Letters to the Editor 951 Tumor-associated and metalloproteinase inducer in prognosis of multiple myeloma

Leukemia (2016) 30, 951–954; doi:10.1038/leu.2015.191 Because it is the M2φ that are mainly implicated to have pro- tumoral role, we first evaluated the extent of TAM infiltration in myeloma patients specifically with M2φ using CD163 marker. Multiple myeloma (MM) largely remains incurable and despite CD163 is a scavenger receptor for hemoglobin–haptoglobin therapeutic advances in the last decade, patients eventually complex and is expressed exclusively on cells of monocyte– relapse or develop resistance to therapies.1 The interaction of MM lineage. We specifically chose CD163 because it is cells with different cell components of the tumor microenviron- abundant, sensitive and accurate cell surface marker for M2φ and ment has a significant role in conferring resistance to therapy and all its subtypes.11,12 in the maintenance of residual disease, ultimately dictating the Freshly isolated stroma (CD138 negative fraction) from MM clinical behavior and prognosis.2 patients was collected after ROBOSEP separation of whole bone Tumor-associated macrophages (TAMs) arise from in situ marrow and was stained with Alexa Fluor 647 anti-Human CD163 maturation of recruited circulating monocytes and constitute a antibody (Biolegend, San Diego, CA, USA, clone RM3/1) and sorted significant part of myeloma tumor microenvironment. Once for CD163-positive cells on a BD FACSAria III cell sorter (BD recruited to microenvironment, they are exposed to tumor- and Biosciences, San Jose, CA, USA). Using this, we found M2φ 3 stroma-secreted factors leading to macrophage activation. infiltration in all 10 patients (4 newly diagnosed MM and 6 relapsed Activated macrophages can be grouped as M1 TAMs (M1φ, MM) with a range of 4–15% of the bone marrow cellularity classically activated) and M2 TAMs (M2φ, alternatively activated) (Figures 1a and b). We confirmed this observation by staining subtypes, which are extremes of a dynamic continuum rather than paraffin-embedded bone marrow sections of MM patients with mutually exclusive cell states and display plasticity in both anti-human CD163 antibody, which showed CD163-positive M2φ phenotype and function. The hallmarks of M1 activation are iNOS in abundance (Novocastra, Newcastle, UK, Clone10D6, 1:200; expression, high level of interleukin (IL)-12, low level of IL-10 2 Figures 1c and d). We further extended our observations to production and thus they have immuno-stimulatory functions. a large number (131) of patient samples from different plasma φ M2 are formed under selective pressure because of the cell disorders including MGUS, SMM and MM by analyzing bidirectional interactions of macrophages with myeloma cells M2φ in human tissue microarray (TMA), which consisted of and mesenchymal stromal cells, which effectively educate these paraffin-embedded bone marrow biopsy sections (Mayo Cohort) macrophages to perform pro-tumoral functions. The hallmarks of represented by three core biopsies per patient. Staining was M2 activation are increased amounts of IL-10 and low amounts of α performed using the ImmPACT VIP Substrate (Vector Labs, IL-12 and - , thus orchestrating a locally Burlingame, CA, USA, SK-4605) kit. A detailed slide staining immunosuppressive environment and allowing the tumor to protocol, TMA construction and patient information are included progress.3 Myelomas are highly vascularized tumors and increased (Supplementary Table S1). TMA slides for CD163 stain were scanned neovascularization imparts poor prognosis. Traditionally, plasma using the Bacus Laboratories Inc. (Lombard, IL, USA) slide scanner. cells and mesenchymal stromal cells are assumed to be major The images were captured at 480 × 751 pixel resolution at × 40 sources of vascular endothelial growth factor (VEGF).2 Current magnification and converted to webslide format for viewing and findings additionally suggest, M2φ as another major contributors of VEGF and the central orchestrators of the angiogenic switch counting. After manual count, the highest number of macrophages observed during progression from MGUS to MM. This is attributed from among the three replicate cores was used to denote the φ number of macrophages for each patient. The data were to increased secretion of VEGF by M2 , which in turn is enhanced fi also by a paracrine loop between myeloma and stromal cells as independently veri ed by a pathologist by counting random cores. fi φ fi the tumor expands.4,5 In addition to promoting an immuno- We nd M2 in ltration in all stages of MM, median number of φ suppressive environment, a pro-tumoral role of M2φ is ascribed to M2 per core in different stages ranged from 67 to 80 with no fi secretion of factors such as IL-6 and IL-10. IL-10 secreted mainly by signi cant difference between different stages (Supplementary φ the M2φ is a myeloma growth factor6 and was recently shown to Table S2), although in some MGUS and SMM patients, the M2 φ enhance in MM through VEGF secretion.7 In addition were smaller as compared with huge star-shaped M2 in MM (data to the cytokines, CD147, which is an extracellular matrix not shown). Groups for survival plotting were created based on 100 metalloproteinase inducer (EMMPRIN), is also implicated in MM as cutoff number for M2φ per core and overall survival was tumor progression with levels increasing from benign to analyzed using Kaplan–Meier curves and compared using the malignant disease.8 CD147 functions to stimulate tumor cells log-rank test. Using 100 M2φ count per core as a cutoff for and adjacent fibroblasts to produce TAM infiltration, we found a significant difference in survival and also stimulates expression of VEGF, which leads to (4100 M2φ predict poor prognosis) within relapsed MM patients angiogenesis.9,10 As induction of angiogenesis through VEGF is (32 vs 6 months, P =0.02; Figures 1e and g) and non-hyperdiploid the common theme for both M2φ and CD147, the primary aim of MM patients (53 vs 22 months, P = 0.01; Figure 1f and h). We did this study is to evaluate the degree of M2φ infiltration and CD147 not find significant difference in survival in other groups expression in the bone marrow of patients with different stages of analyzed. On the basis of our observation and the known pro- MM ranging in spectrum from MGUS to relapsed MM. tumoral role of M2φ TAMs, we speculate that M2φ might have a All patients in this study consented to research under the IRB role in modulating drug resistance and imparting aggressiveness 919-04 protocol. to MM clonal cells.

Accepted article preview online 23 July 2015; advance online publication, 11 August 2015

© 2016 Macmillan Publishers Limited (2016) 947 – 1002 Letters to the Editor 952

Figure 1. TAM infiltration in bone marrow of MM patients and its prognostic significance. (a)Representativeflow cytometric analysis showing scatter plot of cell components of freshly isolated bone marrow stroma of myeloma patient, where the dead population (blue) is stained with propidium iodide and M2φ (red) are stained with CD163 Alexa-fluor 647. (b)Post-sortM2φ for purity analysis are shown. (c)Paraffin-embedded bone marrow biopsy section at high-power field (×40) of myeloma patient probed with anti-human CD163 antibody (VIP substrate stained purple) incubated for 30 min at room temperature to mark M2φ infiltration. Photomicrographs were acquired using Zeiss Axiophot microscope and an Axiocam MRc5 digital camera. (d) Same tissue stained with universal negative control serum. (e–f) TMA comprised of bone marrow biopsy samples of 131 myeloma patients with different stages was stained with CD163 antibody to measure number of M2φ presentineachcore.Kaplan–Meier survival curve showing difference in survival based on number of M2φ present in TMA core within relapsed (e) and non-hyperdiploid (f) subgroups of Mayo cohort. (g–h) Corresponding representative images of TMA core (×10) stained with CD163 antibody showing a core with 4100 M2φ and o100 M2φ for relapsed (g) and non-hyperdiploid subgroups (h).

The interaction between TAMs and tumor cells has recently previously generated in our lab using the Affymetrix (Santa Clara, been shown to promote angiogenesis via induction of CD147 CA, USA) Human Genome U133A array platform and is available expression in tumor cells.13 In MM, CD147 is known to have a role in GEO data sets-GSE6477. In our cohort, we found that CD147 in promoting tumor growth within an acidic microenvironment gene expression is significantly higher in MM as compared with by chaperoning lactate transporters such as MCT1 to the plasma normal plasma cells, MGUS and SMM (Figure 2a). When we used membrane. Moreover, functional studies revealed that the 1 as the cutoff for normalized gene expression level of CD147, we natural CD147 ligand, cyclophilin B stimulates MM growth by also find significant survival difference in the groups based on enhancing MM tumor cell proliferation.8 To evaluate CD147 normalized CD147 gene expression (o1is80monthsvs41is expression in MM, we first used the normalized gene expression 31 months, P = 0.001; Figure 2b). Further when we compared the data for the expression of CD147 and VEGF1 in CD138-positive gene expression of CD147 and VEGF, we found a positive plasma cells in different stages of MM. This data has been correlation between the two as expected (Figure 2c), which was

Leukemia (2016) 947 – 1002 © 2016 Macmillan Publishers Limited Letters to the Editor 953 analyzed using Spearman correlation with Graphpad Prism6 antibody anti-human CD147 (LS Biosciences, Seattle, WA, USA, (GraphPad Software, La Jolla, CA, USA). We further analyzed LS-B3163, 1:500) and compared the expression of CD163 (M2φ protein expression of CD147 by staining the TMA with specific infiltration) in the same patients. For CD147 staining, the Aperio

Figure 2. CD147 expression is increased in MM, has a prognostic significance and is associated with TAM infiltration and VEGF expression. (a) CD147 expression is significantly increased in plasma cells of newly diagnosed MM patients as compared with MGUS and it is significantly increased in all grades of MM when compared with normal plasma cells. (b) Mayo cohort patients show significant survival difference if they are divided into two groups based on CD147 expression level in plasma cells. (c) CD147 and VEGF gene expression levels show significant positive correlation. (d–f) By immnunohistochemistry, M2φ infiltration as measured by CD163 expression shows significant correlation with CD147 expression in all patients (d), which is more pronounced in relapsed MM (e) and non-hyperdiploid (f) groups.

© 2016 Macmillan Publishers Limited Leukemia (2016) 947 – 1002 Letters to the Editor 954 (Leica Biosystems, Buffalo Grove, IL, USA) ScanScope AT Turbo of macrophages in random cores. GA, JM and SS contributed to the acquisition system was used to scan the slides. The images were captured of data. SP drafted the article and all authors read, revised the article critically at 480 × 751 pixel resolution at × 40 magnification and converted for important intellectual content, gave final approval of the version to be to webslide format. For morphometric analysis, the CD163 published and contributed to the interpretation of the data. positivity and CD147 positivity in the corresponding core were 14 quantified by standard protocol as percentage of CD163- and S Panchabhai1, K Kelemen2, G Ahmann1, S Sebastian1, CD147-stained area to total area, calculated as pixel number J Mantei1 and R Fonseca1 by color range tool using Adobe Photoshop CS6 software 1Division of Hematology/Oncology, Mayo Clinic, Scottsdale, (Adobe Systems, San Jose, CA, USA). We find a positive AZ, USA and correlation between CD163 and CD147 expression in our cohort 2Department of Laboratory Medicine and Pathology, Mayo Clinic, (Figure 2d and Supplementary Table S3). This correlation is Scottsdale, AZ, USA particularly notable in relapsed MM (Figure 2e) and non- E-mail: [email protected] hyperdiploid MM (Figure 2f). The positive correlation between the two serves as a basis to further speculate that increased TAMs induce increased CD147 expression as is true in other REFERENCES 13 . 1 Rajkumar SV. Treatment of multiple myeloma. Nat Rev Clin Oncol 2011; 8: Our study reports M2φ infiltration in the marrow of MM patients 479–491. and demonstrates a negative correlation between M2φ infiltra- 2 Berardi S, Ria R, Reale A, De Luisi A, Catacchio I, Moschetta M et al. Multiple tion and MM patient survival particularly with relapsed and myeloma macrophages: pivotal players in the tumor microenvironment. J Oncol aggressive disease. Our study supports two earlier studies, which 2013; 2013: 183602. suggest TAMs in bone marrow and TAM-derived CD163 in serum 3 Asimakopoulos F, Kim J, Denu RA, Hope C, Jensen JL, Ollar SJ et al. Macrophages as prognostic marker in MM.15,16 We also observe that CD147 in multiple myeloma: emerging concepts and therapeutic implications. Leuk 2013; 54: 2112–2121. levels have positive correlation with M2φ infiltration and φ fi 4 Scavelli C, Nico B, Cirulli T, Ria R, Di Pietro G, Mangieri D et al. Vasculogenic negative correlation with patient survival. Both M2 in ltration mimicry by bone marrow macrophages in patients with multiple myeloma. and CD147 expression can be used as prognostic markers 2008; 27:663–674. for relapsed and aggressive MM. Our cohort included five 5 Ribatti D, Moschetta M, Vacca A. Microenvironment and multiple myeloma clinically and biologically different groups (SMM, MGUS, newly spread. Thromb Res 2014; 133: S102–S106. diagnosed, treated and relapsed MM), which meant small sample 6 Gu ZJ, Costes V, Lu ZY, Zhang XG, Pitard V, Moreau JF et al. Interleukin-10 is a sizewithineachgroupmakingin-depthanalysissuchas growth factor for human myeloma cells by induction of an oncostatin M multivariate analysis difficult. These studies need to be repeated autocrine loop. Blood 1996; 88: 3972–3986. in larger data sets and those efforts are currently underway. 7 Alexandrakis MG, Goulidaki N, Pappa CA, Boula A, Psarakis F, Neonakis I et al. Nevertheless, M2φ reduction and targeting CD147 represent Interleukin-10 induces both plasma cell proliferation and angiogenesis in multiple myeloma. Pathol Oncol Res 2015; e-pub ahead of print 6 March promising approaches for treating MM in addition to current 2015. therapies. 8 Arendt BK, Walters DK, Wu X, Tschumper RC, Huddleston PM, Henderson KJ et al. Increased expression of extracellular matrix metalloproteinase inducer (CD147) in multiple myeloma: role in regulation of myeloma cell proliferation. Leukemia CONFLICT OF INTEREST 2012; 26: 2286–2296. FR has sponsored research from AMGEN, Celgene, Onyx and Sanofi. He has received 9 Xiong L, Edwards CK, Zhou L. The biological function and clinical utilization of consulting fees from Bayer, Novartis, Onyx, Pharmacyclics, Sanofi and serves on the CD147 in human diseases: a review of the current scientific literature. Int J Mol Sci advisory committee of Applied Biosciences. He has received a patent for the 2014; 15: 17411–17441. prognostication of MM based on genetic categorization of the disease The remaining 10 Tang Y, Nakada MT, Kesavan P, McCabe F, Millar H, Rafferty P et al. Extracellular authors declare no competing financial interests. matrix metalloproteinase inducer stimulates tumor angiogenesis by elevating vascular endothelial cell growth factor and matrix metalloproteinases. Res 2005; 65: 3193–3199. ACKNOWLEDGEMENTS 11 Lau SK, Chu PG, Weiss LM. CD163: a specific marker of macrophages in paraffin- 122 – We would like to acknowledge Carol Viso and Tammy-Brehm Gibson for help with embedded tissue samples. Am J Clin Pathol 2004; :794 801. the flow cytometry (Mayo Clinic Arizona), Cheryl Prothoroe from Jamie Lee lab in 12 David S, Kroner A. Repertoire of microglial and macrophage responses after spinal 12 – helping us set the immunohistochemistry protocol for tissue microarray staining cord injury. Nat Rev Neurosci 2011; : 388 399. (Mayo Clinic Arizona). We would also like to thank Anthony Blahnik from Pathology 13 Amit-Cohen B-C, Rahat MM, Rahat MA. Tumor cell-macrophage interactions Imaging services for scanning the tissue microarray slides (Mayo Clinic Rochester). RF increase angiogenesis through secretion of EMMPRIN. Front Physiol 2013; 4 is a clinical investigator of the Damon Runyon Cancer Research Fund. This work is : 178. supported by grants R01 CA83724, ECOG CA 21115 T, Predolin Foundation, Mayo 14 Loughlin PM, Cooke TG, George WD, Gray AJ, Stott DI, Going JJ. Quantifying fi Clinic Cancer Center and the Mayo Foundation. tumour-in ltrating lymphocyte subsets: a practical immuno-histochemical method. J Immunol Methods 2007; 321:32–40. 15 Andersen MN, Abildgaard N, Maniecki MB, Møller HJ, Andersen NF. AUTHOR CONTRIBUTIONS Monocyte/macrophage-derived soluble CD163: A novel biomarker in multiple myeloma. Eur J Haematol 2014; 93:41–47. SP and RF contributed to the conception and design of the study. SP performed 16 Suyanı E, Sucak GT, Akyürek N, Sahin S, Baysal NA, Yağcı M et al. Tumor-associated research and analyzed the data. KK provided the histopathological diagnosis of macrophages as a prognostic parameter in multiple myeloma. Ann Hematol 2013; myeloma patients for analysis and independently counted the number 92:669–677.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

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