HubLE I MY MENTOR AND I Perspective The role of IL-34 in the pathogenesis of osteosarcoma

Kristina Schiavone, MSc. & Dominique Heymann, PhD* Department of Oncology and Metabolism, University of Sheffield, Sheffield, UK.

*Corresponding author: Dominique Heymann. Department of Oncology and Metabolism, University of Sheffield, Beech Hill Road, Sheffield, UK. Email: [email protected].

GRAPHICAL ABSTRACT

Kristina Schiavone is individualised treatments for osteosarcoma a PhD student at the patients. department of Professor Dominique Oncology and Heymann is head of Metabolism at the the Inserm European University of Sheffield Associated Laboratory with an MSc in "Sarcoma Research Molecular Medicine. Unit", at the University Her research is directed at understanding of Nantes (France) and the biological functions of the the University of (IL)-34 and its contribution to the Sheffield (UK). His main research topics pathogenesis of osteosarcoma. She hopes are the pathogenesis of bone sarcomas, that her study will lead to the improvement with particular interest in the dialog of bone of current therapies and development of microenvironment and tumour cells. His research aims to improve the knowledge

To cite this article: K Schiavone, D. Heymann. The role of IL-34 in the pathogenesis of osteosarcoma. HubLE My Mentor & I. 1. DOI: 10.13140/RG.2.2.12991.56481

on sarcomas to develop new therapeutic secondary tumours or bone metastases. approaches. Sarcomas and carcinomas differ in the origination of their tissues. Sarcomas ABSTRACT develop from mesodermal tissue such as bone or muscle, while carcinomas Osteosarcoma (OS) is the most common originate from epithelial tissue malignant primary bone tumour occurring (endodermal or ectodermal origin) such as in children and adolescents. Owing to an the lining of the breast, colon or prostate. aggressive local growth pattern, and a high Carcinomas do not originate from bone, predisposition to metastasize this disease but they can metastasize to bone through has an unfavourable prognosis and is one the lymph nodes or the blood. of the leading causes of paediatric cancer associated death. Overall survival of OS Sarcomas are further subdivided into two patients has remained unchanged over the major groups: soft tissue sarcomas and past 30 years highlighting the need for a bone sarcomas. Soft tissue sarcomas better characterization of the disease and develop from cells that surround, support, new therapeutic strategies. A key and protect extremities and organs of the modulator in the pathogenesis of OS is the body including fat, muscle, connective tumour niche. This is a specialised tissue, nerves and blood vessels. Bone microenvironment that promotes the sarcomas (primary bone tumours) emergence of tumour initiating cells and originate from bone and can be of benign provides favourable conditions for their or malignant form. The three main types of growth and development. IL-34 is a novel malignant bone sarcomas are: cytokine with roles in bone development Osteosarcoma (OS), Ewing sarcoma and and the biology of bone sarcomas. IL-34 chondrosarcoma1. While chondrosarcoma shares a common receptor with the is characterized by a high risk of local macrophage colony stimulating factor (M- recurrence and metastasis according its CSF), and both IL-34 and M-CSF signal grading, OS and Ewing sarcoma are through the macrophage colony associated with the development of stimulating factor receptor (MCSF-R). Both metastases to the bone, or more frequently mediate the biology of mononuclear to the lungs2. phagocytic cells and polarize macrophages into immunosuppressive M2 Primary malignant bone tumours are type. In osteosarcoma, IL-34 has been considerably rare and account for only identified as promoting progression by 0.2% of all malignant tumours. They pre- increasing the tissue vasculature and dominate in children and teenagers, while stimulating the recruitment of they increase from middle age for soft macrophages. Consequently, IL-34 tissue sarcomas. Between 1979 and 2007, appears as a pro-metastatic regulator in around 380 people were diagnosed with osteosarcoma. Understanding the key bone sarcomas yearly in England. This roles of IL-34 in bone development and in number is however increasing as the the pathogenesis of OS by targeting the M- population ages3. In children aged up to 14 CSF/IL-34/M-CSFR triad, represents years, almost 55 bone sarcomas are promising therapeutic approaches for OS. diagnosed yearly, and these account for 4% of all malignancies observed. The 5- INTRODUCTION year survival rate for patients with localized forms of sarcomas are 70-75%. Survival Malignant bone tumours are grouped into rates becomes around 30% for metastatic two categories: sarcomas, commonly sarcomas (specifically metastases), referred to as malignant primary bone and this data has not changed over the tumours, and carcinomas known also as past 40 years4.

To cite this article: K Schiavone, D. Heymann. The role of IL-34 in the pathogenesis of osteosarcoma. HubLE My Mentor & I. 1. DOI: 10.13140/RG.2.2.12991.56481

cases originate in the knee, however they Osteosarcoma: Epidemiology can also occur in the axial skeleton (<10% of cases in pediatric age group), most OS is a rare malignancy with an incidence commonly the pelvis. Around 25% of of one to three cases per million annually5. patients present with detectable It exhibits a bimodal age distribution, metastatic disease after 36 months from primarily affecting children and young diagnosis. The most frequent site for people at a peak incidence of around 18 metastases is the , but they can also years. The second peak incidence is seen develop in other bones and soft tissues in older adults following radiotherapy or in sites2. association with Paget disease. OS is the most prevalent form of primary malignant Pathogenesis bone tumours, describing around 56% of bone cancers in children6. Prevalence is The origin of OS remains enigmatic in part higher in males than in females (male to due to their rarity. They are genetically female ratio of 1.5:1). This difference may unstable tumours characterized by be related to the skeletal growth period, multiple chromosomal translocations, which is longer in males than females. amplifications and deletions. Recently, However it peaks earlier in females (12 human sarcomas have been found to years for females versus 16 years for contain fusion genes associated with males), due to the relatively earlier growth cellular motility, and highlight the high spurt experienced by girls2. genetic instability characteristic of OS. Transcriptome sequencing identified two Characteristics recurrent fusion genes, LRP1-SNRNP25 and KCNMB4-CCND3. These genes are Conventional OS is characterized as a known to be associated with cancer high-grade malignant tumour composed progression and where seen to promote of spindle-shaped mesenchymal cells that OS cell migration and invasion7. produce an osteoid matrix (Marina et al., 2004). The current World Health Over the past two decades genome wide Organisation (WHO) identifies three major association studies led to progress in subtypes: osteoblastic, chondroblastic understanding the genetic origins of OS. A and fibroblastis5. This classification is number of alterations and inactivating based on morphology and organisation of mutations have been found to play a role the tumour cell, including components of in initiating tumour development. These the extracellular matrix. Apart from these include mutations in the tumour subtypes, the WHO classification suppressor genes Tp53 and describes two additional histological retinoblastoma (RB1), mutations in c-MYC variants, small cell and telangiectatic OS. and RECQL4 oncogenes, and down- Telangiectatic OS is constituted of blood regulation of the Wnt signaling pathway8. filled cystic spaces separated by thin These mutations lead to specific and rare septa, while small cell osteosarcoma is syndromes with a predisposition to characterized by sheets of round cells that developing OS: inherited syndromes produce an osteoid matrix which is often including hereditary bilateral confused with Ewing sarcoma2. retinoblastoma (mutation of RB1 gene), Li– Fraumeni syndrome (germline mutations of OS generally develops within the the p53 tumor suppressor gene), Bloom metaphysis of long bones, in particular the syndrome (mutation of BLM gene coding distal femur, the proximal humerus and for a DNA helicase), Rothmund–Thomson proximal tibia, since these regions undergo syndrome (mutation of the RECQL4 gene rapid growth phases. Sixty percent of encoding a DNA helicase). OS may also

To cite this article: K Schiavone, D. Heymann. The role of IL-34 in the pathogenesis of osteosarcoma. HubLE My Mentor & I. 1. DOI: 10.13140/RG.2.2.12991.56481

develop in association with multiple further to the survival and propagation of exostoses and Paget’s disease. However, the tumour. the majority of OS occur without any familial predisposition, and the number of cases associated with germline mutations Conventional Therapy is approximately 3%9. It should be noted that these data were drawn from analysis The current therapeutic approach for of small cohorts of tumour samples, and treating OS was established by Rosen et al that none of the mutations are recurrent or in the late 70’s. Following staging specific therefore their use in diagnostics investigations and a diagnostic biopsy, is limited2. initial treatment consists of intensive polychemotherapy with combinations of A number of studies have reported cisplatin, doxorubicin, methotrexate and difficulties in differentiating low-grade OS ifosfamide. These drugs are administered from benign lesions, as both present with prior to radical en-bloc tumour resection11. similar radiographic appearances. Low- The resected tumour is then scored on the grade OS constitutes between 5–7% of all percentage number of residual viable osteosarcomas, and is subdivided into two tumour cells according to the Huvos scale subgroups subject to its location to the (grade I > 50%, grade II from 11-50%, bone cortex; parosteal and low-grade grade III from 1-0%, grade IV: no central OS. Inability to correctly diagnose detectable viable cancer cells)12. In these lesions may lead to inappropriate practice, patients are divided into good treatment. Low-grade OS is characterised responders if <10% of tumour cells are by supernumerary ring found to be viable, and poor responders if encompassing the amplification of >10% of viable tumour cells are present. A 12q13–15, in conjunction recent international EURAMOS-1 study with cyclin dependent kinase 4 (CDK4) and determined the effect on patient survival murine double-minute type 2 (MDM2) gene by altering post-operative chemotherapy region. The incidence of these based on this histological response. Poor amplifications is more prevalent in low responders were randomized between grade OS rather than high-grade classical continuing MAP (methotrexate, OS and thus MDM2 and CDK4 doxorubicin and cisplatin), and MAPIE immunohistochemistry is the current (addition of ifosfamide and etoposide). technique used to differentiate between Results showed that there was no low grade OS and benign fibrous and fibro- significant difference in the outcome osseous lesions, especially in patients with between the regimens of drugs atypical radio-clinical presentation and/or administered and therefore post-operative limited biopsy samples10. chemotherapy is not adaptive according to response13. Radiotherapy is also Although several studies have been administered when surgery is not possible performed to determine the events leading (such as neck, head, spine), or when to the development of OS, its resection margins are considered as pathogenesis still remains unclear. Rather inadequate14. than looking at the genetic origin of osteosarcoma, its pathogenesis can be The Microenvironment In viewed in terms of its cellular origin, and Osteosarcoma how oncogenic events in its precursor cells lead to malignant transformation and Several other theories have been initiation of the tumour. Within tumour proposed to explain the development of sites, the local environment then helps it to cancer cells in bone. One such is based on survive and proliferate thus contributing the ‘seed and soil’ theory initially proposed

To cite this article: K Schiavone, D. Heymann. The role of IL-34 in the pathogenesis of osteosarcoma. HubLE My Mentor & I. 1. DOI: 10.13140/RG.2.2.12991.56481

by Stephen Paget in the late 19th century grafting, following long periods (7-28 and focuses on OS and its local years) of being disease free20. environment15. When tumour cells invade bone, a disruption in the balance between To explain the development of these bone resorption and bone formation secondary-induced primary bone tumours, occurs, creating an inflammatory-like the authors suggest that tumour growth environment that promotes growth of was promoted by MSCs in the inserted tumour cells. A cycle is established scaffold. Perrot et al., also reported a between the tumour cells and their delayed local reappearance of microenvironment, where the functional osteosarcoma. This came after 13 years equilibrium between osteoblasts and from initial diagnosis, and 18 months osteoclasts is deregulated. During bone following a lipofilling procedure. Perrot et remodeling, osteoblasts deposit new bone al investigated the relationship between tissue while osteoclasts resorb bone tumour growth, fat injections, and tissues. OS cells de-regulate the mesenchymal stem/stromal cell like cells microenvironment by activating osteoclast present in fatty tissue. Results showed that differentiation and resorption, which in turn fat grafts and progenitor cells promote stimulate tumour growth by releasing tumour growth, indicating that de- proliferative factors stored in the regulation of tumour niches may reactivate extracellular matrix16. This leads to the tumour proliferation21,22.An important development of the ‘bone niche’, in which element in the bone microenvironment is the bone microenvironment promotes the also the receptor activator of nuclear factor progression of cancer initiating cells and kappa-B ligand (RANKL) and its receptor provides the right conditions for their RANK. RANKL is an essential mediator in survival and development. Cancer osteoclast activation and differentiation, initiating cells are defined as cells with self- and consequently in bone remodelling. renewal ability, tumour-initiating capacity, RANK is expressed by cells of and ability to give rise to more lineage, endothelial cells, and also by OS differentiated progeny17. This not only tumour cells. A reverse correlation occurs in primary malignant sarcomas, but between the expression of RANK and also during the development of secondary overall patient survival suffering from OS bone metastases. but not with the response to chemotherapy has been demonstrated23. A recent study The niche is a highly complex environment has however reported that RANK was not and is not only restricted to bone related detectable in OS tumours. This absence cells. Other types of cells including suggests that any autocrine RANKL/RANK endothelial cells and macrophages are signalling in human OS tumour cells is not also present. These set up niches of their operative, and anti-RANKL therapy would own, a ‘vascular niche’ and an ‘immune not directly affect the tumour24. Pre-clinical niche’, which contribute to the tumour investigations demonstrated that RANKL microenvironment by modifying the blockade by , or soluble vascularization and altering the local RANK delivery has a strong impact on the immunity respectively18,19. Additionally, tumour development25,26. In other cancer these niches play a role in keeping cancer cell types, tumour-infiltrating regulatory T cells dormant and triggering the cells appear the main source of RANKL development of tumours both locally or to and may be a strong regulator of local distant organs by metastasis. This has immunity27. Therefore inhibition of RANKL been demonstrated in studies of OS that can be an effective way to target the bone developed from benign lesions after microenvironment. This has been patients underwent bone curettage and demonstrated by using , a human monoclonal antibody specific to

To cite this article: K Schiavone, D. Heymann. The role of IL-34 in the pathogenesis of osteosarcoma. HubLE My Mentor & I. 1. DOI: 10.13140/RG.2.2.12991.56481

RANKL, which has been shown to prevent extravasation, survival and proliferation of tumour-induced bone resorption and cancer cells at metastatic sites31. skeletal complications of metastatic bone disease arising from breast cancer28. Tumour vascularization acts as an entry point for tumour cells to disseminate to Tumour Associated Macrophages other parts of the body and promote metastasis29. Additionally, TAMs Cells of the immune system penetrate accumulate in conditions of hypoxia within tumours to regulate the effects of the tumour and up-regulate the expression inflammation and immunity29. The most of hypoxia-inducible factors, which in turn abundant immune cells are macrophages, triggers transcription of various growth which were originally thought to generate factors including VEGF33 anti-tumour activities by recruiting helper T-cells. However, clinical and experimental In many cancers, the presence of TAMs data have shown that certain macrophage leads to poor prognosis. However, it has phenotypes, are correlated with enhanced also been recorded that the expression of tumour progression, induction of TAM-associated genes in pre-treatment angiogenesis and promotion of biopsies of OS, correlated with a lower risk immunosuppression30,31 of metastases. The authors observed an expression of macrophage-associated Macrophages demonstrate functional genes in hematopoietic cells and not in OS plasticity as a result of signals generated tumour cells. They also found that TAMs in from stromal cells and tumour cells. They post-chemotherapy resections and can differentiate into M1 or M2 type metastatic lesions, led to improved macrophages. M1 macrophages induce survival. The authors reported a inflammatory responses and anti-tumor heterogenous population of M1 and M2 immunity, whilst M2 type induce anti- phenotypes in OS tumours, and that there inflammatory responses and pro- was an association between macrophage tumorigenic properties through the infiltration and higher micro-vessel density. induction of neo-angiogenesis. This suggests that the influx of Macrophages that infiltrate tumours are macrophages may support certain aspects known as tumour associated of tumour growth in OS. However overall, macrophages (TAMs). They resemble in OS, direct or indirect antitumor activity closely M2 type macrophages and are of macrophages outweighs their possible recruited to tumours as a result of tumour supporting effects34. Recently, overexpression of growth factors such as Dumars et al. demonstrated the macrophage colony stimulating factor (M- association of TAM to a better overall CSF), CC ligand 2 (CCL-2) and survival of OS patients35. These authors vascular endothelial observed a dysregulation of the (VEGF)32. The M2 subtype has been seen macrophage balance in favor of M1 cells in to promote the progression of cancer and non-metastatic patients. minimize the efficacy of therapy using a combination of mechanisms. Primarily, The above findings are backed by a clinical they reinforce the presence of cancer cells trial of 662 OS patients using muramyl by inhibiting anti-tumour responses and tripeptide (MTP), a macrophage activating stimulate cell proliferation. Secondly, agent. Addition of this peptide to TAMs regulate angiogenesis by enhancing chemotherapy regimens of doxorubicin, the angiogenic switch, and promoting the cisplatin, and methotrexate resulted in a proliferation of endothelial cells. TAMs statistically significant improvement in 6- contribute to tumour progression by year overall survival, from 70% to 78% assisting in cancer cell invasion, seeding, (P=0.3, hazard ratio = 0.71, 95% CI, 0.52

To cite this article: K Schiavone, D. Heymann. The role of IL-34 in the pathogenesis of osteosarcoma. HubLE My Mentor & I. 1. DOI: 10.13140/RG.2.2.12991.56481

to 0.96)36. It is therefore possible that the M1 and M2 macrophage ratio may Lymphocytes, osteoblasts and stromal regulate metastasis in OS, and that once a cells secrete M-CSF in order to sustain the threshold of either phenotype is reached continuous proliferation of the tumour by a the tumour microenvironment may be direct or indirect effect depending on the changed to one that favors metastases. expression of M-CSFR in cancer cells. M- The results from this study are considered CSF can act as an autocrine, paracrine and controversial, meaning that adjuvant MTP endocrine factor. Increased expression of for OS has not been universally adopted M-CSF has been found in a number of and further investigation into this cancers including breast, pancreatic and mechanism is needed. colorectal cancer. High expression levels of M-CSF in ovarian cancer correlate with Macrophage Colony-Stimulating Factor increased tumour aggressiveness and poorer prognosis39. The macrophage colony-stimulating factor (M-CSF or CSF-1) is a cytokine required M-CSF has also been suspected in the for proliferation, differentiation and survival process of tumour metastasis in breast of cells from the hematopoietic lineage cancer. A recessive null mutation of CSF- including , macrophages, and 1 gene resulted in delayed lung metastasis osteoclasts37. The effects of M-CSF are and tumour progression in a murine breast regulated through a type III tyrosine kinase cancer model. This reduction was receptor called MCSF-R, (also known as c- explained by the authors as arising due to fms, CD115 and CSF-1R) which is lack of TAMs (M1 type). Restoring local encoded by the proto-oncogene c-fms. concentrations of MCS-F, resulted in the The importance of M-CSF has been promotion of tumour development. These demonstrated in vivo using mutant pro-tumoral actions are exerted through osteopetrotic (op/op) mice38. The mice macrophages, suggesting that lack of exhibited a number of skeletal macrophages in tumours of abnormalities (e.g. stunted growth, domed Csf1op/Csf1op mice is primarily due to the skull, stubby appearance of the tarsals, systematic loss of CSF-1, and that other metatarsals, femur and humerus), a chemo attractants are present in mice to toothless phenotype, and deficiencies in recruit macrophages into the tumour site40. macrophages and osteoclasts. This phenotype resulted from a null mutation in Other studies have reported that M-CSF the CSF-1 gene by insertion of a single has the potential to bring about anti- , and led to a deficiency in the tumour responses as well. Rat T9 glioma production of osteoclasts37. Preliminary cells transfected with membrane bound experiments indicated that the above isoforms of macrophage M-CSF (mMCSF; effects might be restored by injecting the a non-secreted isoform of M-CSF) were recombinant form of human M-CSF to the killed by macrophages in a dose op/op mice. This resulted in correction of dependent manner. Killing of mM-CSF the observed osteopetrotic phenotype, as expressing tumour cells by macrophage in well as restoration of the number of vitro occurred through phagocytosis41. macrophages and osteoclasts38. However Although these reports are contradictory, it did not overcome all the defects the tumour promoting actions of M-CSF observed in the op/op mice, indicating that are well documented, and overall it is other variants of M-CSF or other regarded as a “pro-tumor” cytokine. , might be acting in combination Another cytokine which promotes the to regulate the activity of osteoclast. differentiation, proliferation, and functional regulation of monocytes, macrophages M-CSF in Cancer

To cite this article: K Schiavone, D. Heymann. The role of IL-34 in the pathogenesis of osteosarcoma. HubLE My Mentor & I. 1. DOI: 10.13140/RG.2.2.12991.56481

and dendritic cells, is the recently An interesting observation comes from the discovered IL-34. study of Wei et al., in 2010, which provides new insights on the expression patterns of Interleukin-34 M-CSF and IL-34. When studying the expression levels of these two cytokines In 2008, a novel cytokine, Interleukin-34 during mouse development, they showed (IL-34) was discovered by producing that their expression levels differ recombinant forms of from substantially in a spatiotemporal manner. cDNA’s encoding both secreted proteins Thus IL-34 although it has overlapping and extracellular domains of trans- functions with M-CSF, could be acting to membrane proteins. They transfected coordinate the cellular communication these into HEK 293T cells and screened network between osteoblasts, their biological activities through a number macrophages and osteoclasts at the of cell-based assays. They demonstrated microenvironment level rather than at the that IL-34 transduced signaling pathways systemic level. This however, needs to be through MCSF-R receptor. They also elucidated further since it might have showed that IL-34 induced the formation further implications on the bone niche and of colony forming unit macrophages in how IL-34 plays a role in maintaining the human bone marrow cultures with the survival and proliferation of sarcoma cells. same effectiveness as M-CSF42. In of this study, it was hypothesized that IL-34 IL-34 is highly expressed in post-natal and shared common features with M-CSF and adult brains and concurrently, MCSF-R is revealed a functional overlap. also highly present in early development, but dramatically decreases, almost Functional studies demonstrated that both undetected, in adult brains. This high M-CSF and IL-34 stimulate macrophage expression of IL-34 in adult brains without differentiation and up-regulate monocyte expression of its receptor, suggested that activity42,43. However several phenotypic other receptors for this cytokine exists45. differences were observed in the resulting The receptor tyrosine phosphatase macrophages. These differences (RPTPβ/ζ) was recently identified on the predicted that IL-34 uses an alternative glioblastoma cell line U251, as another binding mode from M-CSF on binding to receptor for IL-34 through its cell surface the MCSF-R receptor. Structural analysis chondroitin sulfate (CS) chains. By using showed that IL-34 and M-CSF bind to the IL-34 affinity chromatography of extracellular domain of MCSF-R in a solubilized mouse brain membrane similar way, but through two distinct followed by mass spectrometric analysis, contact points. Binding of IL-34 or M-CSF Nandi et al reported that IL-34 selectively to MCSF-R leads to receptor dimerization binds to cell surface PTP-ζ and initiates and differential auto-phosphorylation on downstream signalling leading to inhibition its eight tyrosine residues44 (Figure 1A). M- of cell proliferation and motility. They also CSF/MCSF-R and IL-34/MCSF-R crystals showed that IL-34 binding to PTP-ζ is have a similar shape, but the IL-34/M- dependent on the presence of CS chains. CSFR complex is more stable. Chihara et Similarly, syndecan-1, also a proteoglycan al. showed some differences in the kinetics with CS chains, was able to modulate IL- of MCSF-R phosphorylations and in the 34-induced M-CSFR signalling pathways. nature and intensity of phosphorylated Syndecan-1 also increases the migration tyrosine residues after IL-34 binding, partly M2 macrophages induced by IL-34. In explaining the differences in the signaling addition, it was proved that IL-34 induced pathways they elicit, and also revealing in myeloid cell migration, is dependent on part their functional overlap. syndecan-146. Therefore in addition to MCSF-R, RPTPβ/ζ and syndecan-1 are

To cite this article: K Schiavone, D. Heymann. The role of IL-34 in the pathogenesis of osteosarcoma. HubLE My Mentor & I. 1. DOI: 10.13140/RG.2.2.12991.56481

key regulators of IL-34 activity, and may dependent on M-CSF and RANKL, a TNF play a role in bone inflammatory diseases family cytokine. The role of M-CSF in and bone sarcoma development (Fig 1B osteoclastogenesis, as demonstrated in and 1C). the osteopetrotic op/op mice, was previously described38. Similarly IL-34 can contribute to osteoclastogenesis. It was Role of IL-34 And M-CSF In Bone demonstrated that IL-34 expression was Biology able to recover the main defects observed in op/op mice42. Using in vitro murine and In addition to the major role played by human models of osteotogenesis47, IL-34 these cytokines in macrophage was able to support RANKL-induced proliferation and differentiation, M-CSF osteoclastogenesis in the absence of M- and IL-34 play a central part in bone CSF. IL-34 stimulated RANKL-induced remodeling through osteoclastogenesis, osteoclastogenesis by promoting the the process by which osteoclasts break adhesion and proliferation of osteoclasts down bone. In association with RANKL, IL- progenitors, solidifying further the 34 can replace M-CSF to induce hypothesis that M-CSF and IL-34 exhibit a osteoclast formation by stimulating the functional overlap. proliferation and adhesion of osteoclast precursors. IL-34 can completely Similar to M-CSF, IL-34 was shown to substitute for M-CSF during this process, promote the differentiation of monocytes thus defining a novel pathway for into immunosuppressive M2 osteoclastogenesis. IL-34 was highly macrophages48 and to up-regulate IL-6 expressed in osteoclast-like cells found in and P-10/CXCL10, IL- giant cell tumours of bone. In contrast to 8/CXCL8 and MCP-1/CCL2 in human osteoblasts, osteoclasts showed very whole blood49. Recently, it was also strong staining for IL-34, suggesting a demonstrated that both cytokines induced potential role in the pathogenesis of bone similar effects on macrophage sarcomas by promoting osteoclast differentiation ability but was associated 47 formation . Osteoclastogenesis and the with a different polarization potential differentiation of osteoclasts is mainly (A) (B) (C)

Figure1: The receptors of Interleukin-34. Diagram showing the three proposed receptors for IL-34. (A) MCSF-R Receptor; IL-34 and M-CSF bind to the MCSF-R receptor by two distinct

To cite this article: K Schiavone, D. Heymann. The role of IL-34 in the pathogenesis of osteosarcoma. HubLE My Mentor & I. 1. DOI: 10.13140/RG.2.2.12991.56481

contact points. Binding induces the auto-phosphorylation of different tyrosine residues (red for M-CSF and green for IL-34) and subsequently differential biological responses (B) Syndecan- 1; a proteoglycan with CS chains, able to regulate IL-34-induced M-CSFR signalling pathways. Syndecan-1 also increases the migration of M2 macrophages induced by IL-34. (C) RPTPβ/ζ; Binding of IL-34 to PTP-ζ is dependent on the presence of CS chains. IL-34 selectively binds to cell surface PTP-ζ and initiates downstream signalling leading to inhibition of cell proliferation and motility. MCSF-R, RPTPβ/ζ and syndecan-1 are key regulators of IL-34 activity, and may play a role in bone inflammatory diseases and bone sarcoma development.

This suggests both IL-34 and M-CSF are endothelium respectively. IL-34 stimulated key regulators of inflammation. IL-34 the proliferation of endothelial cells and overlaps with the roles played by M-CSF in vascular cord formation in cultures of inflammation in degenerative bone ECFCs, and increased the number of diseases such as rheumatoid arthritis and adherent monocytes in co-cultures with periodontal inflammation. The role of IL-34 HUVECs. More importantly were the in rheumatoid arthritis was confirmed results from in vivo studies using mouse when cytokine levels where significantly models inoculated with OS cells higher in the synovial fluids of rheumatoid overexpressing IL-34. In comparison to arthritis patients in comparison to control OS cells, OS cells overexpressing osteoarthritis patients, and correlated with IL-34 resulted in larger primary tumours leucocyte number and inflammation and increased number of lung metastases. intensity51,52. In periodontal inflammation, In vivo, IL-34 increased the recruitment of IL-34 was found to be expressed in M2 TAMs into the tumour tissue55. gingival fibroblasts, and its expression enhanced by the pro-inflammatory IL-34 also functions in an autocrine cytokines TNF-a and IL-1b They also manner by acting on CSFR-1 expressing confirmed once more that IL-34 was able tumour cells and TAMs. IL-34 induces the to support RANKL-induced phosphorylation of CSFR-1 which in turn osteoclastogenesis of bone marrow leads to activation of C/EBPb via the AKT macrophages independently of M-CSF53. mediated pathway. This enhances the pro- tumourigenic and immunosuppressive The Role Of IL-34 In Osteosarcoma functions of TAMs which consequently contributed to therapeutic resistance in IL-34 is expressed in a number of cancers cancers. This helps overcome strict such as breast, ovarian, colorectal, lung, conditions of chemotherapy such as that skin, and brain54 and plays an important observed in lung cancers. IL-34 was role in the tumour microenvironment detected at high levels in cells rendered through alternations of the bone, immune resistant to chemotherapeutic agents56,57. and vascular niches. One of the main In addition, expression of IL-34 was mechanisms by which IL-34 acts on correlated with tumour progression and cancer cells, is by recruiting M2-polarised poor survival in lung cancer patients57. macrophages and promoting formation of Recently, Franze et al. showed that IL-34 new blood vessels and extravasation of was able to support pro-tumourigenic immune cells. This has been demonstrated signal in colon cancer58. IL-34 expression in osteosarcoma where in vitro effects in tumours may therefore be a critical were studied using endothelial cell prognostic biomarker correlating with precursors (ECFC) and human umbilical tumour malignancy. In light of the pro- vascular endothelial cells (HUVEC) to tumourigenic functions described above, determine the role IL-34 in angiogenesis Il-34 makes an attractive therapeutic and the adhesion of monocyte to target. Inhibition of CSF-1 targeting alone,

To cite this article: K Schiavone, D. Heymann. The role of IL-34 in the pathogenesis of osteosarcoma. HubLE My Mentor & I. 1. DOI: 10.13140/RG.2.2.12991.56481

will be insufficient to block the signaling of signaling pathway that controls the IL-34 since IL-34 binds to several other production of IL-34 in the tumour receptors including syndecan-1 and microenvironment may be effective in RPTPβ/ζ which are frequently expressed in multi-agent chemotherapy. various cancers. Thus targeting the

Table 1: A comparison of the characteristics of Interleukin-34 (IL-34) and macrophage colony stimulating factor (M-CSF)59

IL-34 CSF-1 Location Chromosome 1

Size 242 aa, 27.5 KDa 554 aa, 60 KDa

Isoforms 2 isoforms 3 isoforms

N-glycosylated homodimer Homodimer or heterodimer, Subunit structure disulfide linked

Extracellular space, extracellular exosome, cell Gene Product Extracellular space membrane, perinuclear region of cytoplasm, plasma membrane

mRNA expression Predominantly expressed in *

M-CSFR Receptors Syndecan-1 MCSF-R RPTPβ/ζ * for IL-34 and M-CSF. Both cytokines are also detected in a range of other tissues including , brain, lung, , , , testis, ovary, , prostate and colon.

CONCLUSION AND FUTURE a cytokine playing important roles in bone DIRECTIONS biology and in the pathophysiology of Over the past few years, increasing diseases. IL-34 increases evidence has solidified the role of IL-34 as monocyte/macrophage survival and

To cite this article: K Schiavone, D. Heymann. The role of IL-34 in the pathogenesis of osteosarcoma. HubLE My Mentor & I. 1. DOI: 10.13140/RG.2.2.12991.56481

viability, and can substitute for MCS-F in Monoclonal Antibody; TAM, Tumour RANK-L induced osteoclastogenesis. Associated Macrophages; M-CSF, Primarily, IL-34 uses the MSCF-R receptor Macrophage- Colony Stimulating Factor; CSF- to induce the activation of downstream 1, Colony Stimulating Factor-1; CCL-2, signaling pathways. Additionally, two other Chemokine ligand 2; VEGF, Vascular Endothelial Growth Factor; MTP, Murayml receptors for IL-34 are present: syndecan- Tripeptide; MCSF-R, Macrophage-Colony 1 and RPTPβ/ζ. These receptors however, Stimulating Factor Receptor; FGF, Fibroblast are not specific to the cytokine, and thus a Growth Factor; TGF, Transforming Growth receptor specific for IL-34 is still expected factor; mMCS-F, Membrane Macrophage- to be recognized. IL-34 has been shown to Colony Stimulating Factor; IL-34, Interleukin- play important roles in the pathogenicity of 34; cDNA, Complementary DNA; TNFα, diseases associated with inflammation, Tumour Necrosis Factor Alpha; IL-1β, whilst in cancers, Il-34 is also found to be Interleukin – 1 Beta; ECFC, Endothelial Colony a key regulator. In osteosarcoma, Il-34 Forming Cells; HUVEC, Human Umbilical Vein promotes tumour progression and Endothelial Cells; RPTPβ/ζ, Receptor Protein Tyrosine Phosphatase Beta/Zeta; CS, metastasis by macrophage recruitment, Chondroitin Sulfate. promoting angiogenesis and chemotherapeutic resistance. Studies are also increasingly suggesting that IL-34 REFERENCES might be specifically coordinating the communication network at the tumour 1. Brown, H. K., Schiavone, K., microenvironment. The future of IL-34 lies Gouin, F., Heymann, M. & in exploring new therapeutic strategies to Heymann, D. Biology of Bone inhibit signaling pathways and/or Sarcomas and New controlling its production. By selectively Therapeutic Developments. blocking IL-34 or MCS-F, this approach Calcif. Tissue Int. 102, 174–195 may provide more flexibility than blocking (1234). MCSF-R alone. Therapeutic combination 2. Marina, N., Gebhardt, M., Teot, between anti IL-34 and conventional L., & Gorlick, R. (2004). Biology chemotherapy can then be envisaged. and therapeutic advances for Thus, blocking IL-34 is a highly promising pediatric osteosarcoma. The area for improving drug therapies and Oncologist, 9(4), 422–441. personalized treatments for osteosarcoma 3. National Cancer Intelligence patients, especially for those with Network (2010) Bone Sarcomas: metastatic disease. Incidence and Survival Rates in England. Retrieved from URL CONFLICT OF INTEREST http://www.ncin.org.uk/publicat None Reported. ions/data_briefings/bone_sarco mas_incidence_and_survival

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To cite this article: K Schiavone, D. Heymann. The role of IL-34 in the pathogenesis of osteosarcoma. HubLE My Mentor & I. 1. DOI: 10.13140/RG.2.2.12991.56481

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To cite this article: K Schiavone, D. Heymann. The role of IL-34 in the pathogenesis of osteosarcoma. HubLE My Mentor & I. 1. DOI: 10.13140/RG.2.2.12991.56481