Activated T cells recruit exosomes secreted by dendritic cells ...
Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation
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).
Oncotarget3www.impactjournals.com/oncotarget
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
003�� OHYHOV� VHFUHWHG� E\� ERWK� +XPDQ� SULPDU\�2&V�DQG�5DZ������FHOOV�ZHUH�TXDQWL¿HG�UHVSHFWLYHO\�E\�+XPDQ�003���(/,6$�DVVD\V� �,QYLWURJHQ�� DQG�PRXVH�727$/�003�±HQ]\PH�OLQNHG� LPPXQRVRUEHQW� DVVD\V��5'�6\VWHPV��0LQQHDSROLV��01���
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� �$,5&�� WR�3�7�� �µ6SHFLDO�3URJUDP�Molecular Clinical Oncology—5 per mille’ n. 9980,
���������
CONFLICTS OF INTEREST
7KH�DXWKRUV�GHFODUH�QR�FRPSHWLQJ�¿QDQFLDO�LQWHUHVWV�
REFERENCES
��� 5DMH�1�DQG�5RRGPDQ�*'��$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����������������������
���� +VX�+��/DFH\�'/��'XQVWDQ�&5��6RORY\HY�,��&RORPEHUR�$�� 7LPPV� (�� 7DQ� +/�� (OOLRWW� *�� .HOOH\� 0-�� 6DURVL� ,��:DQJ�/��;LD�;=��(OOLRWW�5��&KLX�/��%ODFN�7��6FXOO\�6��HW�DO��7XPRU�QHFURVLV�IDFWRU�UHFHSWRU�IDPLO\�PHPEHU�5$1.�mediates osteoclast differentiation and activation induced
E\�RVWHRSURWHJHULQ�OLJDQG��3URF�1DWO�$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$1&(��RVWHRSURWHJHULQ�F\WRNLQH�axis to trigger bone destruction and promote tumor
SURJUHVVLRQ��3URF�1DWO�$FDG�6FL�8�6�$�����������������
11586.
���� *LXOLDQL�1��%DWDLOOH�5��0DQFLQL�&��/D]]DUHWWL�0�DQG�%DULOOH�6��0\HORPD�FHOOV�LQGXFH�LPEDODQFH�LQ�WKH�RVWHRSURWHJHULQ�osteoprotegerin ligand system in the human bone marrow
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�$�� ������97:10905-10910.
���� 3HLQDGR�+��/DYRWVKNLQ�6�DQG�/\GHQ�'��7KH�VHFUHWHG�IDFWRUV�responsible for pre-metastatic niche formation: old sayings
and new thoughts. Semin Cancer Biol. 2011; 21:139-146.
���� &RUUDGR�&��5DLPRQGR�6��&KLHVL�$��&LFFLD�)��'H�/HR�*�DQG� $OHVVDQGUR� 5�� ([RVRPHV� DV� LQWHUFHOOXODU� VLJQDOLQJ�organelles involved in health and disease: basic science and
FOLQLFDO�DSSOLFDWLRQV��,QW�-�0RO�6FL������������������������� /RJR]]L�0��'H�0LOLWR�$��/XJLQL�/��%RUJKL�0��&DODEUR�/��
6SDGD�0��3HUGLFFKLR�0��0DULQR�0/��)HGHULFL�&��,HVVL�(��%UDPELOOD�'��9HQWXUL�*��/R]XSRQH�)��6DQWLQDPL�0��+XEHU�9��0DLR�0��HW�DO��+LJK�OHYHOV�RI�H[RVRPHV�H[SUHVVLQJ�&'���DQG�FDYHROLQ���LQ�SODVPD�RI�PHODQRPD�SDWLHQWV��3/R6�2QH��2009; 4:e5219.
���� 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
WR�VHFRQGDU\�FHOOV��(XU�-�&DQFHU������������������������� .DKOHUW� &� DQG� .DOOXUL� 5�� ([RVRPHV� LQ� WXPRU�
PLFURHQYLURQPHQW� LQÀXHQFH� FDQFHU� SURJUHVVLRQ� DQG�PHWDVWDVLV��-�0RO�0HG��%HUO��������������������
���� :DQJ� -�� +HQGUL[� $�� +HUQRW� 6�� /HPDLUH�0�� 'H� %UX\QH�(�� 9DQ� 9DOFNHQERUJK� (�� /DKRXWWH� 7�� 'H� :HYHU� 2��9DQGHUNHUNHQ�.�DQG�0HQX�(��%RQH�PDUURZ�VWURPDO�FHOO�derived exosomes as communicators in drug resistance in
multiple myeloma cells. Blood. 2014; 124:555-566.
���� $UHQGW� %.�� :DOWHUV� '.�� :X� ;�� 7VFKXPSHU� 5&� DQG�-HOLQHN�')��0XOWLSOH�P\HORPD�GHOO�GHULYHG�PLFURYHVLFOHV�are enriched in CD147 expression and enhance tumor cell
proliferation. Oncotarget. 2014; 5:5686-5699.
���� 6DYLQD�$��9LGDO�0�DQG�&RORPER�0,��7KH�H[RVRPH�SDWKZD\�LQ� .���� FHOOV� LV� UHJXODWHG� E\� 5DE���� -� &HOO� 6FL�� ������115:2505-2515.
���� 6]\PF]\N�.+��)UHHPDQ�7$��$GDPV�&6��6ULQLYDV�9�DQG�6WHLQEHFN�0-��$FWLYH�FDVSDVH���LV�UHTXLUHG�IRU�RVWHRFODVW�GLIIHUHQWLDWLRQ��-�&HOO�3K\VLRO��������������������
���� <DFFRE\� 6�� :H]HPDQ� 0-�� +HQGHUVRQ� $�� &RWWOHU�)R[�0�� <L� 4�� %DUORJLH� %� DQG� (SVWHLQ� -�� &DQFHU� DQG� WKH�microenvironment: myeloma-osteoclast interactions as a
PRGHO��&DQFHU�5HV������������������������� 5RRGPDQ� *'�� 3DWKRJHQHVLV� RI� P\HORPD� ERQH� GLVHDVH��
Blood Cells Mol Dis. 2004; 32:290-292.
���� 1RWR� 5�� 6DQWDQJHOR� 0*�� 5LFDJQR� 6�� 0DQJLRQH� 05��
Oncotarget17www.impactjournals.com/oncotarget
/HYDQWLQR� 0�� 3H]]XOOR� 0�� 0DUWRUDQD� 9�� &XSDQH� $��Bolognesi M and Manno M. The tempered polymerization
RI�KXPDQ�QHXURVHUSLQ��3/R6�2QH����������H���������� (VFUHYHQWH�&��.HOOHU�6��$OWHYRJW�3�DQG�&RVWD�-��,QWHUDFWLRQ�
and uptake of exosomes by ovarian cancer cells. BMC
Cancer. 2011; 11:108.
���� )HQJ�'��=KDR�:/��<H�<<��%DL�;&��/LX�54��&KDQJ�/)��=KRX�4�DQG�6XL�6)��&HOOXODU�LQWHUQDOL]DWLRQ�RI�H[RVRPHV�RFFXUV�WKURXJK�SKDJRF\WRVLV��7UDI¿F�������������������
���� /LX�<�� =KX�;-�� =HQJ�&��:X� 3+��:DQJ�+;��&KHQ� =&�DQG�/L�4%��0LFURYHVLFOHV�VHFUHWHG�IURP�KXPDQ�PXOWLSOH�P\HORPD�FHOOV�SURPRWH�DQJLRJHQHVLV��$FWD�3KDUPDFRO�6LQ��2014; 35:230-238.
���� &ROOLQ�2VGRE\�3��5RWKH�/��$QGHUVRQ�)��1HOVRQ�0��0DORQH\�:�DQG�2VGRE\�3��5HFHSWRU�DFWLYDWRU�RI�1)�NDSSD�%�DQG�osteoprotegerin expression by human microvascular
HQGRWKHOLDO� FHOOV�� UHJXODWLRQ� E\� LQÀDPPDWRU\� F\WRNLQHV��DQG�UROH�LQ�KXPDQ�RVWHRFODVWRJHQHVLV��-�%LRO�&KHP��������276:20659-20672.
���� %MRUNVWUDQG�%��,DFREHOOL�6��+HJHQEDUW�8��*UXEHU�$��*UHLQL[�+��9ROLQ�/��1DUQL�)��0XVWR�3��%HNVDF�0��%RVL�$��0LORQH�*�� &RUUDGLQL� 3�� *ROGVFKPLGW� +�� GH�:LWWH� 7��0RUULV� &��1LHGHUZLHVHU�'��HW�DO��7DQGHP�DXWRORJRXV�UHGXFHG�LQWHQVLW\�conditioning allogeneic stem-cell transplantation versus
autologous transplantation in myeloma: long-term follow-
XS��-�&OLQ�2QFRO������������������������� 0LWVLDGHV� &6��0F0LOOLQ� ':�� .OLSSHO� 6�� +LGHVKLPD� 7��
&KDXKDQ�'��5LFKDUGVRQ� 3*��0XQVKL�1&� DQG�$QGHUVRQ�.&��7KH�UROH�RI�WKH�ERQH�PDUURZ�PLFURHQYLURQPHQW�LQ�WKH�SDWKRSK\VLRORJ\� RI�P\HORPD� DQG� LWV� VLJQL¿FDQFH� LQ� WKH�GHYHORSPHQW�RI�PRUH�HIIHFWLYH�WKHUDSLHV��+HPDWRO�2QFRO�&OLQ�1RUWK�$P����������������������YLL�YLLL�
���� %DVDN� *:�� 6ULYDVWDYD� $6�� 0DOKRWUD� 5� DQG� &DUULHU� (��0XOWLSOH� P\HORPD� ERQH� PDUURZ� QLFKH�� &XUU� 3KDUP�Biotechnol. 2009; 10:345-346.
���� 5RFFDUR�$0��6DFFR�$��0DLVR�3��$]DE�$.��7DL�<7��5HDJDQ�0��$]DE�)��)ORUHV�/0��&DPSLJRWWR�)��:HOOHU�(��$QGHUVRQ�.&�� 6FDGGHQ� '7� DQG� *KREULDO� ,0�� %0� PHVHQFK\PDO�stromal cell-derived exosomes facilitate multiple myeloma
SURJUHVVLRQ��-�&OLQ�,QYHVW�������������������������� 8PH]X�7��7DGRNRUR�+��$]XPD�.��<RVKL]DZD�6��2K\DVKLNL�
.� DQG� 2K\DVKLNL� -+�� ([RVRPDO� PL5����E� VKHG� IURP�hypoxic multiple myeloma cells enhances angiogenesis by
WDUJHWLQJ�IDFWRU�LQKLELWLQJ�+,)����%ORRG�����������������3757.
���� 5DLPRQGL� /�� $PRGLR� 1�� 'L� 0DUWLQR�07�� $OWRPDUH� (��/HRWWD� 0�� &DUDFFLROR� '�� *XOOD� $�� 1HUL� $�� 7DYHUQD� 6��'¶$TXLOD� 3�� $OHVVDQGUR� 5�� *LRUGDQR� $�� 7DJOLDIHUUL� 3�DQG� 7DVVRQH� 3�� 7DUJHWLQJ� RI� PXOWLSOH� P\HORPD�UHODWHG�DQJLRJHQHVLV�E\�PL5����D��S�PLPLFV��in vitro and in vivo
anti-tumor activity. Oncotarget. 2014; 5:3039-3054.
���� 6WRUWL� 3�� %RO]RQL�0�� 'RQRIULR� *�� $LUROGL� ,�� *XDVFR� '��7RVFDQL�'��0DUWHOOD�(��/D]]DUHWWL�0��0DQFLQL�&��$JQHOOL�/��3DWUHQH�.��0DLJD�6��)UDQFHVFKL�9��&ROOD�6��$QGHUVRQ�
-��1HUL�$�� 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.
���� $PRGLR� 1�� 'L�0DUWLQR�07�� 1HUL� $�� 7DJOLDIHUUL� 3� DQG�7DVVRQH�3��1RQ�FRGLQJ�51$��D�QRYHO�RSSRUWXQLW\�IRU�WKH�personalized treatment of multiple myeloma. Expert Opin
Biol Ther. 2013; 13 Suppl 1:S125-137.
���� /HRWWD� 0�� %LDPRQWH� /�� 5DLPRQGL� /�� 5RQFKHWWL� '�� 'L�0DUWLQR� 07�� %RWWD� &�� /HRQH� (�� 3LWDUL� 05�� 1HUL� $��*LRUGDQR�$��7DJOLDIHUUL�3��7DVVRQH�3�DQG�$PRGLR�1��$�S���'HSHQGHQW�7XPRU�6XSSUHVVRU�1HWZRUN�,V�,QGXFHG�E\�6HOHFWLYH� PL5����D��S� ,QKLELWLRQ� LQ�0XOWLSOH�0\HORPD�&HOOV�� -RXUQDO� RI� &HOOXODU� 3K\VLRORJ\�� ������ ���������2116.
���� $PRGLR�1��/HRWWD�0��%HOOL]]L�'��'L�0DUWLQR�07��'¶$TXLOD�3��/LRQHWWL�0��)DELDQL�)��/HRQH�(��*XOOD�$0��3DVVDULQR�*��&DUDJOLD�0��1HJULQL�0��1HUL�$��*LRUGDQR�$��7DJOLDIHUUL�3� DQG� 7DVVRQH� 3�� '1$�GHPHWK\ODWLQJ� DQG� DQWL�WXPRU�DFWLYLW\�RI�V\QWKHWLF�PL5���E�PLPLFV�LQ�PXOWLSOH�P\HORPD��Oncotarget. 2012; 3:1246-1258.
���� $PRGLR�1��%HOOL]]L�'��/HRWWD�0��5DLPRQGL�/��%LDPRQWH�/��'¶$TXLOD�3��'L�0DUWLQR�07��&DOLPHUL�7��5RVVL�0��/LRQHWWL�0��/HRQH�(��3DVVDULQR�*��1HUL�$��*LRUGDQR�$��7DJOLDIHUUL�3�DQG�7DVVRQH�3��PL5���E�LQGXFHV�62&6���H[SUHVVLRQ�E\�promoter demethylation and negatively regulates migration
of multiple myeloma and endothelial cells. Cell Cycle.
2013; 12:3650-3662.
���� 7DJOLDIHUUL�3��5RVVL�0��'L�0DUWLQR�07��$PRGLR�1��/HRQH�(��*XOOD�$��1HUL�$�DQG�7DVVRQH�3��3URPLVHV�DQG�FKDOOHQJHV�RI�0LFUR51$�EDVHG�WUHDWPHQW�RI�PXOWLSOH�P\HORPD��&XUU�Cancer Drug Targets. 2012; 12:838-846.
���� <DFFRE\�6��3HDUVH�51��-RKQVRQ�&/��%DUORJLH�%��&KRL�<�DQG�(SVWHLQ�-��0\HORPD�LQWHUDFWV�ZLWK� WKH�ERQH�PDUURZ�microenvironment to induce osteoclastogenesis and is
GHSHQGHQW� RQ� RVWHRFODVW� DFWLYLW\�� %U� -� +DHPDWRO�� ������116:278-290.
���� :ULJKW�/0��0DORQH\�:��<X�;��.LQGOH�/��&ROOLQ�2VGRE\�3�DQG�2VGRE\�3��6WURPDO�FHOO�GHULYHG�IDFWRU���ELQGLQJ�WR�LWV�FKHPRNLQH�UHFHSWRU�&;&5��RQ�SUHFXUVRU�FHOOV�SURPRWHV�the chemotactic recruitment, development and survival of
human osteoclasts. Bone. 2005; 36:840-853.
���� GH�0HO�6��/LP�6+��7XQJ�0/�DQG�&KQJ�:-��,PSOLFDWLRQV�RI�KHWHURJHQHLW\�LQ�PXOWLSOH�P\HORPD��%LRPHG�5HV�,QW��������2014:232546.
���� $LPHV�57�DQG�4XLJOH\�-3��0DWUL[�PHWDOORSURWHLQDVH���LV�an interstitial collagenase. Inhibitor-free enzyme catalyzes
WKH� FOHDYDJH� RI� FROODJHQ� ¿EULOV� DQG� VROXEOH� QDWLYH� W\SH�,� FROODJHQ� JHQHUDWLQJ� WKH� VSHFL¿F� ����� DQG� ����OHQJWK�IUDJPHQWV��-�%LRO�&KHP����������������������
���� .XVDQR� .�� 0L\DXUD� &�� ,QDGD� 0�� 7DPXUD� 7�� ,WR� $��1DJDVH� +�� .DPRL� .� DQG� 6XGD� 7�� 5HJXODWLRQ� RI�PDWUL[� PHWDOORSURWHLQDVHV� �003���� ���� ���� DQG� �����by interleukin-1 and interleukin-6 in mouse calvaria:
DVVRFLDWLRQ� RI� 003� LQGXFWLRQ� ZLWK� ERQH� UHVRUSWLRQ��
Oncotarget18www.impactjournals.com/oncotarget
Endocrinology. 1998; 139:1338-1345.
���� &RVWD�$*��&XVDQR�1(��6LOYD�%&��&UHPHUV�6�DQG�%LOH]LNLDQ�-3��&DWKHSVLQ�.��LWV�VNHOHWDO�DFWLRQV�DQG�UROH�DV�D�WKHUDSHXWLF�WDUJHW�LQ�RVWHRSRURVLV��1DW�5HY�5KHXPDWRO��������������456.
���� *LXOLDQL�1��5L]]ROL�9�DQG�5RRGPDQ�*'��0XOWLSOH�P\HORPD�ERQH� GLVHDVH�� 3DWKRSK\VLRORJ\� RI� RVWHREODVW� LQKLELWLRQ��Blood. 2006; 108:3992-3996.
���� 5D¿HL�6�DQG�.RPDURYD�69��0ROHFXODU�VLJQDOLQJ�SDWKZD\V�mediating osteoclastogenesis induced by prostate cancer
cells. BMC Cancer. 2013; 13:605.
���� 7HLWHOEDXP�6/��7KH�RVWHRFODVW�DQG�LWV�XQLTXH�F\WRVNHOHWRQ��$QQ�1�<�$FDG�6FL�������������������
���� &KDPEHUV�7-�DQG�)XOOHU�.��+RZ�DUH�RVWHRFODVWV�LQGXFHG�WR�UHVRUE�ERQH"�$QQ�1�<�$FDG�6FL�����������������
���� 7HLWHOEDXP�6/��%RQH�UHVRUSWLRQ�E\�RVWHRFODVWV��6FLHQFH��2000; 289(5484):1504-1508.
���� %KDUWL�$&�DQG�$JJDUZDO�%%��5DQNLQJ�WKH�UROH�RI�5$1.�ligand in apoptosis. Apoptosis. 2004; 9:677-690.
���� ;LQJ�/��6FKZDU]�(0�DQG�%R\FH�%)��2VWHRFODVW�SUHFXUVRUV��5$1./�5$1.��DQG�LPPXQRORJ\��,PPXQRO�5HY��������208:19-29.
���� +DUVKPDQ� 6:�� &DQHOOD� $�� &LDUODULHOOR� 3'�� 5RFFL�$�� $JDUZDO� .�� 6PLWK� (0�� 7DODEHUH� 7�� (IHEHUD� <$��+RIPHLVWHU� &&�� %HQVRQ� '0�� -U��� 3DXODLWLV� 0(�� )UHLWDV�0$�DQG�3LFKLRUUL�)��&KDUDFWHUL]DWLRQ�RI�PXOWLSOH�P\HORPD�YHVLFOHV� E\� ODEHO�IUHH� UHODWLYH� TXDQWLWDWLRQ�� 3URWHRPLFV��2013; 13:3013-3029.
���� &DL�'��&DR�-��/L�=��=KHQJ�;��<DR�<��/L�:�DQG�<XDQ�=��Up-regulation of bone marrow stromal protein 2 (BST2)
in breast cancer with bone metastasis. BMC Cancer. 2009;
9:102.
���� 7DYHUQD� 6�� )OXJ\� $�� 6DLHYD� /�� .RKQ� (&�� 6DQWRUR� $��0HUDYLJOLD� 6�� 'H� /HR� *� DQG� $OHVVDQGUR� 5�� 5ROH� RI�exosomes released by chronic myelogenous leukemia cells
LQ�DQJLRJHQHVLV��,QW�-�&DQFHU�������������������������� 7KHU\�&��$PLJRUHQD�6��5DSRVR�*�DQG�&OD\WRQ�$��,VRODWLRQ�
and characterization of exosomes from cell culture
VXSHUQDWDQWV�DQG�ELRORJLFDO�ÀXLGV��&XUU�3URWRF�&HOO�%LRO��2006; Chapter 3:Unit 3 22.
���� &RUUDGR�&��5DLPRQGR�6��6DLHYD�/��)OXJ\�$0��'H�/HR�*�DQG�$OHVVDQGUR�5��([RVRPH�PHGLDWHG�FURVVWDON�EHWZHHQ�chronic myelogenous leukemia cells and human bone
marrow stromal cells triggers an interleukin 8-dependent
VXUYLYDO�RI�OHXNHPLD�FHOOV��&DQFHU�/HWW���������������������� 7DYHUQD�6��$PRGHR�9��6DLHYD�/��5XVVR�$��*LDOORPEDUGR�
0��'H�/HR�*�DQG�$OHVVDQGUR�5��([RVRPDO� VKXWWOLQJ�RI�PL5����� LQ� HQGRWKHOLDO� FHOOV� PRGXODWHV� DGKHVLYH� DQG�migratory abilities of chronic myelogenous leukemia cells.
Mol Cancer. 2014; 13:169.