q, Wang Q, Lu XH. Complement membrane attack complex is related with immune-mediated necrotizing myopathy. Int J Clin Exp Pathol. 2014;7:4143–9.PubMedPubMedCentralGoogle Scholar
  9. 9.
    Labelle P, Reilly CM, Naydan DK, Labelle AL. Immunohistochemical characteristics of normal canine eyes. Vet Pathol. 2012;49:860–9.CrossRefPubMedGoogle Scholar
  10. 10.
    Zhu LJ, Dai L, Zheng DH, Mo YQ, Ou-Yang X, Wei XN, et al. Upregulation of tumor necrosis factor receptor-associated factor 6 correlated with synovitis severity in rheumatoid arthritis. Arthritis Res Ther. 2012;14:R133.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Furuta H, Osawa K, Shin M, Ishikawa A, Matsuo K, Khan M, et al. Selective inhibition of NF-kappaB suppresses bone invasion by oral squamous cell carcinoma in vivo. Int J Cancer. 2012;131:E625–35.CrossRefPubMedGoogle Scholar
  12. 12.
    Tang CH, Chuang JY, Fong YC, Maa MC, Way TD, Hung CH. Bone-derived SDF-1 stimulates IL-6 release via CXCR4, ERK and NF-kappaB pathways and promotes osteoclastogenesis in human oral cancer cells. Carcinogenesis. 2008;29:1483–92.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Chen YC, Sosnoski DM, Mastro AM. Breast cancer metastasis to the bone: mechanisms of bone loss. Breast Cancer Res. 2010;12:215.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    14 Bodnar M, Szylberg L, Kazmierczak W, Marszalek A. Tumor progression driven by pathways activating matrix metalloproteinases and their inhibitors. J Oral Pathol Med 2014.Google Scholar
  15. 15.
    Chakravarthi BV, Pathi SS, Goswami MT, Cieslik M, Zheng H, Nallasivam S, et al. The miR-124-Prolyl Hydroxylase P4HA1-MMP1 axis plays a critical role in prostate cancer progression. Oncotarget. 2014;5:6654–69.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Balbin M, Pendas AM, Uria JA, Jimenez MG, Freije JP, Lopez-Otin C. Expression and regulation of collagenase-3 (MMP-13) in human malignant tumors. Apmis. 1999;107:45–53.CrossRefPubMedGoogle Scholar
  17. 17.
    McDonald L, Ferrari N, Terry A, Bell M, Mohammed ZM, Orange C, et al. RUNX2 correlates with subtype-specific breast cancer in a human tissue microarray, and ectopic expression of Runx2 perturbs differentiation in the mouse mammary gland. Dis Model Mech. 2014;7:525–34.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Baniwal SK, Khalid O, Gabet Y, Shah RR, Purcell DJ, Mav D, et al. Runx2 transcriptome of prostate cancer cells: Insights into invasiveness and bone metastasis. Mol Cancer. 2010;9:258.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Pratap J, Lian JB, Javed A, Barnes GL, van Wijnen AJ, Stein JL, et al. Regulatory roles of Runx2 in metastatic tumor and cancer cell interactions with bone. Cancer Metastasis Rev. 2006;25:589–600.CrossRefPubMedGoogle Scholar
  20. 20.
    Zhang JH, Wang J, Tang J, Barnett B, Dickson J, Hahsimoto N, et al. Bone sialoprotein promotes bone metastasis of a non-bone-seeking clone of human breast cancer cells. Anticancer Res. 2004;24:1361–8.PubMedGoogle Scholar
  21. 21.
    Waltregny D, Bellahcene A, de Leval X, Florkin B, Weidle U, Castronovo V. Increased expression of bone sialoprotein in bone metastases compared with visceral metastases in human breast and prostate cancers. J Bone Miner Res. 2000;15:834–43.CrossRefPubMedGoogle Scholar
  22. 22.
    Ogbureke KU, Nikitakis NG, Warburton G, Ord RA, Sauk JJ, Waller JL, et al. Up-regulation of SIBLING proteins and correlation with cognate MMP expression in oral cancer. Oral Oncol. 2007;43:920–32.CrossRefPubMedGoogle Scholar
  23. 23.
    Salem AM, Zohny SF, Abd EM, Hamdy R. Predictive value of osteocalcin and beta-CrossLaps in metastatic breast cancer. Clin Biochem. 2007;40:1201–8.CrossRefPubMedGoogle Scholar
  24. 24.
    Gardner TA, Lee SJ, Lee SD, Li X, Shirakawa T, Kwon DD, et al. Differential expression of osteocalcin during the metastatic progression of prostate cancer. Oncol Rep. 2009;21:903–8.PubMedGoogle Scholar
  25. 25.
    Huang WC, Xie Z, Konaka H, Sodek J, Zhau HE, Chung LW. Human osteocalcin and bone sialoprotein mediating osteomimicry of prostate cancer cells: role of cAMP-dependent protein kinase a signaling pathway. Cancer Res. 2005;65:2303–13.CrossRefPubMedGoogle Scholar
  26. 26.
    Moskaluk CA, Baras AS, Mancuso SA, Fan H, Davidson RJ, Dirks DC, et al. Development and characterization of xenograft model systems for adenoid cystic carcinoma. Lab Invest. 2011;91:1480–90.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Krishna M. Diagnosis of metastatic neoplasms: an immunohistochemical approach. Arch Pathol Lab Med. 2010;134:207–15.PubMedGoogle Scholar
  28. 28.
    Pu Y, Wang L, Wu H, Feng Z, Wang Y, Guo C. High MMP-21 expression in metastatic lymph nodes predicts unfavorable overall survival for oral squamous cell carcinoma patients with lymphatic metastasis. Oncol Rep. 2014;31:2644–50.PubMedGoogle Scholar
  29. 29.
    Sakashita T, Homma A, Suzuki S, Hatakeyama H, Kano S, Mizumachi T, et al. Prognostic value of cyclin D1 expression in tumor-free surgical margins in head and neck squamous cell carcinomas. Acta Otolaryngol. 2013;133:984–91.CrossRefPubMedGoogle Scholar
  30. 30.
    Liao CT, Wallace CG, Lee LY, Hsueh C, Lin CY, Fan KH, et al. Clinical evidence of field cancerization in patients with oral cavity cancer in a betel quid chewing area. Oral Oncol. 2014;50:721–31.CrossRefPubMedGoogle Scholar
  31. 31.
    Luo Y, Yu M, Grady WM. Field cancerization in the colon: a role for aberrant DNA methylation? Gastroenterol Rep (Oxf). 2014;2:16–20.CrossRefGoogle Scholar
  32. 32.
    Torezan LA, Festa-Neto C. Cutaneous field cancerization: clinical, histopathological and therapeutic aspects. An Bras Dermatol. 2013;88:775–86.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    33 Lochhead P, Chan AT, Nishihara R, Fuchs CS, Beck AH, Giovannucci E, Ogino S. Etiologic field effect: reappraisal of the field effect concept in cancer predisposition and progression. Mod Pathol 2014.Google Scholar
  34. 34.
    Roodman GD. Mechanisms of bone metastasis. N Engl J Med. 2004;350:1655–64.CrossRefPubMedGoogle Scholar
  35. 35.
    Thomas RJ, Guise TA, Yin JJ, Elliott J, Horwood NJ, Martin TJ, et al. Breast cancer cells interact with osteoblasts to support osteoclast formation. Endocrinology. 1999;140:4451–8.Google Scholar
  36. 36.
    Ackerman D, Simon MC. Hypoxia, lipids, and cancer: surviving the harsh tumor microenvironment. Trends Cell Biol. 2014;24:472–8.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Ji RC. Hypoxia and lymphangiogenesis in tumor microenvironment and metastasis. Cancer Lett. 2014;346:6–16.CrossRefPubMedGoogle Scholar
  38. 38.
    Spivak-Kroizman TR, Hostetter G, Posner R, Aziz M, Hu C, Demeure MJ, et al. Hypoxia triggers hedgehog-mediated tumor-stromal interactions in pancreatic cancer. Cancer Res. 2013;73:3235–47.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Zeng W, Liu P, Pan W, Singh SR, Wei Y. Hypoxia and hypoxia inducible factors in tumor metabolism. Cancer Lett 2014Google Scholar
  40. 40.
    Hasmim M, Messai Y, Noman MZ. Chouaib S [Tumor hypoxia: a key player in the regulation of stromal and anti-tumor responses]. Med Sci (Paris). 2014;30:422–8.CrossRefGoogle Scholar
  41. 41.
    Tsai YP, Wu KJ. Hypoxia-regulated target genes implicated in tumor metastasis. J Biomed Sci. 2012;19:102.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Ren Y, Hao P, Dutta B, Cheow ES, Sim KH, Gan CS, et al. Hypoxia modulates A431 cellular pathways association to tumor radioresistance and enhanced migration revealed by comprehensive proteomic and functional studies. Mol Cell Proteomics. 2013;12:485–98.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Huan Bian
    • 1
  • Shuai Zhang
    • 1
  • Huanhuan Wu
    • 1
  • Yixiang Wang
    • 2
  1. 1.Department of Oral and Maxillofacial SurgeryPeking University School and Hospital of StomatologyHaidian DistrictChina
  2. 2.Central LaboratoryPeking University School and Hospital of StomatologyHaidian DistrictChina

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