Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

18
Oncotarget 1 www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget/ Oncotarget, Advance Publications 2015 Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation Lavinia Raimondi 1 , Angela De Luca 1 , Nicola Amodio 2 , Mauro Manno 3 , Samuele Raccosta 3 , Simona Taverna 4 , Daniele Bellavia 1 , Flores Naselli 4 , Simona Fontana 4 , Odessa Schillaci 4 , Roberto Giardino 5 , Milena Fini 6 , Pierfrancesco Tassone 2 , Alessandra Santoro 7 , Giacomo De Leo 4 , Gianluca Giavaresi 1,6 and Riccardo Alessandro 4,8 1 Laboratory of Tissue Engineering - Innovative Technology Platforms for Tissue Engineering (PON01-00829), Rizzoli Orthopedic Institute, Palermo, Italy 2 Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, T. Campanella Cancer Center, Salvatore Venuta University Campus, Catanzaro, Italy 3 Institute of Biophysics, National Research Council of Italy, Palermo, Italy 4 Section of Biology and Genetics, Department of Biopathology and Medical Biotechnology, University of Palermo, Italy 5 Rizzoli Orthopedic Institute, Bologna, Italy 6 Laboratory of Preclinical and Surgical Studies, Rizzoli Orthopedic Institute, Bologna, Italy 7 'LYLVLRQH GL (PDWRORJLD $2 2VSHGDOL 5LXQLWL 9LOOD 6R¿D&HUYHOOR 3DOHUPR 8 Institute of Biomedicine and Molecular Immunology (IBIM), National Research Council of Italy, Palermo, Italy Correspondence to: Riccardo Alessandro, email: [email protected] Keywords: exosomes, multiple myeloma, osteoclasts, tumor microenvironment Received: February 02, 2015 Accepted: March 26, 2015 Published: April 12, 2015 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived H[RVRPHV SOD\ D VLJQL¿FDQW SURGLIIHUHQWLDWLYH UROH LQ PXULQH 5DZ FHOOV DQG human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient’s sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell- derived exosomes. INTRODUCTION More than 70% of patients affected by multiple myeloma (MM) develop severe osteolytic bone disease, which negatively affects the quality of life due to the occurring pathological fractures, spinal cord compression, pain and hypercalcemia [1, 2]. Even though, rapid advances in the therapeutic options in bone disease treatment have been accomplished in the recent years, the progression of MM bone disease is rarely

Transcript of Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

Oncotarget1www.impactjournals.com/oncotarget

www.impactjournals.com/oncotarget/ Oncotarget, Advance Publications 2015

Involvement of multiple myeloma cell-derived exosomes in

osteoclast differentiation

Lavinia Raimondi1, Angela De Luca1, Nicola Amodio2, Mauro Manno3, Samuele

Raccosta3, Simona Taverna4, Daniele Bellavia1, Flores Naselli4, Simona Fontana4,

Odessa Schillaci4, Roberto Giardino5, Milena Fini6, Pierfrancesco Tassone2,

Alessandra Santoro7, Giacomo De Leo4, Gianluca Giavaresi1,6 and Riccardo

Alessandro4,8

1 Laboratory of Tissue Engineering - Innovative Technology Platforms for Tissue Engineering (PON01-00829), Rizzoli

Orthopedic Institute, Palermo, Italy

2 Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, T. Campanella

Cancer Center, Salvatore Venuta University Campus, Catanzaro, Italy

3 Institute of Biophysics, National Research Council of Italy, Palermo, Italy

4 Section of Biology and Genetics, Department of Biopathology and Medical Biotechnology, University of Palermo, Italy

5 Rizzoli Orthopedic Institute, Bologna, Italy

6 Laboratory of Preclinical and Surgical Studies, Rizzoli Orthopedic Institute, Bologna, Italy

7 'LYLVLRQH�GL�(PDWRORJLD�$�2��2VSHGDOL�5LXQLWL�9LOOD�6R¿D�&HUYHOOR��3DOHUPR8

Institute of Biomedicine and Molecular Immunology (IBIM), National Research Council of Italy, Palermo, Italy

Correspondence to: Riccardo Alessandro, email: [email protected]: exosomes, multiple myeloma, osteoclasts, tumor microenvironmentReceived: February 02, 2015 Accepted: March 26, 2015 Published: April 12, 2015

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

ABSTRACTBone disease is the most frequent complication in multiple myeloma (MM)

resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM

bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and

bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in

skeletal disorders. Since exosomes have been described for their functional role in

cancer progression, we here investigate whether MM cell-derived exosomes may be

involved in OCs differentiation. We show that MM cells produce exosomes which are

actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation.

MM cell-derived exosomes positively modulate pre-osteoclast migration, through

the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived

H[RVRPHV�SOD\�D�VLJQL¿FDQW�SUR�GLIIHUHQWLDWLYH�UROH�LQ�PXULQH�5DZ������FHOOV�DQG�human primary osteoclasts, inducing the expression of osteoclast markers such as

Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant

Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes

differentiate in multinuclear OCs able to excavate authentic resorption lacunae.

Similar results were obtained with exosomes derived from MM patient’s sera. Our

data indicate that MM-exosomes modulate OCs function and differentiation. Further

studies are needed to identify the OCs activating factors transported by MM cell-

derived exosomes.

INTRODUCTION

More than 70% of patients affected by multiple

myeloma (MM) develop severe osteolytic bone

disease, which negatively affects the quality of life due

to the occurring pathological fractures, spinal cord

compression, pain and hypercalcemia [1, 2]. Even

though, rapid advances in the therapeutic options in bone

disease treatment have been accomplished in the recent

years, the progression of MM bone disease is rarely

Oncotarget2www.impactjournals.com/oncotarget

halted. Additional studies to understand the complex

PHFKDQLVPV� DGRSWHG� E\�00� FHOOV� WR� LQÀXHQFH� WXPRU�microenvironment will be needed to overcome the limits

of current pharmacology [3-6].

MM bone disease is sustained by a supportive

microenvironment based on complex cross-talk involving

stromal, immune, endothelial and bone cells, as well as

extracellular matrix components. In bone marrow, MM

cells modify the normal microenvironmental conditions

DQG�LQ�WXUQ�UHTXLUH�KRVW�PRGL¿FDWLRQV�IRU�WKHLU�VXUYLYDO�>�@��In contrast to normal bone remodelling, during multiple

myeloma progression the functional balance between

RVWHRFODVWV� �2&V�� DQG� RVWHREODVWV� �2%V�� LV� GH¿QLWLYHO\�perturbed. Differently by other metastatic diseases such

as breast and prostate cancer, where both OCs and OBs

activity is increased, in MM increased osteoclastic activity

is the major element of bone disease [2, 8]. The increased

number and activity of OCs further promote MM

progression by both direct and indirect mechanisms, thus

maintaining a vicious cycle between bone destruction and

tumor cell survival [9]. MM cells directly stimulate OCs

differentiation and activation by secreting local osteoclast

activating factors (OAFs) and indirectly by stimulating the

cells of tumor microenvironment, such as bone marrow

stromal cells (BMSCs) and OBs, to secrete OAFs [9].

Among the main cytokines involved in this crosstalk are:

LQWHUOHXNLQ����LQWHUOHXNLQ��ȕ��LQWHUOHXNLQ����71)�Į��7*)��ȕ��DFWLYLQ�$��DQG�GLFNNRSI��'..����>�����@��7KH�UHFHSWRU�DFWLYDWRU�RI�1)�ț%��5$1.���LWV�OLJDQG�5$1./��DQG�WKH�GHFR\�UHFHSWRU�IRU�5$1./��RVWHRSURWHJHULQ��23*���SOD\�a crucial role in bone remodelling, inducing differentiation

and survival of osteoclasts [13]. In MM, deregulation

RI�5$1./� WR�23*� UDWLR� OHDGV� WR� W\SLFDO� RVWHRO\VLV� RI�ERQH�GLVHDVH��KRZHYHU��WKH�GLUHFW�5$1./�H[SUHVVLRQ�E\�human myeloma cells is controversial and several reports

suggested that human MM cells do not directly produce

5$1./�EXW�UDWKHU�LQGXFH�LWV�H[SUHVVLRQ�LQ�VWURPDO�FHOOV�[14-16].

In the last years, new actors of tumor

PLFURHQYLURQPHQW�FURVVWDON�KDYH�EHHQ�LGHQWL¿HG��Exosomes are small membrane vesicles (40-

150 nm) of endocytic origin secreted by most cells and

thoroughly described for their functional relevance in

cancer, being now recognized as a novel mode of cell-cell

communication during tumor growth and progression. A

number of studies have showed that exosomes released

by cancer cells may affect survival, apoptosis, invasion,

angiogenesis and resistance to chemotherapy as well as

prepare the metastatic niche [17].

These nanovesicles actively transport and transfer

LQIRUPDWLRQ� VXFK�DV�SURWHLQV��PLFUR51$V�DQG�P51$V�WR� WDUJHW� FHOOV�� WKXV� LQÀXHQFLQJ� WKHLU� EHKDYLRXU� DQG�strongly modifying the entire microenvironment [18].

Several reports showed that the serum of patients

affected by cancer is characterized by high levels of

exosomes and their quantity seems to correlate with the

malignant behaviour of cancer [19-21]. In MM, cell-

derived microvesicles are considered mediators for

myeloma angiogenesis, while BMSC-derived exosomes

VLJQL¿FDQWO\�DFW�RQ�YLDELOLW\��VXUYLYDO��PLJUDWLRQ�DQG�GUXJ�resistance of MM cells [22, 23]. On these premises, we

hypothesized that MM cell-derived exosomes might play

a relevant functional role in OCs differentiation. MM cell-

GHULYHG�H[RVRPHV�ZHUH�¿UVWO\�LVRODWHG�DQG�FKDUDFWHUL]HG�in vitro and then their biological effects were evaluated in

PXULQH�PDFURSKDJH�5DZ������FHOOV�DQG�KXPDQ�SULPDU\�osteoclasts. Our results clearly show that multiple

myeloma cells release exosomes that in turn support

both viability and migration of osteoclast precursors

(pOCs) as well as their function and differentiation in

giant and multinucleated osteoclasts. Similar results were

obtained with exosomes derived from MM patient’s sera.

In summary, a more detailed understanding about the

molecular mechanisms underlying exosomes-mediated

bone disease may open new opportunities for combinatory

therapeutical approaches as well as could lead to the

LGHQWL¿FDWLRQ�RI�ERQH�GLVHDVH�ELRPDUNHUV�LQ�00�

RESULTS

MM-derived Exosomes Characterization and Internalization in Raw264.7 cells

Exosomes produced by three MM cell lines (U266,

00�6�DQG�230���ZHUH�FKDUDFWHUL]HG�E\�ZHVWHUQ�EORW�analysis. Figure 1A (upper panel) shows that U266- and

MM1s-cell derived exosomes abundantly expressed Alix

DQG�&'����ZKLOH�&DOQH[LQ��DQ�XELTXLWRXVO\�H[SUHVVHG�(5�protein, was exclusively found in cellular fractions

(Figure 1A, lower panel). Similar results were obtained

ZLWK�230��GHULYHG�H[RVRPHV��6XSSO��)LJXUH��$���7KH�'/6�DQDO\VLV�VKRZHG�DQ�DYHUDJH�K\GURG\QDPLF�GLDPHWHU�of about 100 nm for U266- and MM1s-cell-derived

H[RVRPHV�DQG����QP�IRU�230��GHULYHG�H[RVRPHV��)LJXUH�1B; Suppl. Figure 1B). We then tested the activity of

acetylcholinesterase, an enzyme known to be enriched in

exosomes, and we observed an increased activity in the

extracellular nanovesicles (Figure 1C; Suppl. Figure 1C)

[24].

00�FHOO�GHULYHG�H[RVRPHV� ODEHOHG�ZLWK�3.+����were internalized by the murine macrophage cell line

5DZ������DIWHU�LQFXEDWLRQ�RI���KRXUV�DW����&��)LJXUH��$�shows a typical perinuclear localization of internalized

H[RVRPHV��7KH�XS�WDNH�RI�H[RVRPHV�LQ�5DZ������FHOOV�ZDV�LQKLELWHG�E\�LQFXEDWLRQ�DW���&��)LJXUH��%���DV�ZHOO�DV�E\�(,3$�WUHDWPHQW��)LJXUH��&���6HPL�TXDQWLWDWLYH�DQDO\VLV�RI� 3.+���� ÀXRUHVFHQFH� LQWHQVLW\� LQ� WKH� F\WRSODVP� RI�5DZ������FHOOV�FRQ¿UPHG�WKH�LPDJLQJ�GDWD��6XSSO��)LJXUH�2).

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Figure 1: Characterization of exosomes released by multiple myeloma cells. A. Western blotting analysis of Alix, CD63 and

Calnexin in both U266, MM1s-derived exosomes and cellular lysates. B.�'\QDPLF�OLJKW�VFDWWHULQJ��'/6��DQDO\VLV�RI�8����DQG�00�V�derived exosomes C. Acetylcholinesterase assay of exosomes and cell lysates obtained from U266 and MM1s cells.

Figure 2: Uptake of multiple myeloma cell-derived exosomes by osteoclasts precursors. A. Analysis at confocal microscopy

RI�5DZ������FHOOV�WUHDWHG�IRU���KRXUV�ZLWK����ȝJ�PO�RI�8�����00�V�DQG�230��H[RVRPHV��5DZ������FHOOV�ZHUH�VWDLQHG�ZLWK�$FWLQ�JUHHQ��JUHHQ��� QXFOHDU� FRXQWHUVWDLQLQJ�ZDV� SHUIRUPHG� XVLQJ�+RHVFKW� �EOXH�� DQG� H[RVRPHV�ZHUH� ODEHOOHG�ZLWK� 3.+��� �UHG���B. To evaluate

ZKHWKHU�H[RVRPHV�XSWDNH�ZDV�D�ELRORJLFDOO\�DFWLYH�SURFHVV��5DZ������FHOOV�WUHDWHG�ZLWK����ȝJ�PO�RI�8�����00�V�DQG�230��H[RVRPHV�ZHUH�LQFXEDWHG�DW���&�C.�7R�HYDOXDWH�ZKHWKHU�H[RVRPHV�XSWDNH�ZDV�PHGLDWHG�E\�HQGRF\WRVLV�LQ�DQ�HQHUJ\�GHSHQGHQW�SURFHVV���5DZ������FHOOV�ZHUH�WUHDWHG�IRU���KRXU�ZLWK����ȝJ�PO�RI�H[RVRPHV�DQG�(,3$�����ȝ0���6FDOH�EDU� ����ȝP�

Oncotarget4www.impactjournals.com/oncotarget

MM cell-derived exosomes support migration of pOCs cells

Since, in bone disease, myeloma cells exert relevant

effects on recruitment and proliferation of OC progenitors,

here we investigated if MM cell-derived exosomes may

modulate the proliferative and migratory properties

RI� 5DZ������ FHOOV�� &HOO� YLDELOLW\� DQDO\VLV� VKRZHG� WKDW�U266- and MM1s-derived exosomes induced only a slight

LQFUHDVH�LQ�5DZ������FHOO�SUROLIHUDWLRQ�ZLWKLQ����KRXUV�(Suppl. Figure 3A, upper panel) and a decrease after 6

days of exposure when induction of mature osteoclasts

differentiation occurred (Suppl. Fig.3A, lower panel).

230��GHULYHG� H[RVRPHV� GLG� QRW� DIIHFW�5DZ������ FHOO�viability (Suppl. Figure 3B).

The role of MM cell-derived exosomes on osteoclast

precursors (pOCs) migration was investigated by a

WUDQVZHOO�FKDPEHU�FKHPRWD[LV�DVVD\��1RWDEO\��ZH�IRXQG�

that a 24h pretreatment of human pOCs with U266 and

MM1s cell-derived exosomes increased their migratory

attitudes (Figure 3A, upper panel), presumably via an

LQFUHDVH�RI�&;&5��H[SUHVVLRQ��)LJXUH��%��6LPLODUO\��WKH�QXPEHU�RI�5DZ������FHOOV�PLJUDWHG�

across the 8-µm pore-size membrane increased when cells

were pretreated for 24h with U266- or MM1s cell-derived

exosomes (Figure 3C, left panel).

Finally, both human pOCs (Figure 3A, lower panel)

DQG�5DZ������FHOOV��)LJXUH��&��ULJKW�SDQHO��ZHUH�LQGXFHG�to migrate by MM exosomes, when MM vesicles were

DGGHG�WR�WKH�ERWWRP�RI�WKH�WUDQVZHOOV��230��FHOO�GHULYHG�H[RVRPHV� DIIHFWHG�5DZ������PLJUDWLRQ� OHVV� HI¿FLHQWO\��6XSSO��)LJXUH��&��XSSHU�DQG�ORZHU�SDQHO���7KHVH�¿QGLQJV�indicate that exosomes released by myeloma cells can

LQÀXHQFH�ERWK�PXULQH�DQG�KXPDQ�S2&V�PLJUDWLRQ�

Figure 3: Multiple myeloma cell-derived exosomes induce migration of osteoclasts precursors. A. Migration assay of

KXPDQ�S2&V�XQWUHDWHG�RU�SUHWUHDWHG�IRU����KRXUV�ZLWK����ȝJ�PO�RI�8����DQG�00�V�H[RVRPHV��XSSHU�SDQHO���LQ�WKH�ORZHU�SDQHO�LV�VKRZQ�WKH�PRWLOLW\�RI�KXPDQ�S2&V�VWLPXODWHG�E\�DGGLWLRQ�RI����ȝJ�PO�RI�8����DQG�00�V�H[RVRPHV�LQ�WKH�ERWWRP�FKDPEHU�RI�WKH�WUDQVZHOO��ORZHU�panel). Sidak test: *,EXO-U266 vs Untreated (*p������������(;2�00�V�vs�8QWUHDWHG���p < 0.05). B.�)ORZ�F\WRPHWU\�DQDO\VLV�RI�&;&5��LQ�KXPDQ�S2&V�XQWUHDWHG��FRQWURO��RU�SUHWUHDWHG�IRU����KRXUV�ZLWK����ȝJ�PO�RI�8����DQG�00�V�H[RVRPHV�C.�0LJUDWLRQ�DVVD\�RI�5DZ������FHOOV�XQWUHDWHG�RU�SUHWUHDWHG�IRU����KRXUV�ZLWK����ȝJ�PO�RI�8����DQG�00�V�H[RVRPHV��OHIW�SDQHO���,Q�WKH�ULJKW�SDQHO�LV�VKRZQ�WKH�PRWLOLW\�RI�5DZ������FHOOV�VWLPXODWHG�E\�DGGLWLRQ�RI����ȝJ�PO�RI�8����DQG�00�V�H[RVRPHV�LQ�WKH�ERWWRP�FKDPEHU�RI�WKH�WUDQVZHOO��6LGDN�WHVW��*,EXO-U266 vs Untreated (*p�������������(;2�00�V�vs�8QWUHDWHG���p < 0.05).

Oncotarget5www.impactjournals.com/oncotarget

Effect of multiple myeloma cell-derived exosomes on osteoclast gene expression

T57�3&5� DQDO\VLV� UHYHDOHG� WKDW� VWLPXODWLRQ� RI�PXULQH�PDFURSKDJHV�ZLWK�5$1./�LQFUHDVHG��DV�DOUHDG\�NQRZQ��WKH�H[SUHVVLRQ�RI�2&V�VSHFL¿F�PDUNHUV��QRWDEO\��U266 and MM1s exosomes markedly induced expression

RI� 75$3� DQG� &76.� DV� FRPSDUHG� WR� FRQWURO� FHOOV�� ,Q�SDUWLFXODU��003��P51$� H[SUHVVLRQ� ZDV� VLJQL¿FDQWO\�KLJKHU� LQ� 5DZ������ FHOOV� WUHDWHG� ZLWK� H[RVRPHV� DV�FRPSDUHG� WR� XQWUHDWHG� FHOOV� RU� 5$1./�WUHDWHG� FHOOV�(Figure 4A). Consistently, we observed a dose dependent

HIIHFW� RI� 00� FHOO�GHULYHG� H[RVRPHV� RQ� WKH� P51$�levels of OCs markers as well as a reduced effect of the

corresponding conditioned medium deprived of exosomes

(Suppl. Figure 4A-4B).

7KH�UHVXOWV�ZHUH�FRQ¿UPHG�DOVR�E\�ZHVWHUQ�EORWWLQJ�DQDO\VLV�IRU�&76.�DQG�003���)LJXUH��%���7KH�LQFUHDVH�RI�H[SUHVVLRQ�ZDV�VWDWLVWLFDOO\�VLJQL¿FDQW�EXW�OHVV�HYLGHQW�

LQ� FHOOV� WUHDWHG� ZLWK� 230�� H[RVRPHV� �6XSSO�� )LJXUH��'���7DNLQJ�LQ�DFFRXQW� WKDW�003��SURWHLQ�VHFUHWLRQ�LV�increased in differentiated OCs, we investigated whether

MM cell-derived exosomes were able to increase murine

003��VHFUHWLRQ�LQ�SUH�2&V�FHOOV��)LJXUH��&�VKRZV�WKDW�5DZ������FHOOV�WUHDWHG�ZLWK�8����DQG�00�V�H[RVRPHV�VLJQL¿FDQWO\�UHOHDVHG�P003��SURWHLQ�LQ�FXOWXUH�PHGLXP��$OO�WRJHWKHU�WKHVH�¿QGLQJV�LQGLFDWH�WKDW�00�FHOO�GHULYHG�H[RVRPHV�GLUHFWO\�LQGXFH�WKH�H[SUHVVLRQ�RI�VSHFL¿F�2&V�markers and modulate the secretion of proteases involved

in bone resorption activity.

Multiple myeloma cell-derived exosomes induce osteoclast formation and stimulate bone resorption activity in mature osteoclast-like cells

We evaluated if exosomes released by myeloma

cells might control OCs formation and OCs activity.

75$3�VWDLQLQJ�DQG�WKH�IRUPDWLRQ�RI�PXOWLQXFOHDWHG�FHOOV�

)LJXUH����0XOWLSOH�P\HORPD�FHOO�GHULYHG�H[RVRPHV�LQFUHDVH�H[SUHVVLRQ�RI�RVWHRFODVWV�VSHFL¿F�PDUNHUV���A. Quantitative

57�3&5�RI�P75$3��P&76.�DQG�P003��LQ�5DZ������FHOOV�XQWUHDWHG�RU�WUHDWHG�IRU�VL[�GD\V�ZLWK���QJ?PO�RI�KU5$1./��SRVLWLYH�FRQWURO��RU�ZLWK����ȝJ�PO�RI�8�����OHIW�SDQHO��DQG�00�V�H[RVRPHV��ULJKW�SDQHO���9DOXHV�DUH�H[SUHVVHG�DV�IROG�RI�FRQWURO�DQG�DUH�WKH�PHDQ�RI�WKUHH�different experiments. Sidak test: *�(;2�8����00�V�vs Untreated (*p�������������KU5$1./�vs�8QWUHDWHG���p < 0.05); + B. Western blotting

DQDO\VLV�RI�&76.�DQG�003��LQ�5DZ������FHOOV�XQWUHDWHG�RU�WUHDWHG�IRU�VL[�GD\V�ZLWK���QJ?PO�RI�KU5$1./��SRVLWLYH�FRQWURO��RU�ZLWK����ȝJ�PO�RI�8����DQG�00�V�H[RVRPHV��ȕ�DFWLQ�ZDV�XVHG�DV�ORDGLQJ�FRQWURO��C.�0XULQH�0HWDOORSURWHLQDVH����P003���SURWHLQ�OHYHOV�LQ�WKH�FRQGLWLRQHG�PHGLXP�GHULYHG�IURP�5DZ������FHOOV�XQWUHDWHG�RU�WUHDWHG�IRU�VL[�GD\V�ZLWK���QJ?PO�RI�KU5$1./��SRVLWLYH�FRQWURO��RU�ZLWK����ȝJ�PO�RI�8����DQG�00�V�H[RVRPHV�ZHUH�GHWHUPLQHG�E\�(/,6$��'DWD�SUHVHQWHG�DUH�WKH�PHDQ�RI�WKUHH�GLIIHUHQW�H[SHULPHQWV��6LGDN�WHVW��*�(;2�8����00�V�vs Untreated (*p������������KU5$1./�vs�8QWUHDWHG���p < 0.05).

Oncotarget6www.impactjournals.com/oncotarget

were used as markers of differentiated cells. As showed in

)LJXUH��$��WUHDWPHQW�RI�5DZ������FHOOV�ZLWK�H[RJHQRXV�5$1./� FRQ¿UPHG� WKH� SUHVHQFH� RI� 75$3�SRVLWLYH�multinucleated OCs (Figure 5A, lower panel). Importantly,

in the presence of MM cell-derived exosomes, the number

RI�75$3�SRVLWLYH�PXOWLQXFOHDWHG�2&V�ZDV�VLJQL¿FDQWO\�higher compared to control cells (Figure 5A, upper panel).

The strongest effect was observed with U266-derived

exosomes, reaching a ten fold increase in the size of

multinucleated OCs compared to control cells. We next

LQYHVWLJDWHG�WKH�DELOLW\�RI�5DZ�������FHOO�GHULYHG�2&V�WR�resorb bone by stimulating for 6 days OCs formation on

V\QWKHWLF�GHQWLQH�GLVFV�ZLWK�5$1./�RU�00�FHOO�GHULYHG�

exosomes (Figure 5B, upper and lower panel). After

removal of adherent cells, dentine discs were examined

IRU� WKH� SUHVHQFH� RI� UHVRUSWLRQ� ODFXQDH� E\� GDUN� ¿HOG�microscopy. OCs induced by MM cell-derived exosomes

had a marked ability to resorb dentin substrate (dark

areas); in particular, stimulation with exosomes increased

not only the number of pits but also the digestion areas,

which resulted larger compared to control cells (Figure 5B,

lower panel). In Figure 5C is shown a scanning electron

microscopy (SEM) image of a typical bone resorption

area obtained by OCs cultured on synthetic dentine disc

and treated with MM cell-derived exosomes. To further

FRQ¿UP�WKH�H[RVRPH�LQGXFHG�SKHQRW\SH�RI�WKH�FHOOV��ZH�

Figure 5: Exosomes produced by multiple myeloma cells induce osteoclast formation in RAW 264.7 cells and promote formation of lacunae on dentine slices. A.�5DZ������FHOOV�LQFXEDWHG�ZLWK���QJ?PO�RI�KU5$1./��SRVLWLYH�FRQWURO��RU�ZLWK����ȝJ�PO�RI�8����DQG�00�V�H[RVRPHV�IRU�VL[�GD\V��DQG�WKHQ�VWDLQHG�IRU�75$3�H[SUHVVLRQ��75$3�SRVLWLYH�FHOOV�ZHUH�SKRWRJUDSKHG�DQG�FRXQWHG��/RZHU�SDQHO�VKRZV�UHSUHVHQWDWLYH�SLFWXUHV�RI�75$3+5DZ������FHOOV��RULJLQDO�PDJQL¿FDWLRQ���[���'DWD�SUHVHQWHG�DUH�WKH�PHDQ�RI�WKUHH�different experiments. Sidak test: *�(;2�8����00�V�vs Untreated (*p������������KU5$1./�vs�8QWUHDWHG���p < 0.05); B. Bone resorption

DELOLW\�RI�5DZ������FHOOV�WUHDWHG�IRU�VL[�GD\V�ZLWK���QJ?PO�RI�KU5$1./��SRVLWLYH�FRQWURO��RU�ZLWK����ȝJ�PO�RI�8����DQG�00�V�H[RVRPHV�was evaluated by resorption pit assay on dentine discs. Data relative to the resorbed areas by mature osteoclasts are represented as fold

LQFUHDVH�RI�5DZ������FHOOV�XQWUHDWHG��EODFN�FROXPQ����DUELWUDU\�XQLW���'DWD�SUHVHQWHG�DUH�WKH�PHDQ�RI�WKUHH�VHSDUDWH�H[SHULPHQWV��6LGDN�WHVW�� �(;2�8����00�V�vs Untreated (*p������������KU5$1./�vs�8QWUHDWHG���S����������/RZHU�SDQHO�VKRZV�UHSUHVHQWDWLYH�SLFWXUHV�RI�ODFXQDH��GDUN�DUHDV��IRUPHG�E\�FHOOV�WUHDWHG�ZLWK�KU5$1./�RU�00�H[RVRPHV��RULJLQDO�PDJQL¿FDWLRQ���[���C. 5HSUHVHQWDWLYH�VFDQQLQJ�HOHFWURQ�PLFURVFRS\��6(0��LPDJH�RI�UHVRUSWLRQ�SLWV�JHQHUDWHG�E\�5DZ������FHOOV�FXOWXUHG�RQ�GHQWLQH�VOLFHV�DQG�WUHDWHG�ZLWK�00�H[RVRPHV��6FDOH�EDU� ����ȝP��D.�&RQIRFDO�PLFURVFRS\�RI�5DZ������FHOOV�FXOWXUHG�IRU�VL[�GD\V�ZLWK����ȝJ�PO�RI�8�����RU�00�V�GHULYHG�H[RVRPHV�DQG�¿[HG��DFWLQ�¿ODPHQWV�LQ�SRGRVRPHV�ZHUH�ODEHOHG�XVLQJ�$OH[D�)OXRU®�����SKDOORLGLQ��JUHHQ��DQG�QXFOHL�ZHUH�FRXQWHUVWDLQHG�ZLWK�'$3,��EOXH���6FDOH�EDU� ����ȝP�

Oncotarget7www.impactjournals.com/oncotarget

also observed osteoclast morphology by staining cells with

$OH[D�ÀXRU�����SKDOORLGLQ�WKDW�GHWHFWV�DFWLQ�¿ODPHQWV�LQ�podosomes. As evident in Figure 5D, osteoclasts induced

by MM cell-derived exosomes exhibited features of

IXQFWLRQDO�UHVRUSWLYH�FHOOV�VXFK�DV�DFWLQ�ULQJV��7R�¿QDOO\�FRQ¿UP� WKDW� WKH� HIIHFW� RI� 00�FHOO� GHULYHG� H[RVRPHV�RQ�RVWHRFODVWRJHQHVLV�ZDV�FHOO�W\SH�VSHFL¿F��ZH�WUHDWHG�5DZ������FHOOV�ZLWK�H[RVRPHV�GHULYHG�E\�6:����FHOOV��a highly aggressive metastatic colorectal cancer cell

line which usually not metastasize to bone. Exosomes

produced by SW620 cells were characterized by western

EORW�DQDO\VLV��6XSSO��)LJXUH��$��DQG�'/6�DQDO\VLV��6XSSO��

)LJXUH��%���WKXV�FRQ¿UPLQJ�H[RVRPH�LGHQWLW\�The results showed in Figure 6A clearly indicate

WKDW� WKH� H[SUHVVLRQ� RI� 2&V� VSHFL¿F� PDUNHUV� GLG� QRW�change after SW620 cell-derived exosomes treatment;

DOVR� P003�� VHFUHWHG� E\� 2&V� FXOWXUHG� ZLWK� 6:����FHOO�GHULYHG�H[RVRPHV�ZDV�QRW�VLJQL¿FDWLYH��)LJXUH��%���)XUWKHUPRUH��75$3�VWDLQLQJ�DVVD\�UHYHDOHG�WKDW�6:����exosomes did not increase OCs formation (Figure 6C). In

summary, these data demonstrate that MM cell-derived

H[RVRPHV�VSHFL¿FDOO\�PHGLDWH�WKH�GLIIHUHQWLDWLRQ�RI�SUH�OCs, promoting their bone resorptive activity.

Figure 6: Colorectal cancer cell-derived exosomes not affect osteoclastogenesis of RAW 264.7 precursors. A. Quantitative

57�3&5�RI�P75$3��P&67.�DQG�P003��LQ�5DZ������FHOOV�XQWUHDWHG�RU�WUHDWHG�IRU�VL[�GD\V�ZLWK���QJ?PO�RI�KU5$1./��SRVLWLYH�FRQWURO��RU�ZLWK����ȝJ�PO�RI�6:����H[RVRPHV��9DOXHV�DUH�H[SUHVVHG�DV�IROG�RI�FRQWURO�DQG�DUH�WKH�PHDQ�RI�WKUHH�GLIIHUHQW�H[SHULPHQWV��6LGDN�WHVW��*,EXO-SW620 vs Untreated (*p������������KU5$1./�vs Untreated (p < 0.05); B.�0XULQH�0HWDOORSURWHLQDVH����P003���SURWHLQ�OHYHOV�LQ�WKH�FRQGLWLRQHG�PHGLXP�GHULYHG�IURP�5DZ������FHOOV�XQWUHDWHG�RU�WUHDWHG�IRU�VL[�GD\V�ZLWK���QJ�PO�RI�KU5$1./��SRVLWLYH�FRQWURO��RU�ZLWK����ȝJ�PO�RI�6:����H[RVRPHV�ZHUH�GHWHUPLQHG�E\�HQ]\PH�OLQNHG�LPPXQRDGVRUEHQW�DVVD\��(/,6$���'DWD�SUHVHQWHG�DUH�WKH�PHDQ�of three separate experiments. Sidak test: *,EXO-SW620 vs Untreated (p������������KU5$1./�vs�8QWUHDWHG���p < 0.05); statistically not

VLJQL¿FDWLYH��QV��C.�5$:�������FHOOV�LQFXEDWHG�ZLWK���QJ�PO�RI�KU5$1./��SRVLWLYH�FRQWURO��RU�ZLWK����ȝJ�PO�RI�6:����IRU�VL[�GD\V��DQG�WKHQ�VWDLQHG�IRU�75$3�H[SUHVVLRQ��75$3�SRVLWLYH�FHOOV��L�H���WKRVH�FRQWDLQLQJ�WKUHH�QXFOHL��ZHUH�SKRWRJUDSKHG�DQG�FRXQWHG��/RZHU�SDQHO�VKRZV�UHSUHVHQWDWLYH�SLFWXUHV�RI�75$3+5DZ������FHOOV��RULJLQDO�PDJQL¿FDWLRQ���[���'DWD�SUHVHQWHG�DUH�WKH�PHDQ�RI�WKUHH�VHSDUDWH�experiments. Sidak test: *,EXO-SW620 vs Untreated (*p������������KU5$1./�vs�8QWUHDWHG���p����������VWDWLVWLFDOO\�QRW�VLJQL¿FDWLYH��QV�

Oncotarget8www.impactjournals.com/oncotarget

Multiple myeloma cell-derived exosomes inhibit apoptosis of pOCs cells and promote their survival

:H�QH[W�H[DPLQHG��E\�$QQH[LQ�9�VWDLQLQJ��ZKHWKHU�DSRSWRWLF� HYHQWV� RFFXUUHG� LQ� 5DZ������ FHOOV� FXOWXUHG�with MM cell-derived exosomes. As shown in Figure 7A,

00�FHOO�GHULYHG� H[RVRPHV�� DV�ZHOO� DV� KU5$1./�� GLG�QRW� LQGXFH� DSRSWRVLV� LQ�5DZ������ FHOOV�� GLIIHUHQWO\� E\�treatment with DMSO, a known apoptogen and activator

RI�FDVSDVH���DFWLYLW\��1RWDEO\��ZH�IRXQG�WKDW�WUHDWPHQW�RI�cells with MM cell-derived exosomes was associated with

FDVSDVH���DFWLYDWLRQ�DV�UHYHDOHG�E\�LQFUHDVHG�FDVSDVH�����activity (Figure 7B).

$V�6]\PF]\N�.+�DQG�FROOHDJXHV�>��@�UHSRUWHG�WKDW�LQGXFWLRQ�RI�GLIIHUHQWLDWLRQ�LQ�5DZ������FHOOV�LV�GHSHQGHQW�on caspase-3 activity, we next investigated if OCs

differentiation induced by MM cell-derived exosomes

may require caspase-3 activation. We pharmacologically

LQKLELWHG�FDVSDVH���DFWLYLW\�LQ�5DZ������FHOOV�H[SRVHG�WR�MM cell-derived exosomes (Suppl. Figure 6A) and then

ZH�DQDO\]HG�75$3�DFWLYLW\�� LQWHUHVWLQJO\��ZH�REVHUYHG�WKDW� LQKLELWLRQ� RI� FDVSDVH��� DFWLYLW\� UHGXFHG� 75$3�P51$�H[SUHVVLRQ� LQ�5DZ������FHOOV� WUHDWHG�ZLWK�00�cell-derived exosomes (Figure 7C) as well as the number

RI�75$3�SRVLWLYH�FHOOV�DIWHU���GD\V�RI�WUHDWPHQW��6XSSO��Figure 6B).

Western blotting analysis revealed an increase

of anti-apoptotic protein BCl-Xl levels, survivin levels

DQG� SKRVSKR�$.7� OHYHOV� �)LJXUH� �'��� 2XU� GDWD� ZHUH�FRQ¿UPHG�E\� WKH� ODFN� RI�781(/� VWDLQLQJ� DQG� QXFOHDU�FRQGHQVDWLRQ�LQ�5DZ�������FHOOV�WUHDWHG�ZLWK�H[RVRPHV��GDWD�QRW�VKRZQ���7DNHQ�WRJHWKHU��WKHVH�¿QGLQJV�SURYLGH�

Figure 7: Exosomes released by multiple myeloma cells support osteoclast precursors survival and inhibit apoptosis. A.�)DFV�DQDO\VLV�RI�$QQH[LQ�9�LRGLGH�VWDLQLQJ�RI�5DZ������FHOOV�WUHDWHG�IRU����KRXUV�ZLWK���QJ?PO�KU5$1./�����'062�RU����ȝJ�PO�of U266- and MM1s-derived exosomes B.�&DVSDVH�����DFWLYLW\�DVVD\�ZDV�SHUIRUPHG�LQ�5DZ������FHOOV�XQWUHDWHG�RU�WUHDWHG�IRU����KRXUV�ZLWK���QJ?PO�RI�KU5$1./�DQG�ZLWK����ȝJ�PO�RI�8�����DQG�00�V�GHULYHG�H[RVRPHV��/XPLQHVFHQFH�ZDV�PHDVXUHG�XVLQJ�WKH�&DVSDVH�����*OR�DVVD\��5HVXOWV�DUH�H[SUHVVHG�DV�SHUFHQWDJH�RI�FDVSDVH�DFWLYLW\�FRPSDUHG�WR�XQWUHDWHG�FHOOV��$YHUDJHG�YDOXHV�RI�WKUHH�LQGHSHQGHQW�experiments are plotted including ±S.D. Sidak test: *�(;2�8����00�V�vs Untreated (*p������������KU5$1./�vs�8QWUHDWHG���S����������C. 4XDQWLWDWLYH�57�3&5�RI�P75$3�LQ�5DZ������FHOOV�XQWUHDWHG�RU�SUHWUHDWHG�ZLWK�D�FDVSDVH���LQKLELWRU�����0�=�'(9'�)0.���K�EHIRUH�DQG�GXULQJ�KU5$1./�RU�00�H[RVRPHV�WUHDWPHQW��9DOXHV�DUH�H[SUHVVHG�DV�IROG�RI�FRQWURO�DQG�DUH�WKH�PHDQ�RI�WKUHH�GLIIHUHQW�H[SHULPHQWV��Sidak test: *�(;2�8����00�V���='(9'�vs�(;2�8����00�V��*p������������KU5$1./�='(9'�vs�KU5$1./���S����������+ D. Western

EORWWLQJ�DQDO\VLV�RI�S�$.7��727�$.7��%FO�[O�DQG�6XUYLYLQ�LQ�5DZ������FHOOV�XQWUHDWHG�RU�WUHDWHG�ZLWK���QJ?PO�RI�KU5$1./��SRVLWLYH�FRQWURO��RU�ZLWK����ȝJ�PO�RI�8����RU�00�V�H[RVRPHV��([R�8����00�V���ȕ�DFWLQ�ZDV�XVHG�DV�ORDGLQJ�FRQWURO�

Oncotarget9www.impactjournals.com/oncotarget

evidence that MM cell-derived exosomes exert pro-

differentiative effects on pOCs cells by inhibiting

apoptotic mechanisms.

Exosomes isolated from plasma of patients with multiple myeloma modulate osteoclasts differentiation

To further strengthen our results on the role of

MM cell-derived exosomes on osteoclastogenesis, we

assessed the effect of exosomes isolated from plasma of

��00�SDWLHQWV�RQ�5DZ������FHOOV�GLIIHUHQWLDWLRQ��)LUVW��WR�characterize MM patient-derived exosomes, we performed

'\QDPLF�/LJKW�6FDWWHULQJ�$QDO\VLV�DQG�ZH�IRXQG�WKDW�VL]H�of serum exosomes was in accordance with the reported

size range of exosomes (Suppl. Figure 5C). We then

examined the effect of MM patient-derived exosomes on

RVWHRFODVWRJHQHVLV��5DZ������FHOOV�LQFXEDWHG�ZLWK�00�patient-derived exosomes for 6 days, differentiated into

PDWXUH�75$3�SRVLWLYH�PXOWLQXFOHDWHG�RVWHRFODVW��)LJXUH��$��OHIW�DQG�ULJKW�SDQHO���LQ�DGGLWLRQ��VHFUHWLRQ�RI�P003��

Figure 8: Exosomes in plasma of patients with multiple myeloma promote osteoclasts differentiation from Raw264.7 precursors and regulate bone resorption activity of osteoclasts. A.�5DZ������FHOOV�ZHUH�LQFXEDWHG�ZLWK���QJ?PO�RI�KU5$1./��SRVLWLYH�FRQWURO��RU�ZLWK����ȝJ�PO�RI�H[RVRPHV�LVRODWHG�IURP�SODVPD�RI�WZR�SDWLHQWV�ZLWK�00�IRU�VL[�GD\V���DQG�WKHQ�VWDLQHG�IRU�75$3�H[SUHVVLRQ��75$3�SRVLWLYH�FHOOV��L�H��� WKRVH�FRQWDLQLQJ�WKUHH�QXFOHL��ZHUH�SKRWRJUDSKHG�DQG�FRXQWHG��5LJKW�SDQHO�VKRZV�UHSUHVHQWDWLYH�SLFWXUHV�RI�5DZ������FHOOV�VWDLQHG�IRU�75$3��RULJLQDO�PDJQL¿FDWLRQ���[���'DWD�SUHVHQWHG�DUH�WKH�PHDQ�RI�WKUHH�VHSDUDWH�H[SHULPHQWV��6LGDN�test: �(;2�3DWLHQW������vs Untreated (*p������������KU5$1./�vs Untreated (p < 0.05); B.�0XULQH�0HWDOORSURWHLQDVH����P003���SURWHLQ�OHYHOV�LQ�WKH�FRQGLWLRQHG�PHGLXP�GHULYHG�IURP�5DZ������FHOOV�XQWUHDWHG�RU�WUHDWHG�IRU�VL[�GD\V�ZLWK���QJ?PO�RI�KU5$1./��SRVLWLYH�FRQWURO��RU�ZLWK����ȝJ�PO�RI�H[RVRPHV�LVRODWHG�IURP�SODVPD�RI�WZR�SDWLHQWV�ZLWK�00�ZHUH�GHWHUPLQHG�E\�HQ]\PH�OLQNHG�LPPXQRDGVRUEHQW�DVVD\��(/,6$���'DWD�SUHVHQWHG�DUH�WKH�PHDQ�RI�WKUHH�VHSDUDWH�H[SHULPHQWV��6LGDN�WHVW��*�(;2�3DWLHQW������vs Untreated (*p������������KU5$1./�vs�8QWUHDWHG���p < 0.05); C.�%RQH�UHVRUSWLRQ�DELOLW\�RI�5DZ������FHOOV�WUHDWHG�IRU�VL[�GD\V�ZLWK���QJ?PO�RI�KU5$1./��SRVLWLYH�FRQWURO��RU�ZLWK����ȝJ�PO�RI�H[RVRPHV�LVRODWHG�IURP�SODVPD�RI�WZR�SDWLHQWV�ZLWK�00��ZDV�HYDOXDWHG�E\�UHVRUSWLRQ�SLW�DVVD\�RQ�GHQWLQH�GLVFV��'DWD�UHODWLYH�WR�WKH�UHVRUEHG�DUHDV�E\�PDWXUH�RVWHRFODVWV�DUH�UHSUHVHQWHG�DV�IROG�LQFUHDVH�RI�5DZ������FHOOV�XQWUHDWHG��EODFN�FROXPQ����DUELWUDU\�unit). Data presented are the mean of three separate experiments. Sidak test: *�(;2�3DWLHQW������vs Untreated (*p������������KU5$1./�vs

Untreated (p����������5LJKW�SDQHO�VKRZV�UHSUHVHQWDWLYH�SLFWXUHV�RI�ODFXQDH��GDUN�DUHDV��IRUPHG�E\�FHOOV�WUHDWHG�ZLWK�UK5$1./�RU�H[RVRPHV�IURP�00�SDWLHQWV��RULJLQDO�PDJQL¿FDWLRQ���[���

Oncotarget10www.impactjournals.com/oncotarget

LQFUHDVHG�VLJQL¿FDQWO\�ZKHQ�5DZ������FHOOV�ZHUH�H[SRVHG�WR�H[RVRPHV��)LJXUH��%���:H�¿QDOO\�LQYHVWLJDWHG�LI�00�patient-derived exosomes played a role on the ability of

PDWXUH�RVWHRFODVWV�WR�UHVRUE�ERQH��1RWDEO\��WUHDWPHQW�ZLWK�00�SDWLHQW�GHULYHG�H[RVRPHV�VLJQL¿FDQWO\�LQFUHDVHG�WKH�formation of resorption pits as measured by the amounts of

SLWV�DQG�RYHUDOO�DUHD�FRPSDUHG�ZLWK�XQWUHDWHG�5DZ������cells (Figure 8C, left and right panel). Taken together,

our results are in line with in vitro data using MM cell

line-derived exosomes and demonstrate that MM patient-

derived exosomes induce differentiation and modulate

UHVRUSWLRQ�DFWLYLW\�RI�5DZ������FHOOV�

Multiple myeloma cell-derived exosomes affect osteoclastogenesis in human primary osteoclasts

To evaluate the effects of MM cell-derived

exosomes in a more physiological system, we analyzed the

role of MM exosomes also on human primary osteoclasts.

Both pOCs and mature OCs were prepared from human

3%0&V�REWDLQHG�IURP�WKUHH�KHDOWK\�GLIIHUHQW�GRQRUV�>��@��in detail, we studied the effects of U266- and MM1s-

exosomes on differentiation of OCs cultured in complete

OCs medium (differentiation medium) and in OCs

PHGLXP�ODFNLQJ�5$1./�0&6�)�GH[DPHWKDVRQH��EDVDO�medium). As shown in Figure 9A, pOCs treated with

Figure 9: Exosomes produced by multiple myeloma cells induce osteoclast formation in human primary pOCs and promote formation of lacunae on dentine slices. A.�+XPDQ�S2&V�ZHUH�FXOWXUHG�XQWUHDWHG�RU�WUHDWHG�ZLWK����ȝJ�PO�RI�8����RU�00�V�H[RVRPHV�LQ�EDVDO�PHGLXP�DQG�GLIIHUHQWLDWLRQ�PHGLXP��DQG�WKHQ�VWDLQHG�IRU�75$3�H[SUHVVLRQ��75$3�SRVLWLYH�FHOOV�ZHUH�SKRWRJUDSKHG�DQG�FRXQWHG��ORZHU�SDQHO���8SSHU�SDQHO�VKRZV�UHSUHVHQWDWLYH�SLFWXUHV�RI�KXPDQ�2&V�VWDLQHG�IRU�75$3��RULJLQDO�PDJQL¿FDWLRQ���[���'DWD�presented are the mean of three different experiments. Sidak test: *�(;2�8����00�V�vs Untreated (*p < 0.05); B.�4XDQWLWDWLYH�57�3&5�RI�K75$3�DQG�K&67.�LQ�KXPDQ�S2&V�FXOWXUHG�XQWUHDWHG�RU�WUHDWHG�ZLWK����ȝJ�PO�RI�8����RU�00�V�H[RVRPHV�LQ�EDVDO�PHGLXP�DQG�GLIIHUHQWLDWLRQ�PHGLXP��9DOXHV�DUH�H[SUHVVHG�DV�IROG�RI�XQWUHDWHG�FHOOV��FRQWURO��DQG�DUH�WKH�PHDQ�RI�WKUHH�GLIIHUHQW�H[SHULPHQWV��6LGDN�WHVW��*�(;2�8����00�V�vs Untreated (*p < 0.05); C.�(/,6$�DVVD\�RI�KXPDQ�003��VHFUHWHG�LQ�WKH�FRQGLWLRQHG�PHGLXP�GHULYHG�IURP�KXPDQ�2&V�XQWUHDWHG�RU�WUHDWHG�ZLWK����ȝJ�PO�RI�8����RU�00�V�H[RVRPHV�LQ�EDVDO�PHGLXP�DQG�GLIIHUHQWLDWLRQ�PHGLXP��'DWD�SUHVHQWHG�DUH�WKH�PHDQ�RI�WKUHH�VHSDUDWH�H[SHULPHQWV��6LGDN�WHVW�� �(;2�8����00�V�vs Untreated (*p < 0.05); D. Bone resorption ability of human OCs

XQWUHDWHG�RU�WUHDWHG�ZLWK����ȝJ�PO�RI�8����RU�00�V�H[RVRPHV�LQ�EDVDO�PHGLXP�DQG�GLIIHUHQWLDWLRQ�PHGLXP�ZDV�HYDOXDWHG�E\�UHVRUSWLRQ�pit assay on dentine discs. Data relative to the resorbed areas by mature OCs are represented as fold increase of human OCs untreated

�EODFN�FROXPQ����DUELWUDU\�XQLW���'DWD�SUHVHQWHG�DUH�WKH�PHDQ�RI�WKUHH�VHSDUDWH�H[SHULPHQWV��6LGDN�WHVW�� �(;2�8����00�V�vs Untreated

(*p < 0.05).

Oncotarget11www.impactjournals.com/oncotarget

U266- and MM1s- exosomes differentiated into large,

multinucleated OCs, even in basal medium; the number

RI�75$3�SRVLWLYH�PDWXUH�2&V�ZDV�VLJQL¿FDQWO\�KLJKHU�compared to control cells.

$FFRUGLQJO\�� T57�3&5� DQDO\VLV� VKRZHG� WKDW�treatment of human pOCs with U266 and MM1s exosomes

LQFUHDVHG�WKH�H[SUHVVLRQ�RI�75$3�DQG�&67.��DV�FRPSDUHG�WR�FRQWURO�FHOOV� �)LJXUH��%��� IXUWKHUPRUH��(/,6$�DVVD\�UHYHDOHG� WKDW� KXPDQ� 003�� SURWHLQ� VHFUHWLRQ� ZDV�increased in differentiated OCs treated with MM exosomes

(Figure 9C). Furthermore, mature OCs treated with U266-

exosomes strongly formed resorption pits on synthetic

dentine discs, both in basal and differentiation medium

�)LJXUH��'���:H�¿QDOO\�XVHG�DV�QRUPDO�FRQWUROV��H[RVRPHV�derived from human peripheral blood mononuclear cells

from healthy donors; osteoclastogenesis assays performed

RQ�ERWK�KXPDQ�S2&V�DQG�PXULQH�5DZ������FHOOV�VKRZHG�WKDW�3%0&V�H[RVRPHV�ZHUH�QRW�DEOH�WR�DIIHFW�VLJQL¿FDQWO\�OCs differentiation (Suppl. Fig.7).

DISCUSSION

Multiple myeloma bone disease (MBD) is

characterized by osteolytic bone destruction leading

to impairment of patients’s quality of life. Increased

osteoclast activity and suppressed osteoblast function

is the main mechanism of MBD. In detail, osteolytic

lesions are consequential to osteoclasts recruitment,

differentiation and function, which can be induced in

the local bone marrow microenvironment by tumor

cells; furthermore, osteoclastogenesis promote myeloma

progression by both direct cell-cell communications as

well as indirectly by soluble factors released by local

bone marrow microenvironment cells [27]. Within the

tumor microenvironment, the release of exosomes from

cancer cells may confer relevance in cancer growth and

progression [2, 21].

In the current study, we focused on the role played

by MM cell-derived exosomes, as well as by MM

patient’s exosomes, on the process of bone remodelling

by performing in vitro assay with both human primary

RVWHRFODVWV�DQG�PXULQH�SUH�RVWHRFODVWLF�5DZ������FHOOV��Exosomes from three different MM cell types (U266,

00�V�DQG�230���DQG�IURP�00�SDWLHQWV�ZHUH�LVRODWHG�and characterized for their biochemical and dimensional

properties and the results obtained were in agreement

with those from other groups [24, 28]. We also showed

that MM exosomes were able to be actively internalized

E\�5DZ������FHOOV��ZKLOH�XSWDNH�LQKLELWLRQ�LQ�WKH�SUHVHQFH�RI� ��HWK\O�1�LVRSURS\O� DPLORULGH� �(,3$�� VXJJHVWHG� WKH�involvement of additional endocytic pathways [29, 30].

5HFHQW�GDWD�UHSRUWHG�WKDW�KXPDQ�PXOWLSOH�P\HORPD�cells secrete microvesicles able to promote in vitro and in vivo angiogenesis by stimulating the endothelial cells to

SUROLIHUDWH��LQYDGH�DQG�VHFUHWH�,/���DQG�9(*)�>��@��,Q�WKH�bone marrow niche, OCs develop and function in close

association with microvascular endothelial cells which,

in turn, are critically involved in bone development and

UHPRGHOOLQJ��LQÀXHQFLQJ�2&V�UHFUXLWPHQW��IRUPDWLRQ�DQG�activity [32]. The crosstalk between bone marrow stromal

cells (BMSCs) and MM cells supports the proliferation,

survival, migration, and drug resistance of MM cells as

well as osteoclastogenesis and angiogenesis [34,35].

Several factors produced as a result of MM-BMSC cell

interactions alter OCs and OBs activities; MM cells

directly stimulate OCs differentiation by secreting OC-

activating factors and, indirectly, by stimulating BMSCs

secretion of additional OC-activating factors [33-35].

5RFFDUR�$0�DQG�FROOHDJXHV�KDYH�UHFHQWO\�GHPRQVWUDWHG�that BMSCs of patients with MM release exosomes that

may be transferred to MM cells thus inducing MM cell

growth and promoting dissemination. The proteomic

analysis of MM BMSCs exosomes evidenced elevated

expression of oncogenic proteins, cytokines and protein

kinases compared with exosomes from normal BMSCs

[36].

In another study, exosomes obtained from BMSCs

induced survival and drug resistance of human MM

cells and the authors speculated on the possibility that

BMCSs and myeloma cells may also communicate

WKURXJK�PXWXDO�H[FKDQJH�RI�H[RVRPHV�>��@��5HFHQWO\��LW�has been showed that multiple myeloma cells in chronic

hypoxic bone marrow secrete more exosomes than the

parental cells under normoxia; interestingly, exosomal

PL5����E� UHJXODWHG� WKH� +,)��� VLJQDOLQJ� LQ� +89(&V�E\�HQKDQFLQJ�DQJLRJHQHVLV�>��@��7KHVH�¿QGLQJV�DFTXLUH�greater importance if we consider that hypoxia triggers

angiogenic events within the microenvironment [38]

and that inhibition of hypoxia in MM cells impair pro-

RVWHRFODVWRJHQLF�F\WRNLQHV�H[SUHVVLRQ�ZLWK�D�VLJQL¿FDWLYH�reduction of bone loss [39]. Furthermore, the analysis

RI� GHUHJXODWHG� PLFUR51$V�� DV� ZHOO� DV� RI� QRQ�FRGLQJ�51$V�>��@�LV�HPHUJLQJ�DV�D�QRYHO�DSSURDFK�WR�GLVFORVH�the regulation of tumor suppressor or tumor promoting

pathways in tumor cells [41-44].

Overall, our results here summarized support

the hypothesis that exosomes are important mediators

of crosstalk between MM cells and BM microenvironment.

7KH� ¿QGLQJV� GHVFULEHG� LQ� RXU� VWXG\�� SURYLGH� WKH� ¿UVW�evidence, at our knowledge, that exosomes derived from

P\HORPD� FHOOV� GLUHFWO\� LQÀXHQFH� 2&V� GLIIHUHQWLDWLRQ�and function. Bone resorption is the result of sequential

stages characterized by pOCs migration, invasion and

homing from the peripheral circulation to bone, where

eventually takes place differentiation of pre-OCs into

multinucleated OCs, responsible of bone remodelling.

1RWDEO\��LQ�0%'��P\HORPD�FHOOV�H[HUW�D�VLJQL¿FDQW�HIIHFW�on recruitment, proliferation and differentiation of OC

progenitors [26, 45]. Even if the mechanism of osteoclast

precursor recruitment remains poorly understood, several

chemoattractants have been show to play critical role in

controlling the migration of monocyte-lineage precursors

Oncotarget12www.impactjournals.com/oncotarget

from peripheral circulation to sites of bone resorption [46].

Consistent with these observations, we investigated the

potential role of MM cell-derived exosomes on migration

RI�S2&V�FHOOV��2XU�¿QGLQJV�FOHDUO\�LQGLFDWHG�WKDW�H[RVRPHV�LVRODWHG�E\�8����DQG�00�V�FHOOV�LQÀXHQFHG�SRVLWLYHO\�both murine and human pOCs migration, inducing a

VWURQJ�H[SUHVVLRQ�RI�&;&5��LQ�KXPDQ�S2&V��230��FHOO�derived exosomes exerted minor effects on the phenomena

GHVFULEHG� DERYH�� ,Q� DGGLWLRQ�� 230��FHOO� GHULYHG�exosomes affected osteoclast differentiation and function

PRUH�ZHDNO\�FRPSDUHG�WR�8����00�V�H[RVRPHV��7KLV�could be explained by well-known heterogeneity in

myeloma [47]. Since MM cells can colonize bone marrow

microenvironment where they induce osteolytic lesions,

we investigated the potential role of MM cell-derived

exosomes on OCs differentiation. To address this issue,

ZH�DQDO\]HG�WKH�H[SUHVVLRQ�RI�RVWHRFODVW�VSHFL¿F�PDUNHUV�LQ� 5DZ������ FHOOV� FXOWXUHG� IRU� �� GD\V� ZLWK�00� FHOO�GHULYHG�H[RVRPHV�RU�ZLWK�5$1./��D�ZHOO�NQRZQ�LQGXFHU�of OCs differentiation, used as positive control [13]. In

parallel, we evaluated the effects of MM cell-derived

exosomes on human primary pOCs cultured in basal or

complete OCs medium. In particular, we examined the

H[SUHVVLRQ�RI�&76.�� D� SURWHRO\WLF� HQ]\PH� UHVSRQVLEOH�RI�ERQH�UHVRUSWLRQ��003���D�JHODWLQDVH�VHFUHWHG�E\�2&V�PDWXUH�LQYROYHG�LQ�ERQH�UHVRUSWLRQ��DQG��¿QDOO\��75$3��D�regulator of bone resorption through degradation of bone

matrix phosphoproteins, highly expressed in terminally

differentiated OCs [48-50].

We found that the expression of OCs markers, such

DV�VHFUHWLRQ�RI�P003���ZDV�VLJQL¿FDQWO\� LQFUHDVHG�LQ�5DZ������ FHOOV� DQG� KXPDQ�2&V� H[SRVHG� WR�00�FHOO�derived exosomes; these results demonstrate that in

addition to affect migration of osteoclast precursors,

MM cell-derived exosomes induce their differentiation

in mature OCs and control secretion of factors involved

in bone resorption activity. Data from literature reported

that MM cells stimulate OCs development from precursor

cells as well as OCs activity through production of key

RVWHRFODVWRJHQLF� IDFWRUV� >���� ��@�� +RZHYHU�� D� GLUHFW�effect of MM cells on recruitment and differentiation

of pOCs has not clearly demonstrated. In our study,

addition of multiple myeloma derived exosomes to

osteoclast precursors resulted in formation of functional

osteoclasts. These latter were evidenced by the presence

RI�PXOWLQXFOHDWHG�75$3�SRVLWLYH�FHOOV�DQG�FKDUDFWHUL]HG�by OCs actin rings, indicative of unique cell adhesion

structures established at sites of OCs attachment to the

ERQH� VXUIDFH� >�����@��1RWDEO\��2&V�� IRUPHG� IROORZLQJ�treatment with MM-derived exosomes, resemble mature

resorptive cells able to excavate lacunae in vitro, similar

to the structures formed when the cells degrade bone in vivo [55]. Of importance, the results obtained appear to be

FHOO�W\SH�VSHFL¿F��DV�H[RVRPHV�GHULYHG�E\�WKH�PHWDVWDWLF�colorectal cancer cell line SW620 did not elicit the same

effects.

Several studies showed that pathways correlated

with OCs differentiation promote OCs survival, activate

3,��.LQDVH�$.7� VLJQDOLQJ� SDWKZD\� DQG� LQFUHDVH� WKH�expression of anti-apoptotic genes [56, 57]. Interestingly,

6]\PF]\N�.+�HW�DO�GHPRQVWUDWHG�WKDW�DFWLYH�FDVSDVH���is required for OCs differentiation, suggesting a non-

apoptotic role for caspase-3 in pOC cells. In particular,

authors showed that caspase-3 is an early signalling event

WKDW�DIIHFW�75$3�H[SUHVVLRQ�DQG�WKH\�REVHUYHG�D�GHFUHDVHG�number of functional OCs in procaspase-3 knockout mice

as well as the lack of differentiation in OCs in response

WR�5$1./�ZKHQ�FDVSDVH���DFWLYLW\�ZDV�LQKLELWHG�>��@��In our study, we provided evidence that MM cell-derived

H[RVRPHV�LQGXFHG�FDVSDVH���DFWLYDWLRQ�LQ�5DZ������FHOOV��even in absence of a clear apoptosis; notably, treatment

ZLWK�00�FHOO�GHULYHG� H[RVRPHV� IDLOV� WR� LQGXFH�75$3�DFWLYLW\�LQ�5DZ������FHOOV� WKDW�ZHUH�SKDUPDFRORJLFDOO\�inhibited for caspase-3 activity.

)XUWKHUPRUH��00�GHULYHG�H[RVRPHV�DFWLYDWHG�$.7�pathway which in turn, as it is well known, enhanced

survival and anti-apoptotic gene expression in OCs.

Importantly, our in vitro� REVHUYDWLRQV� ZHUH� FRQ¿UPHG�ex vivo by using exosomes isolated from MM patients.

Exosomes isolated and characterized from plasma of MM

patients in fact positively modulated the differentiation

and activity of OCs, thus adding a potential clinical

VLJQL¿FDQFH�WR�RXU�UHVXOWV��An important question that remains to be addressed

is to understand how MM cell-derived exosomes may

act on osteoclastogenesis; to address this issue, future

studies will be necessary to identify the exosomal factors

responsible for the processes we described.

1RWDEO\�� D� UHFHQW� VWXG\� GHVFULEHG� WKH� SURWHRPLF�content of exosomes derived from MM1s and U266

FHOO� OLQHV�� FRPSDULQJ� WKH� SURWHLQ� LGHQWL¿HG� EHWZHHQ�the vesicles and cellular lysates of each cell line [58].

The authors detected the exclusive presence in the

exosomal compartment of bone marrow stromal protein

2 (BST-2), previously described as strongly expressed

in bone metastatic breast cancer [59]. Taken together

WKH� UHVXOWV� UHSRUWHG� LQ� WKLV� VWXG\� VLJQL¿FDQWO\� HQKDQFH�our understanding about intercellular communication in

MBD by demonstrating that MM cells release biologically

active exosomes responsible, inside the metastatic niche,

of pOCs recruitment, migration and differentiation also

through apoptotic inhibition.

MATERIALS AND METHODS

Cell lines and reagents

8�����00�V�DQG�230��FHOO�OLQHV�ZHUH�SXUFKDVHG�from ATCC®��/*&�6WDQGDUGV�6�U�O�6HVWR�6DQ�*LRYDQQL��0,�� ,WDO\�� DQG� JURZQ� LQ� 530,������ �*LEFR�� /LIH�

Oncotarget13www.impactjournals.com/oncotarget

Technologies, USA) supplemented with 10% Fetal Bovine

6HUXP��)%6��/RQ]D�*URXS��%DVHO��6ZLW]HUODQG���0XULQH�PDFURSKDJH�5DZ������FHOOV�ZHUH�SXUFKDVHG�

from ATCC®�DQG�FXOWXUHG�LQ�'XOEHFFR¶V�PRGL¿HG�(DJOH¶V�medium (DMEM), supplemented with 10% FBS. To

LQGXFH� GLIIHUHQWLDWLRQ�� FHOOV� ZHUH� WUHDWHG� ZLWK� ��QJ�PO� RI� KXPDQ� UHFRPELQDQW�5$1.�/LJDQG� �*LEFR��/LIH�Technologies, USA) for 6 days in DMEM, supplemented

ZLWK�����)%6��SUHYLRXVO\�XOWUDFHQWULIXJDWHG��5DZ������cells OC medium). Alternately, cells were treated for

��GD\V�ZLWK����J�PO�RI�00�FHOO�GHULYHG�H[RVRPHV�� LQ�DMEM, supplemented with 10% of ultracentrifugated

)%6� �VHH� EHORZ��� ���1�(WK\O�1�LVRSURS\O�� DPLORULGH��(,3$�� ZDV� SXUFKDVHG� IURP� 6LJPD� $OGULFK�,WDO\��&DVSDVH���LQKLELWRU�=�'(9'�)0.�ZDV�SXUFKDVHG�IURP�5'�V\VWHPV��

Isolation of human peripheral blood mononuclear cells

+XPDQ�EORRG�VDPSOHV�ZHUH�REWDLQHG�IURP�KHDOWK\�donors, after written informed consent obtained in

DFFRUGDQFH�ZLWK� WKH�'HFODUDWLRQ�RI�+HOVLQNL�JXLGHOLQHV�DQG� 8QLYHUVLW\� RI� 3DOHUPR� (WKLFV� FRPPLWWHH�� +XPDQ�SHULSKHUDO� EORRG� PRQRQXFOHDU� FHOOV� �3%0&V�� ZHUH�LVRODWHG� XVLQJ� WKH� )LFROO�3DTXH� �*(� +HDOWKFDUH� %LR�Science, Uppsala, Sweden) separation technique.

Preparation of human primary pOC and OCs

3%0&V�ZHUH�FXOWXUHG�DW����[��6FHOOV�PO�Į�0(0�supplemented with 10% FBS previously ultracentrifuged,

��QJ�PO� RI� KXPDQ� UHFRPELQDQW� 5$1.� /LJDQG��*LEFR�� /LIH� 7HFKQRORJLHV�� 86$��� ��QJ�PO� RI� KXPDQ�0�&6)� �*LEFR�� /LIH� 7HFKQRORJLHV�� 86$��� DQG� ��Q0�GH[DPHWKDVRQH� �6LJPD�$OGULFK� ,WDO\�� �+XPDQ� 2&�medium). After 2-4 days, the culture were washed with

Į�0(0� PHGLXP� WR� UHPRYH� QRQ� DGKHUHQW� FHOOV�� 7KH�UHPDLQLQJ� FHOOV�ZHUH�PRQRQXFOHDWHG�� H[SUHVVHG�75$3�and were considered committed pre-osteoclast cells.

For human osteoclastogenesis assays, OC medium was

added and the cultures were continued for additional 6-10

days, at the end of the period they contained large mature

multinucleated OCs. The culture period was 16 days for

ERWK�75$3�VWDLQLQJ�DVVD\��ERQH�UHVRUSWLRQ�DVVD\�DQG�T57�3&5�DQDO\VLV�

([RVRPH�SXUL¿FDWLRQ

Exosomes released by both multiple myeloma cells

�8�����00�V� DQG�230�� FHOOV�� GXULQJ� D� ��� K� FXOWXUH�SHULRG� DQG� 3%0&V� GXULQJ� D� ��K� FXOWXUH� SHULRG�� ZHUH�isolated from conditioned culture medium supplemented

with 10% FBS previously ultracentrifuged by differential

FHQWULIXJDWLRQV�DV�SUHYLRXVO\�GHVFULEHG�>��@��7R�FRQ¿UP�YHVLFOHV�LGHQWLW\�DV�H[RVRPHV��YHVLFOHV�ZHUH�SXUL¿HG�RQ�D�����VXFURVH�'�2�FXVKLRQ��9HVLFOHV� FRQWDLQHG� LQ� WKH�sucrose cushion were recovered, washed, ultracentrifuged

IRU� ��� PLQ� LQ� 3%6� DQG� FROOHFWHG� IRU� XVH�� ([RVRPH�protein content was determined by the Bradford assay

[61]. Exosomes isolated from human blood samples

were obtained from two diagnosed MM patients.

Informed consent was obtained from patients, according

WR� WKH�'HFODUDWLRQ�RI�+HOVLQNL�DQG�ZLWK�KRVSLWDO�(WKLFV�Committee approval. Serum were isolated using the Ficoll-

3DTXH� �*(� +HOWKFDUH�%LR� 6FLHQFH�� 8SSVDOD�� 6ZHGHQ��separation technique, according to the manufacturer’s

instructions. Exosomes isolated from human serum

were prepared as described above. The activity of

acetylcholinesterase, an exosome marker protein, was

determined as described by Savina et al. [24].

Uptake of multiple myeloma exosomes by Raw264.7 cells

MM cell-derived exosomes (isolated from

8����� 00�V� DQG� 230��� ZHUH� ODEHOHG� ZLWK� 3.+���(Sigma-Aldrich, Italy), according to the manufacturer’s

LQVWUXFWLRQV�� %ULHÀ\�� H[RVRPHV� FROOHFWHG� DIWHU� WKH�100,000×g ultracentrifugation, were incubated with

3.+���IRU����PLQ�DW�URRP�WHPSHUDWXUH��/DEHOHG�H[RVRPHV�ZHUH�ZDVKHG�LQ�3%6��FHQWULIXJDWHG��UHVXVSHQGHG�LQ�ORZ�VHUXP�PHGLXP�DQG�LQFXEDWHG�ZLWK�5DZ������FHOOV�IRU�����KRXUV�DW����&�RU���&��,Q�D�VHW�RI�H[SHULPHQWV��5DZ������FHOOV�ZHUH�SUHWUHDWHG�ZLWK����ȝ0�(,3$��D�NQRZQ�LQKLELWRU�of exosomes uptake, for 1 hour. After incubation, cells

ZHUH�SURFHVVHG�DV�SUHYLRXVO\�GHVFULEHG�>��@��5DZ������FHOOV�ZHUH�VWDLQHG�ZLWK�$FWLQ*UHHQTM�����5HDG\�3UREHV5 5HDJHQW��/LIH�7HFKQRORJLHV��86$��WKDW�ELQGV�)�DFWLQ�ZLWK�KLJK�DI¿QLW\��1XFOHL�ZHUH�VWDLQHG�ZLWK�+RHFKVW��0ROHFXODU�3UREHV��/LIH�7HFKQRORJLHV��86$��DQG�DQDO\VHG�E\�FRQIRFDO�PLFURVFRS\��1LNRQ�(FOLSVH�7i).

Western blotting and antibodies

6'6�3$*(�(OHFWURSKRUHVLV�DQG�:HVWHUQ�%ORWWLQJ�ZHUH�SHUIRUPHG�DV�SUHYLRXVO\�GHVFULEHG�>��@��%ULHÀ\��FHOOV�were lysed for an 1 hr in lysis buffer containing 15mM

7ULV�+&O�S+��������P0�1D&O����P0�.&O���P0�('7$�������7ULWRQ�;�����DQG�3URWHDVH�,QKLELWRU�&RFNWDLO�����;��Sigma–Aldrich, USA). Cell lysates (30µg per lane) were

VHSDUDWHG�XVLQJ�������1RYH[�%LV�7ULV�6'6�DFU\ODPLGH�JHOV��,QYLWURJHQ��/LIH�7HFKQRORJLHV��86$���WUDQVIHUUHG�RQ�1LWURFHOOXORVH�PHPEUDQHV��,QYLWURJHQ��/LIH�7HFKQRORJLHV��USA), and immunoblotted with the primary antibodies.

The antibodies against the following proteins were used:

%FO�;/��VF��������VXUYLYLQ��VF���������003���VF��������&'����VF���������&DOQH[LQ��VF���������ȕ�DFWLQ��VF��������and all secondary antibodies were obtained from Santa

Oncotarget14www.impactjournals.com/oncotarget

Cruz Biotechnology (Santa Cruz Biotechnology, Inc.,

6DQWD�&UX]��&$��86$���3KRVSKR�$NW��6HU������$NW��SDQ��(C67E7), Alix (2171S) were obtained from Cell Signaling

�%HYHUO\��0$���&DWKHSVLQ�.��DE��������ZDV�REWDLQHG�IURP�$EFDP��$EFDP��&DPEULGJH��8.���&KHPLOXPLQHVFHQFH�was detected using AmershamTM�(&/TM Western Blotting

'HWHFWLRQ�5HDJHQWV��*(�+HDOKWFDUH��8.��

Dynamic Light Scattering (DLS) analysis

([RVRPH�VL]H�GLVWULEXWLRQ�ZDV�GHWHUPLQHG�E\�'/6�experiments. Collected exosome samples were diluted

30 times to avoid inter-particle interaction and placed at

���&�LQ�D�WKHUPRVWDWHG�FHOO�FRPSDUWPHQW�RI�D�%URRNKDYHQ�Instruments BI200-SM goniometer, equipped with a

solid-state laser tuned at 532 nm. Scattered intensity

autocorrelation functions g2(t) were measured by using

a Brookhaven BI-9000 correlator and analyzed in order

WR� GHWHUPLQH� WKH� GLVWULEXWLRQ� 3�'�� RI� WKH� GLIIXVLRQ�FRHI¿FLHQW� '� E\� XVLQJ� D� FRQVWUDLQHG� UHJXODUL]DWLRQ�method or alternatively a gamma distribution [28]. The

size distribution, namely the distribution of hydrodynamic

diameter Dh, was derived by using the Stokes-Einstein

UHODWLRQ��'� ��N%7����ʌȘ�'K���ZKHUH�'�LV�WKH�GLIIXVLRQ�FRHI¿FLHQW�� N%� LV� WKH� %ROW]PDQ� FRQVWDQW�� Ș� LV� WKH�medium viscosity and T is the temperature. The mean

hydrodynamic diameter of exosomes was calculated

E\� ¿WWLQJ� D� *DXVVLDQ� IXQFWLRQ� WR� WKH� PHDVXUHG� VL]H�distribution.

Cell viability

&HOO� YLDELOLW\� RI� 5DZ������ FHOOV� VWLPXODWHG� ZLWK�MM cell-derived exosomes was evaluated by MTT

�6LJPD�$OGULFK��,WDO\���5DZ������FHOOV�ZHUH�VHHGHG�LQ����ZHOO�SODWHV�DW�D�GHQVLW\�RI������FHOOV�FP2 per well and then

FXOWXUHG�ZLWK�00�FHOO�GHULYHG�H[RVRPHV��KU5$1./�RU�a MM-conditioned medium that was previously deprived

RI�H[RVRPHV��9LDELOLW\�DW�GLIIHUHQW�WLPH�SRLQWV�����KRXUV�and 7 days) was assessed by measuring the absorbance at

����QP�XVLQJ�DQ�(/,6$�SODWH�UHDGHU��(/����$EVRUEDQFH�0LFURSODWH�5HDGHU��%LR7HN�,QVWUXPHQWV��8�6���

Migration assay

0LJUDWLRQ� ZDV� GHWHUPLQHG� XVLQJ� ���SRUH� ¿OWHUV�for the Transwell migration assay (BD Biosciences),

according to the manufacturer’s instructions and as

SUHYLRXVO\�GHVFULEHG�>��@��%ULHÀ\��PXULQH�5DZ������FHOOV�and human pre-osteoclast cells (1x105 FHOOV�ZHOO��WUHDWHG�IRU���KUV�ZLWK�00�FHOO�GHULYHG�H[RVRPHV�����J�PO��ZHUH�washed and resuspended in DMEM high glucose medium

supplemented with 1% of ultracentifuged FBS. Cells were

then placed in the upper migration chambers, whereas the

lower chamber contained DMEM high glucose medium

supplemented with 10% ultracentifugated FBS. Filters

ZHUH�UHPRYHG�DIWHU����KRXUV��¿[HG�LQ�PHWKDQRO�DQG�FHOOV�VWDLQHG� ZLWK� 'LII�4XLFN� �0HGLRQ� 'LDJQRVWLFV� *PE+��Düdingen, Switzerland). Alternatively, pre-osteoclast cells

ZHUH�QRW�WUHDWHG�DQG�00�FHOO�GHULYHG�H[RVRPHV�����J�ml) were added directly in the lower chamber contained

DMEM high glucose medium supplemented with 10%

ultracentifugated FBS. Two independent experiments were

SHUIRUPHG�LQ�WULSOLFDWH��FHOOV�IURP���GLIIHUHQW�¿HOGV�ZHUH�counted for each condition.

RNA extraction and real-time PCR

7RWDO�51$�IURP�ERWK�5DZ������FHOOV�DQG�+XPDQ�SULPDU\� RVWHRFODVW� FHOOV� ZDV� H[WUDFWHG� XVLQJ� 75,]RO�5HDJHQW���,QYLWURJHQ��/LIH�7HFKQRORJLHV��86$��DFFRUGLQJ�WR� WKH�PDQXIDFWXUHU¶V� SURWRFRO�� T57±3&5�ZDV� XVHG� WR�FRQ¿UP� WKH� H[SUHVVLRQ� OHYHOV� RI� P51$V�� )RU� P51$�GHWHFWLRQ��ROLJR�G7�SULPHG�F'1$�ZDV�REWDLQHG�XVLQJ�WKH�+LJK�&DSDFLW\� F'1$� 5HYHUVH� 7UDQVFULSWLRQ� .LWV� �$%�Applied Biosystems, USA) and then used as template to

TXDQWLI\�P51$�OHYHOV�E\�)DVW�6<%55�*UHHQ�0DVWHU�0L[��$%�$SSOLHG�%LRV\VWHP��86$���3ULPHUV�DUH�UHSRUWHG�LQ�6XSSOHPHQWDO�7DEOH����5HODWLYH�FKDQJHV�LQ�JHQH�H[SUHVVLRQ�between control and treated samples were determined with

WKH�ǻǻ&W�PHWKRG��)LQDO�YDOXHV�ZHUH�H[SUHVVHG�DV�IROG�RI�induction.

TRAP staining assay

+XPDQ� SULPDU\� RVWHRFODVWV� DQG� 5DZ������ FHOOV�cultured in OC medium alone or with MM cell-derived

H[RVRPHV�����J�PO��ZHUH�VWDLQHG�IRU�GHWHFWLRQ�RI�WDUWUDWH�UHVLVWDQW�DFLG�SKRVSKDWDVH��75$3��DFWLYLW\��DFFRUGLQJ�WR�WKH�PDQXIDFWXUHU¶V�SURWRFRO��$FLG�3KRVSKDWDVH��/HXNRF\WH��75$3��.LW��6LJPD±$OGULFK��86$��DQG�HYDOXDWHG�E\�OLJKW�PLFURVFRS\��0XOWLQXFOHDWHG�75$3��FHOOV�FRQWDLQLQJ�PRUH�than three nuclei were scored as mature osteoclasts. Three

independent experiments were performed in triplicate;

FHOOV� IURP� �� GLIIHUHQW� ¿HOGV� ZHUH� FRXQWHG� IRU� HDFK�condition.

Bone resorption assay

+XPDQ� SULPDU\� RVWHRFODVWV� DQG� 5DZ������ FHOOV�were seeded at a density of 5x104� FHOOV�PO� LQ� ���ZHOO�SODWHV�RQ�RUJDQLF�GHQWLQH�GLVFV��'HQWLQH�'LVFV��3DQWHF��Torino, Italy) and cultured in OC medium alone or with

00�FHOO�GHULYHG�H[RVRPHV�����J�PO���7KH�GHQWLQH�GLVFV��at the end of the culture duration, were rinsed with 70%

VRGLXP� K\SRFKORULWH� DQG� ¿[HG� LQ� ��� JOXWDUDOGHK\GH��The resorption pits were stained using 1% toluidine blue

DQG� REVHUYHG�ZLWK� D� OLJKW�PLFURVFRSH� �/HLFD�'0�����

Oncotarget15www.impactjournals.com/oncotarget

0LFURV\VWHPV��*HUPDQ\��DW�D���;�PDJQL¿FDWLRQ��7KUHH�¿HOGV�RI�HDFK�GHQWLQ�GLVF�IRU�HDFK�H[SHULPHQWDO�SRLQW�ZHUH�scored in three independent experiments. The number of

WKH�SLWV�ZDV�FDOFXODWHG�E\�1,+�LPDJH-�VRIWZDUH�DQDO\VLV��KWWWS��UVEZHE�QLK�JRY�LM����6XUIDFH�RI�GHQWLQ�VDPSOHV�KDYH�EHHQ�REVHUYHG�XVLQJ�=HLVV�(92�'+���VFDQVLRQ�HOHFWURQ�microscopy (SEM).

Cytoskeletal organization

7R� DVVHVV� F\WRVNHOHWRQ� RUJDQL]DWLRQ�� 5DZ� ������cells were seeded on coverslips, cultured for 6 days with

HLWKHU�KU5$1./����QJ�PO��RU�00�FHOO�GHULYHG�H[RVRPHV�����J�PO���¿[HG�LQ����SDUDIRUPDOGHK\GH�DQG�VWDLQHG�ZLWK�SKDOORLGLQ±$OH[D� ÀXRU� ���� �/LIH� 7HFKQRORJLHV�� 86$���$QDO\VLV�ZDV�SHUIRUPHG�DW�FRQIRFDO�PLFURVFRS\��1LNRQ�Eclipse Ti).

Caspases assay

+XPDQ� SULPDU\� RVWHRFODVWV� DQG� 5DZ������ FHOOV�FXOWXUHG�IRU���K�LQ�2&�PHGLXP�DORQH�RU�ZLWK����J�PO�MM cell-derived exosomes were analyzed for activity of

FDVSDVH�����E\�&DVSDVH�*OR®�����$VVD\�6\VWHP��3URPHJD��0DQQKHLP�� *HUPDQ\�� DFFRUGLQJ� WR� PDQXIDFWXUHU¶V�instructions. Fluorescence and luminescence was

UHFRUGHG� XVLQJ� *OR0D[� 0XOWL�PLFURSODWH� UHDGHU��3URPHJD�� 0DQQKHLP�� *HUPDQ\��� &DVSDVH��� DFWLYLW\�ZDV�SKDUPDFRORJLFDOO\�LQKLELWHG�XVLQJ����0�=�'(9'�)0.��5'�V\VWHPV���7KH�LQKLELWRU�ZDV�DGGHG��K�EHIRUH�and during osteoclast differentiation treatment. These

inhibitors are peptide substrates that compete for active

site binding based on the substrate preference of caspase-3.

Annexin V apoptosis assays

$QQH[LQ�9�DSRSWRVLV�DVVD\V�ZHUH�SHUIRUPHG�XVLQJ�$QQH[LQ�9�),7&�FRQMXJDWHG��/LIH�7HFKQRORJLHV��86$���The stained cells were immediately analyzed on a CyFlow

6SDFH�F\WRPHWHU��3DUWHF�&\)ORZ5�Space Flow cytometer).

Flow cytometric analysis

+XPDQ� SULPDU\� RVWHRFODVWV� WUHDWHG� IRU� ��K� ZLWK�00� FHOO�GHULYHG� H[RVRPHV� ����J�PO��� ZHUH� VWDLQHG�ZLWK�3(�FRQMXJDWHG�PRXVH�DQWL�+XPDQ�&;&5�� �FORQH���*���5'�6\VWHP���7KH�VWDLQHG�FHOOV�ZHUH�LPPHGLDWHO\�DQDO\]HG�RQ�D�&\)ORZ�VSDFH�F\WRPHWHU��3DUWHF�&\)ORZ5�

Space Flow cytometer).

ELISA assay

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Statistical analysis

The statistical analysis was performed using the

,%0�6366�6WDWLVWLFV���� VRIWZDUH��'DWD�DUH� UHSRUWHG�DV�PHDQ� �� VWDQGDUG� GHYLDWLRQ� �6'�� DW� D� VLJQL¿FDQW� OHYHO�RI� S� �� ������$IWHU� KDYLQJ� YHUL¿HG� QRUPDO� GLVWULEXWLRQ��.ROPRJURF�6PLUQRY� WHVW�� DQG� KRPRVFHGDVWLFLW\��/HYHQH�WHVW��RI�GDWD��WKH�LQÀXHQFH�RI�00�FHOO�GHULYHG�exosome on various parameters of murine macrophage

5DZ������FHOOV�ZDV�LQYHVWLJDWHG�E\�D�JHQHUDOL]HG�OLQHDU�PRGHO� �*/0�� IRU� UHSHDWHG�PHDVXUHV�ZLWK� µH[SHULPHQW�UHSOLFDWLRQ¶��Q ���DV�D�ZLWKLQ�VXEMHFW�IDFWRU�DQG�µJURXSV¶�as between-subject factor, followed by adjusted Sidak

multiple comparison test.

ACKNOWLEDGEMENTS

This study was developed with the contribution

RI� �1DWLRQDO�2SHUDWLRQDO� 3URJUDPPH� IRU�5HVHDUFK� DQG�&RPSHWLWLYHQHVV�������������321��B������³6YLOXSSR�GL�una piattaforma tecnologica per il trattamento non invasive

di patologie oncologiche e infettive basate sull’uso di

ultrasuoni focalizzati (FUS)”.

This work was supported by a grant from Italian

$VVRFLDWLRQ�IRU�&DQFHU�5HVHDUFK��$,5&��WR�5�$��DQG�E\�8QLYHUVLW\� RI� 3DOHUPR� �,QWHUQDWLRQDO� &RRSHUDWLRQ�� DQG�))5�WR�5�$��DQG�WR�*�'�/�

This study was supported by Italian Association

IRU�&DQFHU�5HVHDUFK� �$,5&�� WR�3�7�� �µ6SHFLDO�3URJUDP�Molecular Clinical Oncology—5 per mille’ n. 9980,

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CONFLICTS OF INTEREST

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REFERENCES

��� 5DMH�1�DQG�5RRGPDQ�*'��$GYDQFHV� LQ� WKH�ELRORJ\�DQG�treatment of bone disease in multiple myeloma. Clin Cancer

5HV������������������������ +DPHHG�$��%UDG\�--��'RZOLQJ�3��&O\QHV�0�DQG�2¶*RUPDQ�

3��%RQH�GLVHDVH�LQ�PXOWLSOH�P\HORPD��SDWKRSK\VLRORJ\�DQG�PDQDJHPHQW��&DQFHU�*URZWK�0HWDVWDVLV����������������

��� 5RVVL�0��'L�0DUWLQR�07��0RUHOOL�(��/HRWWD�0��5L]]R�$��*ULPDOGL�$��0LVVR�*��7DVVRQH�3�DQG�&DUDJOLD�0��0ROHFXODU�

Oncotarget16www.impactjournals.com/oncotarget

targets for the treatment of multiple myeloma. Curr Cancer

Drug Targets. 2012; 12:757-767.

��� 6HEDJ� 0�� &&5�� EORFNDGH� DQG� P\HORPD� ERQH� GLVHDVH��Blood. 2012; 120:1351-1352.

��� 7HUSRV�(��0RXORSRXORV�/$�DQG�'LPRSRXORV�0$��$GYDQFHV�LQ�LPDJLQJ�DQG�WKH�PDQDJHPHQW�RI�P\HORPD�ERQH�GLVHDVH��-�Clin Oncol. 2011; 29:1907-1915.

��� 'L�0DUWLQR�07��/HRQH�(��$PRGLR�1��)RUHVWD�8��/LRQHWWL�0��3LWDUL�05��&DQWD¿R�0(��*XOOD�$��&RQIRUWL�)��0RUHOOL�(��7RPDLQR�9��5RVVL�0��1HJULQL�0��)HUUDULQL�0��&DUDJOLD�0��6KDPPDV�0$��HW�DO��6\QWKHWLF�PL5���D�PLPLFV�DV�D�QRYHO�therapeutic agent for multiple myeloma: in vitro and in vivo

HYLGHQFH��&OLQ�&DQFHU�5HV������������������������ *KREULDO�,0��5HYLVLWLQJ�WUHDWPHQW�SDUDGLJPV�LQ�KLJK�ULVN�

smoldering multiple myeloma: out with the old, in with the

QHZ"�/HXN�/\PSKRPD������������������������ 5RVVL�0��3LWDUL�05��$PRGLR�1��'L�0DUWLQR�07��&RQIRUWL�

)��/HRQH�(��%RWWD�&��3DROLQR�)0��'HO�*LXGLFH�7��,XOLDQR�(�� &DUDJOLD� 0�� )HUUDULQL� 0�� *LRUGDQR� $�� 7DJOLDIHUUL� 3�DQG� 7DVVRQH� 3�� PL5���E� QHJDWLYHO\� UHJXODWHV� KXPDQ�osteoclastic cell differentiation and function: implications

for the treatment of multiple myeloma-related bone disease.

-�&HOO�3K\VLRO������������������������� +HLGHU�8��)OHLVVQHU�&��=DYUVNL�,��.DLVHU�0��+HFKW�0��-DNRE�

&�DQG�6H]HU�2��%RQH�PDUNHUV�LQ�PXOWLSOH�P\HORPD��(XU�-�Cancer. 2006; 42:1544-1553.

���� 6LOEHUPDQQ�5��%RO]RQL�0��6WRUWL�3��*XDVFR�'��%RQRPLQL�6�� =KRX� '�� :X� -�� $QGHUVRQ� -/�� :LQGOH� --�� $YHUVD�)�� 'DYLG� 5RRGPDQ� *� DQG� *LXOLDQL� 1�� %RQH� PDUURZ�PRQRF\WH��PDFURSKDJH�GHULYHG� DFWLYLQ� $� PHGLDWHV� WKH�RVWHRFODVWRJHQLF� HIIHFW� RI� ,/��� LQ� PXOWLSOH� P\HORPD��/HXNHPLD�������������������

���� $EH�0��+LXUD�.��:LOGH�-��6KLR\DVRQR�$��0RUL\DPD�.��+DVKLPRWR�7��.LGR�6��2VKLPD�7��6KLEDWD�+��2]DNL�6��,QRXH�D and Matsumoto T. Osteoclasts enhance myeloma cell

growth and survival via cell-cell contact: a vicious cycle

between bone destruction and myeloma expansion. Blood.

2004; 104:2484-2491.

���� 7LDQ�(��=KDQ�)��:DONHU�5��5DVPXVVHQ�(��0D�<��%DUORJLH�%�DQG�6KDXJKQHVV\�-'��-U��7KH�UROH�RI�WKH�:QW�VLJQDOLQJ�DQWDJRQLVW�'..��LQ�WKH�GHYHORSPHQW�RI�RVWHRO\WLF�OHVLRQV�LQ�PXOWLSOH�P\HORPD��1�(QJO�-�0HG����������������������

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E\�RVWHRSURWHJHULQ�OLJDQG��3URF�1DWO�$FDG�6FL�8�6�$��������96:3540-3545.

���� 3HDUVH�51��6RUGLOOR�(0��<DFFRE\�6��:RQJ�%5��/LDX�')��&ROPDQ�1��0LFKDHOL� -�� (SVWHLQ� -� DQG�&KRL�<��0XOWLSOH�P\HORPD�GLVUXSWV�WKH�75$1&(��RVWHRSURWHJHULQ�F\WRNLQH�axis to trigger bone destruction and promote tumor

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11586.

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environment. Blood. 2001; 98:3527-3533.

���� .LP�1��2GJUHQ�35��.LP�'.��0DUNV�6&��-U��DQG�&KRL�<��Diverse roles of the tumor necrosis factor family member

75$1&(� LQ� VNHOHWDO� SK\VLRORJ\� UHYHDOHG� E\� 75$1&(�GH¿FLHQF\�DQG�SDUWLDO�UHVFXH�E\�D� O\PSKRF\WH�H[SUHVVHG�75$1&(� WUDQVJHQH�� 3URF�1DWO�$FDG� 6FL�8� 6�$�� ������97:10905-10910.

���� 3HLQDGR�+��/DYRWVKNLQ�6�DQG�/\GHQ�'��7KH�VHFUHWHG�IDFWRUV�responsible for pre-metastatic niche formation: old sayings

and new thoughts. Semin Cancer Biol. 2011; 21:139-146.

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���� 2¶%ULHQ�.��5DQL�6��&RUFRUDQ�&��:DOODFH�5��+XJKHV�/��)ULHO�$0��0F'RQQHOO�6��&URZQ�-��5DGRPVNL�0:�DQG�2¶'ULVFROO�/��([RVRPHV�IURP�WULSOH�QHJDWLYH�EUHDVW�FDQFHU�FHOOV�FDQ�transfer phenotypic traits representing their cells of origin

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multiple myeloma cells. Blood. 2014; 124:555-566.

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proliferation. Oncotarget. 2014; 5:5686-5699.

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Blood Cells Mol Dis. 2004; 32:290-292.

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Oncotarget17www.impactjournals.com/oncotarget

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and uptake of exosomes by ovarian cancer cells. BMC

Cancer. 2011; 11:108.

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autologous transplantation in myeloma: long-term follow-

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anti-tumor activity. Oncotarget. 2014; 5:3039-3054.

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-��1HUL�$�� HW� DO��+\SR[LD�LQGXFLEOH� IDFWRU� �+,)���� DOSKD�suppression in myeloma cells blocks tumoral growth in vivo

LQKLELWLQJ�DQJLRJHQHVLV�DQG�ERQH�GHVWUXFWLRQ��/HXNHPLD��2013; 27:1697-1706.

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Biol Ther. 2013; 13 Suppl 1:S125-137.

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of multiple myeloma and endothelial cells. Cell Cycle.

2013; 12:3650-3662.

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human osteoclasts. Bone. 2005; 36:840-853.

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