Dysregulation of macrophage polarization is associated with the metastatic process in osteosarcoma

Clotilde Dumars, Jean-Michel Ngyuen, Aurélie Gaultier, Rachel Lanel, Nadège Corradini, François Gouin, Dominique Heymann, Marie-Françoise Heymann, Clotilde Dumars, Jean-Michel Ngyuen, Aurélie Gaultier, Rachel Lanel, Nadège Corradini, François Gouin, Dominique Heymann, Marie-Françoise Heymann

Abstract

Osteosarcoma (OS) is the most common bone sarcoma in adolescents, and has poor prognosis. A vicious cycle is established between OS cells and their microenvironment in order to facilitate the tumor growth and cell spreading. The present work aims to better characterize the tumor microenvironment in OS in order to identify new therapeutic targets relating to metastatic process. Tissue microarrays of pre-chemotherapy OS biopsies were used for characterizing the tumor niche by immunohistochemistry. Parameters studies included: immune cells (M1, M2-subtypes of tumor-associated macrophages (TAM); T, B lymphocytes; mast cells), vascularization (endothelial, perivascular cells), OPG, RANKL, and mitotic index. Two groups of patients were defined, 22 localized OS (OS Meta-) and 28 metastatic OS (OS Meta+). The OS Meta- group was characterized by a higher infiltration of INOS+ M1-polarizedmacrophages and upregulated OPG immunostaining. OS Meta+ tumors showed a significant increase in CD146+ cells. INOS+ M1-macrophages were correlated with OPG staining, and negatively with the presence of metastases. CD163+ M2-macrophages were positively correlated with CD146+ cells. In multivariate analysis, INOS and OPG were predictive factors for metastasis. An older age, non-metastatic tumor, good response to chemotherapy, and higher macrophage infiltration were significantly associated with better overall survival. TAMs are associated with better overall survival and a dysregulation of M1/M2 polarized-macrophages in favor of M1 subtype was observed in non-metastatic OS.

Keywords: Pathology Section; osteoprotegerin; osteosarcoma; tumour associated macrophage.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors report no declarations of interest.

Figures

Figure 1. Representative immunohistochemical results of CD68,…
Figure 1. Representative immunohistochemical results of CD68, INOS, CD146 and OPG, in primary lesions of OS Meta- and OS Meta+
The immunohistochemical study was performed on tissue micro-arrays and analyzed on digitized images. Macrophages are the main cells in the bone niche; the qualitatively analysis revealed their perivascular location (Bar scale 250 and 50 µm, original magnification x10 and x40 respectively). Quantitative analysis showed a significantly higher infiltration of M1-polarized macrophages in OS Meta- patients [number of INOS+ cells for 1 high power (x40) microscopic field (HPF) estimated on 3 “hot-spots“; median value symbolized with a horizontal bar] (Fisher test, p = 0.001). On the contrary, OS Meta+ had a significantly higher vascular density (CD146 staining) (Wilcoxon test, p = 0.014) and OPG density was significantly higher in the OS Meta- group (Wilcoxon test, p = 0.028). (Semi-quantitative analysis of vascular or cellular density: 0, no staining; 1, 2/3 of the tumor surface). OS Meta-: patients with non-metastatic OS; OS Meta+: patients with metastatic OS
Figure 2. INOS+ M1-polarized macrophages correlate CD3+…
Figure 2. INOS+ M1-polarized macrophages correlate CD3+ CD8+ lymphocytes, OPG, RANKL and the mitotic index and M2-polarized macrophage with the vascularization in primary lesions of OS
A. A positive correlation was found between INOS+ M1-polarized macrophages and CD3+CD8+ lymphocytes, OPG, RANKL and the mitotic index (Spearman correlation, p = 0.00151, p = 0.03131, p = 0.0150, p = 0.00218 respectively). M2-polarized macrophages as determined by CD163+ cells, were correlated with vascularity as determined by CD146+ cell density (Spearman correlation, p = 0.00154). Kaplan-Meier Curve of survival, according to presence of metastasis at diagnosis, and of response to chemotherapy: B. Median overall survival was 5.12 years (95%CI: 4.62-7.12). C. Patients with metastasis (synchronous or metachronous) were associated with significantly lower median overall survival [3.26 years (95%CI: 3.78-7.18)], p < 0.001. D. Patients with a worse histological response to chemotherapy [defined as < 90% of tumor necrosis after neoadjuvant chemotherapy] were associated with worse overall survival [4.61 years (95%CI: 3.78-7.18)], p < 0.01. Meta-: non-metastatic patients; Meta+: metastatic patients; Grade III, I-II according the Huvos score.
Figure 3. Flow chart of inclusion/exclusion criteria
Figure 3. Flow chart of inclusion/exclusion criteria
159 patients were enrolled and after the selective process two groups of patients were defined: 22 non-metastatic patients “OS Meta- group”, and 28 metastatic patients, “OS Meta+ group”.

References

    1. The ESMO / European Sarcoma Network Working Group Bone sarcomas: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2012(23):vii100–vii109.
    1. Mohseny AB, Hogendoorn PCW. Concise review: mesenchymal tumors: when stem cells go mad. Stem Cells. 2011;29:397–403.
    1. Xiao W, Mohseny AB, Hogendoorn PC, Celton-Jansen AM. Mesenchymal stem cell transformation and sarcoma genesis. Clin Sarcoma Res. 2013(3):10.
    1. Messerschmitt PJ, Garcia RM, Abdul-Karim FW, Greenfield EM, Getty PJ. Osteosarcoma. J Am Acad Orthop Surg. 2009(17):515–527.
    1. Grimaud E, Soubigou L, Couillaud S, Coipeau P, Moreau A, Passuti N, Gouin F, Redini F, Heymann D. Receptor activator of nuclear factor kappaB ligand (RANKL)/osteoprotegerin (OPG) ratio is increased in severe osteolysis. Am J Pathol. 2003(163):2021–2031.
    1. Ungefroren H, Sebens S, Seidl D, Lehnert H, Hass R. Interaction of tumor cells with the microenvironment. Cell Commun Signal. 2011(9):18.
    1. Zhu L, McManus MM, Hughes DPM. Understanding the Biology of Bone Sarcoma from Early Initiating Events through Late Events in Metastasis and Disease Progression. Front Oncol. 2013(3):230.
    1. Meads MB, Hazlehurst LA, Dalton WS. The bone marrow microenvironment as a tumor sanctuary and contributor to drug resistance. Clin Cancer Res. 2008(14):2519–2526.
    1. Olechnowicz SWZ, Edwards CM. Contributions of the host microenvironment to cancer-induced bone disease. Cancer Res. 2014(74):1625–1631.
    1. Quail DF, Joyce JA. Microenvironmental regulation of tumor progression and metastasis. Nat Med. 20113(19):1423–1437.
    1. Endo-Munoz L, Evdokiou A, Saunders NA. The role of osteoclasts and tumor-associated macrophages in osteosarcoma metastasis. Biochim Biophys Acta. 2012:1826, 434–442.
    1. Theoleyre S, Mori K, Cherrier B, Passuti N, Gouin F, Rédini F, Heymann D. Phenotypic and functional analysis of lymphocytes infiltrating osteolytic tumors: use as a possible therapeutic approach of osteosarcoma. BMC Cancer. 2005(5):123.
    1. Gordon S, Martinez FO. Alternative activation of macrophages: mechanism and functions. Immunity. 2010(32):593–604.
    1. Lewis CE, Pollard JW. Distinct role of macrophages in different tumor microenvironments. Cancer Res. 2006(66):605–612.
    1. Qian BZ, Pollard JW. Macrophage diversity enhances tumor progression and metastasis. Cell. 2010(141):39–51.
    1. Rogers TL, Holen I. Tumor macrophages as potential targets of bisphosphonates. J Transl Med. 2011(9):177.
    1. Alfranca A, Martinez-Cruzado L, Tornin J, Abarrategi A, Amaral T, de Alava E, Menendez P, Garcia-Castro J, Rodriguez R. Bone microenvironment signals in osteosarcoma development. Cell Mol Life Sci. 2015(72):3097–3113.
    1. Ando K, Heymann MF, Stresing V, Mori K, Rédini F, Heymann D. Current therapeutic strategies and novel approaches in osteosarcoma. Cancers. 2013;5(591):616.
    1. Baud'huin M, Lamoureux F, Duplomb L, Rédini F, Heymann D. RANKL, RANK, osteoprotegerin: key partners of osteoimmunology and vascular diseases. Cell Mol Life Sci. 2007(64):2334–2350.
    1. Fidler IJ. The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited. Nat Rev Cancer. 2003(3):453–458.
    1. Paget S. The distribution of secondary growths in cancer of the breast. 1889. Cancer Metastasis Rev. 1989(8):98–101.
    1. Buddingh EP, Kuijjer ML, Duim RA, Bürger H, Agelopoulos K, Myklebost O, Serra M, Mertens F, Hogendoorn PC, Lankester AC, Cleton-Jansen AM. Tumor-infiltrating macrophages are associated with metastasis suppression in high-grade osteosarcoma: a rationale for treatment with macrophage activating agents. Clin Cancer Res. 2011(17):2110–2119.
    1. Xiao Q, Zhang X, Wu Y, Yang Y. Inhibition of macrophage polarization prohibits growth of human osteosarcoma. Tumor Biol. 2014(35):7611–7616.
    1. Pahl JHW, Kwappenberg KM, Varypataki EM, Santos SJ, Kuijjer ML, Mohamed S, Wijnen JT, van Tol MJ, Cleton-Jansen AM, Egeler RM, Jiskoot W, Lankester AC, Schilham MW. Macrophages inhibit human osteosarcoma cell growth after activation with the bacterial cell wall derivative liposomal muramyl tripeptide in combination with interferon-γ. J Exp Clin Cancer Res. 2014(33):27.
    1. Ségaliny AI, Mohamadi A, Dizier B, Lokajczyk A, Brion R, Lanel R, Amiaud J, Charrier C, Boisson-Vidal C, Heymann D. Interleukin-34 promotes tumor progression and metastatic process in osteosarcoma through induction of angiogenesis and macrophage recruitment. Int J Cancer. 2015(137):73–85.
    1. de Nigris F, Mancini FP, Schiano C, Infante T, Zullo A, Minucci PB, Al-Omran M, Giordano A, Napoli C. Osteosarcoma cells induce endothelial cell proliferation during neo-angiogenesis. J Cell Physiol. 2013(228):846–852.
    1. Schiano C, Grimaldi V, Casamassimi A, Infante T, Esposito A, Giovane A, Napoli C. Different expression of CD146 in human normal and osteosarcoma cell lines. Med Oncol. 2012(29):2998–3002.
    1. Dirkx AE, Oude Egbrink MG, Wagstaff J, Griffioen AW. Monocyte/macrophage infiltration in tumors: modulators of angiogenesis. J Leukoc Biol. 2006;80:1183–1196.
    1. Lin EY, Pollard JW. Tumor-associated macrophages press the angiogenic switch in breast cancer. Cancer Res. 2007(67):5064–5066.
    1. Lin EY, Li JF, Gnatovskiy L, Deng Y, Zhu L, Grzesik DA, Qian H, Xue XN, Pollard JW. Macrophages regulate the angiogenic switch in a mouse model of breast cancer. Cancer Res. 2006(66):11238–11246.
    1. Murdoch C, Muthana M, Coffelt SB, Lewis CE. The role of myeloid cells in the promotion of tumor angiogenesis. Nat Rev Cancer. 2008(8):618–631.
    1. Guo C, Buranych A, Sarkar D, Fisher PB, Wang XY. The role of tumor-associated macrophages in tumor vascularization. Vasc Cell. 2013(5):20.
    1. Ando K, Mori K, Verrecchia F, Baud'huin Rédini F, Heymann D. Molecular alterations associated with osteosarcoma development. Sarcoma. 2012;2012:523432.
    1. Guo M, Cai C, Zhao G, Qiu X, Zhao H, Ma Q, Tian L, Li X, Hu Y, Liao B, Ma B, Fan Q. Hypoxia promotes migration and induces CXCR4 expression via HIF-1α activation in human osteosarcoma. PloS One. 2014(9):e90518.
    1. Lamoureux F, Moriceau G, Picarda G, Rousseau J, Trichet V, Rédini F. Regulation of osteoprotegerin pro- or anti-tumoral activity by bone tumor microenvironment. Biochim Biophys Acta. 2010(1805):17–24.
    1. Avnet S, Longhi A, Salerno M, Halleen JM, Perut F, Granchi D, Ferrari S, Bertoni F, Giunti A, Baldini N. Increased osteoclast activity is associated with aggressiveness of osteosarcoma. Int J Oncol. 2008(33):1231–1238.
    1. Endo-Munoz L, Cumming A, Rickwood D, Wilson D, Cueva C, Ng C, Strutton G, Cassady AI, Evdokiou A, Sommerville S, Dickinson I, Guminski A, Saunders NA. Loss of osteoclasts contributes to development of osteosarcoma pulmonary metastases. Cancer Res. 2010(70):7063–7072.
    1. Guihard P, Danger Y, Brounais B, David E, Brion R, Delecrin J, Richards CD, Chevalier S, Rédini F, Heymann D, Gascan H, Blanchard F. Induction of osteogenesis in mesenchymal stem cells by activated monocytes/macrophages depends on oncostatin M signaling. Stem Cells. 2012(30):762–772.
    1. Heymann MF, Herisson F, Davaine JM, Charrier C, Battaglia S, Passuti N, Lambert G, Gouëffic Y, Heymann D. Role of the OPG/RANK/RANKL triad in calcifications of the atheromatous plaques: comparison between carotid and femoral beds. Cytokine. 2012(58):300–306.
    1. Mosheimer BA, Kaneider NC, Feistritzer C, et al. Expression and function of RANK in human monocyte chemotaxis. Arthritis Rheum. 2004(50):2309–2316.
    1. Mosheimer BA, Kaneider NC, Feistritzer C, Djanani AM, Sturn DH, Patsch JR, Wiedermann CJ. Syndecan-1 is involved in osteoprotegerin-induced chemotaxis in human peripheral blood monocytes. J Clin Endocrinol Metab. 2005(90):2964–2971.
    1. Ando K, Mori K, Corradini N, Redini F, Heymann D. Mifamurtide for the treatment of nonmetastatic osteosarcoma. Expert Opin Pharmacother. 2011(12):285–292.
    1. Chou AJ, Kleinerman ES, Krailo MD, Chen Z, Betcher DL, Healey JH, Conrad EU, 3rd, Nieder ML, Weiner MA, Wells RJ, Womer RB, Meyers PA, Children's Oncology Group Addition of muramyl tripeptide to chemotherapy for patients with newly diagnosed metastatic osteosarcoma: a report from the Children's Oncology Group. Cancer. 2009(115):5339–5348.
    1. Meyers PA, Chou AJ. Muramyl tripeptide-phosphatidyl ethanolamine encapsulated in liposomes (L-MTP-PE) in the treatment of osteosarcoma. Adv Exp Med Biol. 2014(804):307–321.
    1. Cathomas R, Rothermundt C, Bode B, Fuchs B, von Moos R, Schwitter M. RANK ligand blockade with denosumab in combination with sorafenib in chemorefractory osteosarcoma: a possible step forward? Oncology. 2015(88):257–260.
    1. Lamoureux F, Richard P, Wittrant Y, Battaglia S, Pilet P, Trichet V, Blanchard F, Gouin F, Pitard B, Heymann D, Redini F. Therapeutic relevance of osteoprotegerin gene therapy in osteosarcoma: blockade of the vicious cycle between tumor cell proliferation and bone resorption. Cancer Res. 2007(67):7308–7318.
    1. Rousseau J, Escriou V, Lamoureux F, Brion R, Chesneau J, Battaglia S, Amiaud J, Scherman D, Heymann D, Rédini F, Trichet V. Formulated siRNAs targeting Rankl prevent osteolysis and enhance chemotherapeutic response in osteosarcoma models. J Bone Miner Res. 2011(26):2452–2462.
    1. Heymann MF, Brown H, Heymann D. Drugs in early clinical development for the treatment of osteosarcoma. Expert Opin Investig Drugs. 2016(25):1265–1280.
    1. Burningham Z, Hashibe M, Spector L, Schiffman JD. The epidemiology of sarcoma. Clin Sarcoma Res. 2012(2):14.
    1. WHO classification of tumors of soft tissue and bone. IARC Press; 2013.
    1. Rosen G. Preoperative (neoadjuvant) chemotherapy for osteogenic sarcoma: a ten year experience. Orthopedics. 1985(8):659–664.

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