Bone Pain Induced by Multiple Myeloma Is Reduced by ...Microenvironment and Immunology Bone Pain Induced by Multiple Myeloma Is Reduced by Targeting V-ATPase and ASIC3 Masahiro Hiasa1,Tatsuo

Microenvironment and Immunology

Bone Pain Induced by Multiple Myeloma IsReduced by Targeting V-ATPase and ASIC3Masahiro Hiasa1, Tatsuo Okui1, Yohance M. Allette2, Matthew S. Ripsch2,Ge-Hong Sun-Wada3, Hiroki Wakabayashi4, G. David Roodman1,5,Fletcher A.White2, and Toshiyuki Yoneda1

Abstract

Multiple myeloma patients experience severe bone pain(MMBP) that is undertreated and poorly understood. In thisstudy, we studied MMBP in an intratibial mouse xenograftmodel that employs JJN3 human multiple myeloma cells. Inthis model, mice develop MMBP associated in bone withincreased sprouting of calcitonin gene-related peptide-positive(CGRPþ) sensory nerves and in dorsal root ganglia (DRG)with upregulation of phosphorylated ERK1/2 (pERK1/2)and pCREB, two molecular indicators of neuron excitation.We found that JJN3 cells expressed a vacuolar proton pump(V-ATPase) that induced an acidic bone microenvironment.Inhibition of JJN3-colonized bone acidification by a singleinjection of the selective V-ATPase inhibitor, bafilomycin A1,decreased MMBP, CGRPþ sensory neuron sprouting, andpERK1/2 and pCREB expression in DRG. CGRPþ sensorynerves also expressed increased levels of the acid-sensingnociceptor ASIC3. Notably, a single injection of the selective

ASIC3 antagonist APETx2 dramatically reduced MMBP in themodel. Mechanistic investigations in primary DRG neuronscocultured with JJN3 cells showed increased neurite outgrowthand excitation inhibited by bafilomycin A1 or APETx2. Fur-thermore, combining APETx2 with bafilomycin A1 reducedMMBP to a greater extent than either agent alone. Finally,combining bafilomycin A1 with the osteoclast inhibitorzoledronic acid was sufficient to ameliorate MMBP, whichwas refractory to zoledronic acid. Overall, our resultsshow that osteoclasts and multiple myeloma cooperateto induce an acidic bone microenvironment that evokesMMBP as a result of the excitation of ASIC3-activatedsensory neurons. Furthermore, they present a mechanisticrationale for targeting ASIC3 on neurons along with themultiple myeloma-induced acidic bone microenvironmentas a strategy to relieve MMBP in patients. Cancer Res; 77(6);1283–95. �2017 AACR.

IntroductionMultiplemyeloma is amalignant plasma cell disorder account-

ing for approximately 10%of all hematologic cancers (1). Seventypercent of multiple myeloma patients present with severe bonepain associated with osteolytic bone lesions that result fromincreased osteoclastic bone resorption and reduced osteoblasticbone formation (2). Bone pain contributes to increasedmortalityandmorbidity (3). The degree of bone pain inmultiple myelomais often greater than pain experienced with solid tumors (4–6),and bone pain is the most common presenting complaint ofmultiple myeloma patients (7). Control of bone pain is thus amajor goal in the management of multiple myeloma patients.

Despite this, the pathophysiology of bone pain associated withmultiple myeloma (MMBP) is poorly understood, and MMBP isfrequently inadequately treated (3).

Bone is densely innervated by sensory neurons (SN), especiallythe calcitonin gene-related peptide-positive (CGRPþ) SN (8–12),which show pathologic sprouting in the presence of tumors (13,14). Osteoclasts are also increased in cancer-colonized bone andactively secrete protons (Hþ) to destroy mineralized bone via theplasmamembrane a3 isoform vacuolar-Hþ-ATPase (a3V-ATPase)localized in the ruffled borders (15), creating an acidic microen-vironment (pH range¼ 4.0–6.0; refs. 16, 17). Hþ is a well-knownpotent pain-inducingmediator (18, 19), suggesting that the acidicmicroenvironment created by increased Hþ release from bone-resorbing osteoclasts via the a3V-ATPase activates pH-sensitiveSNs to elicit bone pain. Consistent with this notion, specificinhibitors of osteoclastic bone resorption, including bisphospho-nates and denosumab, significantly reduce bone pain in patientswith multiple myeloma (2, 20) and bone metastases of solidcancers (21–23), indicating that osteoclast bone resorption con-tributes to the activation of nociceptive SNs in the bone to inducebone pain.

However, bisphosphonate or denosumab treatment does notprevent the progression of bone pain in bone metastasis atadvanced stages in patients with solid tumors (22, 23), suggestingthat tumor cells also contribute to bone pain. Solid tumor cellsrelease increased the levels of Hþ via various types of plasmamembrane pH regulators, thereby inducing an acidic extracellularmicroenvironment (pH range¼6.5–7.0; refs. 24, 25) that can also

1Division of Hematology and Oncology, Indiana University School of Medicine,Indianapolis, Indiana. 2Department of Anesthesia, Paul and Carole Stark Neu-rosciences Research Institute, Indiana University School of Medicine, Indiana-polis, Indiana. 3Department of Biochemistry, Faculty of PharmaceuticalSciences, Doshisha Women's College, Kyoto, Japan. 4Department of Orthopae-dic Surgery, Mie University Graduate School of Medicine, Mie, Japan. 5TheRoudebusch VA, Indianapolis, Indiana.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Toshiyuki Yoneda, Indiana University School of Med-icine, Walther Hall, R3-C321D, 980 WWalnut St, Indianapolis, IN 46202. Phone:317-274-3530; Fax: 317-274-0396; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-3545

�2017 American Association for Cancer Research.

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excite pH-sensitive nociceptive SNs innervating bone to evokebone pain. Similarly, it has been clinically well recognized thatMMBP in patients at advanced stages is unresponsive to bispho-sphonates or denosumab. Thus,multiplemyeloma cells may alsocontribute to induce MMBP through the creation of acidic extra-cellular microenvironment via plasma membrane pH regulatorsas do solid tumors.

A subpopulation of peripheral nerve fibers, termed nociceptors(18), recognizes and transduces local nociceptive stimuli intoelectrochemical signals. The nociceptors in turn transmit thesenoxious signals to the central nervous system (CNS) and brain viathe dorsal root ganglion (DRG), which is the cell body of primaryafferent SN fibers innervating peripheral tissues and serves as thegateway for peripheral noxious signals to the CNS (26). Thespecific acid-sensing nociceptors related to acid-induced bonepain are the transient receptor potential channel-vanilloid sub-family member 1 (TRPV1) and the acid-sensing ion channel 3(ASIC3; ref. 18). TRPV1 is activated by pH values <6.0 (27),equivalent to the pH under the osteoclast ruffled border (15–17). Previous studies reported that TRPV1 plays a critical role incancer-associated bone pain (28, 29). In contrast, little is knownabout the contributions of ASIC3 tobone pain, despite that ASIC3senses milder pH between 6.5 and 7.0 (30), equivalent to the pHof the bone microenvironment of cancer-involved bone (24, 25).

In this study, we show that Hþ released via V-ATPase bybone-resorbing osteoclasts and JJN3 multiple myeloma cellscreate an acidic bone microenvironment that excites SNs inner-vating bone via activation of ASIC3 to induce MMBP. Further-more, blocking the generation of the acidic bone microenvi-ronment and the activation of ASIC3 on SNs significantlyreduced MMBP and could be a mechanism-based therapeuticapproach for MMBP.

Materials and MethodsReagents

The sources for the reagents were as follows: APETx2 andSB366791 (Tocris); zoledronic acidmonohydrate (Sigma); rabbitmAbs to ERK1/2,CREB, pERK1/2, pCREB, horseradish peroxidase(HRP)-conjugated horse anti-mouse IgG and goat anti-rabbit IgGantibody (Cell Signaling Technology); bafilomycin A1 (Baf A1),rabbit polyclonal antibodies toASIC3, TRPV1, the goat polyclonalantibody against CGRP, andHRP-conjugatedmousemAb againstb-actin (Abcam); Alexa Fluor 488-conjugated donkey anti-rabbitIgG and donkey anti-chicken IgY, and rhodamine red-X (RRX)-conjugated donkey anti-goat IgG antibody (Jackson); rhodaminephalloidin (Life Technologies); and rabbit polyclonal antibodiesto RANKL, IL6, mousemAb toMIP-1a, and HRP-conjugated goatanti-chicken antibody (Santa Cruz Biotechnology).

Cell cultureThe JJN3 human multiple myeloma cell line was provided by

Dr. Nicola Giuliani (Department of Clinical and ExperimentalMedicine, University of Parma, Parma, Italy; ref. 31). JJN3, H929,and U266 multiple myeloma cell lines and primary CD138þ

multiple myeloma cells and CD138� cells (isolated from bonemarrow aspirates of multiple myeloma patients using MACSMicroBead) were cultured in RPMI1640 medium with 5% FBS,100 U/mL penicillin, and 100 mg/mL streptomycin. The mouseosteocytic MLO-A5 and MLO-Y4 (Dr. T. Bellido, Department ofAnatomy and Cell Biology, School of Medicine, Indiana Univer-sity, Indianapolis, IN), mouse osteoblastic MC3T3-E1 and F11

SN, human breast cancer MCF-7, and MDA-MB-231 cells werecultured in a-MEM (Gibco) with 10% FBS, 100 U/mL penicillin,and 100 mg/mL streptomycin. Primary rat DRG SN (1� N) cells(Lonza) were cultured in primary neuron growthmedium (Lonza)with 2% FBS. All cell lines were analyzed and authenticated bytargeted genomic and RNA sequencing. All procedures involvingpatients were performed with written informed consent accordingto the Declaration of Helsinki and under a protocol approved bythe Indiana University (IU) Institutional Review Board.

Animal model of multiple myelomaAll animal studies were approved by the Institutional Animal

Care andUse Committee at IU School of Medicine (Indianapolis,IN). JJN3 cells (5� 105) or PBS (sham)were injected into the righttibia marrow of female C57BL/6 SCID mice (4–6 weeks old)under anesthesia (32).

Behavioral pain testsMechanical allodynia and thermal hyperalgesia were evaluated

by von Frey and plantar tests using the Dynamic Plantar Aesthe-siometer and Plantar Test Instrument (Ugo Basile), respectively(33, 34). These tests are widely used for bone pain assessment inrodents (35). Tests were performed prior to multiple myelomacell injection to determine baseline behaviors and every 5 daysfollowing cell injection. Throughout the experiment, behavioraltests were performed under blind conditions by a single research-er. JJN3 cell injections were performed by a researcher blinded tothe experiment. The investigator performing the behavioral testswas blinded as to the experimental condition of the animals.

Acridine orange accumulationAcridine orange (Molecular Probes), which selectively depo-

sits and fluoresces in acidic environments (36), was injected(1.0 mg/kg) into mice via the tail vein 2 hours prior to sacrifice.Tibiae were excised and acridine orange fluorescence detectedusing an EVOS fluorescence microscope (Advanced Microscopy).

Tartrate-resistant acid phosphatase stainingBone sections were stained for tartrate-resistant acid phospha-

tase (TRAP) activity using a TRACP and ALP Double-Stain Kit(Takara Bio; ref. 32) and analyzed using Magnafire 4.1 software(Optronics) with the IX70 microscope (Olympus).

C-terminal telopeptides of type I collagen measurementSera were collected from mice at the indicated time, and C-

terminal telopeptide of type I collagen (CTx) was measured byRatLaps (CTX-I) EIA Kit (Immunodiagnostic Systems) accordingto the manufacturer's instructions.

Extracellular pH measurementCells (105/96-well)were cultured for 48hours in thepresenceof

adriamycin (30 nmol/L) to prevent cell proliferation and extra-cellular pH (pHe) was measured by FiveEasy pH meter (MettlerToledo) immediately after removal from the CO2 incubator.

Knockdown of a3V-ATPase in JJN3 cellsJJN3 cells were infected with 20 mL of control (sc-108080) or

TCIRG1 (sc-96928-V) shRNA lentiviral particles (Santa CruzBiotechnology) in the presence of 5 mg/mL polybrene. One daylater, cells were cultured in RPMI1640 plus 5% FBS for 7 days inthe presence of 2 mg/mL puromycin (Gibco) to select cells stablyexpressing the shRNAs.

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Neurite outgrowth determination1� N cells (1� 103/6-well) were cocultured with JJN3 cells (5�

103) or alone for 120 hours. Neurite outgrowth was visualized bystaining the cultures with calcein AM (Life Technologies), and thelength of neurites was quantified with a fluorescent microscopeusing Neuron J (37).

Imaging of intracellular Ca2þ influx1� N or F11 SN cells were seeded in FluoroDish (World

Precision) and loaded with fura-2 AM (3 mmol/L, MolecularProbes) for 25 minutes (38). Intracellular calcium was measuredby digital video microfluorimetry, using an intensified CCDcamera coupled to a microscope and MetaFluor software (Molec-ular Devices). Cells were illuminated, and the excitation wave-lengths for fura-2 (340/380 nm) were selected by a filter changer.Inhibitors were added directly into the bathing solution 2 hoursbefore stimulation with 0.1 N hydrochloric acid (HCl).

ImmunoblottingCells or tissues were lysed in lysis buffer (Invitrogen), electro-

phoresed on a 10% SDS-PAGE, and blotted onto PVDF mem-branes (Bio-Rad; ref. 32). After blocking, the membranes wereincubated with primary antibodies overnight at 4�C, and thenwith HRP-conjugated secondary antibodies for 1 hour. Proteinbands were visualized with a SuperSignal West Pico Chemilumi-nescent Substrate (Thermo Fisher Scientific).

IHCBones were fixed, embedded inOCT compound, and sectioned

at 30-mm thickness using a cryostat. After blocking, sections wereincubated with the primary antibodies overnight at 4�C andsecondary antibodies for 60 minutes, mounted with coverslipsin VECTASHIELD anti-fade mounting medium (Vector), andobserved under the Leica TCS SP8 confocal laser scanningmicroscope.

For TRAP andCGRPdouble-staining, sectionswere first stainedwith TRAP and incubated with anti-CGRP antibody (1/200)overnight at 4�C, and then with HRP-conjugated secondary anti-bodies and diaminobenzidine substrate (Vector).

ImmunocytochemistryJJN3 cells (1 � 103/96-well) were fixed with 10% neutral-

buffered formalin, incubatedwith3%BSA-PBSblocking solution,and then with a3V-ATPase antibody (1/200; ref. 39) at 4�Covernight, followed by Alexa Fluor 594 anti-chicken secondaryantibody (1/1,000) and Alexa Fluor 488 phalloidin (1/2,000).Nuclei were counterstained with DAPI.

Statistical analysisData are presented as the mean � SEM. Statistical differences

were determined by using one-way, two-way, two-way repeatedmeasures ANOVA followed by Bonferroni or Dunnet post hoc test,as indicated. All statistical analyses were performed by usingStatcell (OMS). P < 0.05 was considered statistically significant.

ResultsAnimal model of MMBP

Mice intratibially injected with JJN3 cells developed charac-teristic osteolytic lesions on X-ray (Fig. 1A) and mCT (Fig. 1B).These lesions showed aggressive proliferation of JJN3 cells inthe bone marrow (Fig. 1C) with increased TRAPþ osteoclast

bone resorption (Fig. 1D and E) and elevated serum CTx (Fig.1F). We found that JJN3 cells produced osteoclastogenic cyto-kines, including RANKL, IL6, and MIP-1a (Fig. 1G). Interest-ingly, MDA-MB-231 human breast cancer cells (25), used as arepresentative aggressive solid cancer cells that develop osteo-lytic bone lesions to compare with multiple myeloma cells,showed undetectable production of MIP-1a. PBS-injected shammice exhibited no osteolytic lesions.

The right leg of mice harboring JJN3 cells initially displayedmechanical allodynia (Fig. 2A) and thermal hyperalgesia (Fig.2B) at day 20 of postinjection, and these pain behaviorsprogressed in parallel with bone destruction. Sham miceshowed no evidence of MMBP (Fig. 2A and B). Left legs ofJJN3-injected mice had no evidence of MMBP, demonstratingthat MMBP is associated with local JJN3 colonization ratherthan a systemic effect of JJN3 cells. Sprouting of CGRPþ SNs inJJN3-injected bone was increased in parallel with the progres-sion of MMBP (Fig. 2C and D).

5TGM1 multiple myeloma cells intratibially injected intoimmunocompetent C57BL/KaLwRij mice also developed osteo-lytic lesions and induced progressive MMBP (Supplementary Fig.S1A and S1B).

Acidification and MMBP are associated with JJN3-colonizedbone

We next determined whether the JJN3-colonized bone is acidicusing the pH sensor fluorescent dye, acridine orange. No fluores-cence was detected in the right tibiae of shammice (Fig. 2E, left).In contrast, the entire marrow of right tibiae injected with JJN3cells was fluorescent (Fig. 2E, middle), demonstrating that JJN3-colonized bone is acidic. The bone marrow injected with 5TGM1cells was also acidic (Supplementary Fig. S1D, right). We thenassessedwhether the selective V-ATPase inhibitor, Baf A1, blockedestablished MMBP in JJN3-injected mice. Baf A1 inhibited acid-ification by osteoclast bone resorption (40), and we reportedreduced bone pain in breast cancer bonemetastases (41). Repeat-ed injections of Baf A1 inhibited tumor growth (42) and boneresorption (40) that alter the status of bone pain. To examinewhether Baf A1 directly inhibits SN activation and reduces MMBPindependent of inhibition of tumor growth and bone resorption,JJN3-injectedmicemanifestingMMBP and shammice were givena single intraperitoneal injection of Baf A1 at day 25 (arrow in Fig.2A and B) and evaluated for changes in MMBP as a function oftime. Baf A1 treatment blocked acidification in JJN3-colonizedbone (Fig. 2E, right). Importantly, Baf A1 blockade of the acid-ification of JJN3-colonized bone inhibited the progression ofmechanical allodynia (Fig. 2F) and thermal hyperalgesia (Fig.2G) as early as 12 hours after injection. The effects lasted up to 24hours and were lost by 48 hours after injection.

In parallel with noxious behaviors, JJN3-injected mice dem-onstrated increased expression of pERK1/2 and pCREB, which aremolecular indicators for neuronal excitation (43), in their DRGSNs compared with sham mice (Fig. 2H). Similarly, 5TGM1-injected mice also exhibited elevated expression of pERK andpCREB in their DRG SNs (Supplementary Fig. S1C). Importantly,the elevated levels of pERK1/2 and pCREB in DRG SNs in JJN3-injected mice were decreased at 12 hours after Baf A1 injection(Fig. 2H), indicating that SN excitation, as well as noxiousbehaviors, is inhibited by blocking the acidification of JJN3-colonized bone. These results suggest that Hþ protons releasedvia V-ATPase directly excite SNs inbone to evokeMMBP. They also

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suggest that Baf A1 amelioratesMMBPby inhibiting SN excitationin a time period too short to be due to inhibition of JJN3 tumorgrowth and bone resorption.

Expression of V-ATPase and pHe in JJN3 cellsWenext examinedwhether JJN3 cells contribute to acidification

of the bone microenvironment by releasing Hþ via V-ATPase.Humanmultiple myeloma cell lines, JJN3, H929, and U266, andMDA-MB-231 human breast cancer cell line (serve as a positivecontrol) expressed a3V-ATPase (Fig. 3A). Importantly, CD138þ,but not CD138�, cells derived from three multiple myelomapatients expressed a3V-ATPase (Fig. 3B). JJN3 expressed thea3V-ATPase on their plasma membrane (Fig. 3C), as did highlyinvasive MDA-MB-231 cells (44). The pHe of JJN3, H929, andU266 and MDA-MB-231 and CD138þ cells was acidic (Fig. 3D).The human multiple myeloma cells MM.1S expressed undetect-

able a3V-ATPase (Fig. 3A), and the pHe of MM.1S was not acidic(Fig. 3D). The expression of a3V-ATPase in MC3T3-E1 osteoblas-tic and MLO-A5 and MLO-Y4 osteocytic cells was undetectable(Fig. 3A), and their pHewas not acidic (Fig. 3D). Thus, osteoblastsand osteocytes unlikely contribute to extracellular acidification ofmultiple myeloma–colonized bone. Baf A1 significantly blockedextracellular acidification in JJN3 cultures (Fig. 3E) with noinhibition of cell proliferation (Fig. 3F). In contrast, Baf A1showed no effects on pHe in other cells. Furthermore, knockdownof a3V-ATPase in JJN3 cells significantly blocked extracellularacidification (Fig. 3G).

Thus, the a3V-ATPase contributes to the acidification of JJN3cell extracellular microenvironment.

5TGM1 cells also expressed plasma membrane a3V-ATPase,and the pHe of 5TGM1 cells was acidic, which was neutralized byBaf A1 (Supplementary Fig. S1E–S1G).

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Development of osteolytic lesions in JJN3-injected mice. A, Radiographs of osteolyticlesions (arrowheads). B, Three-dimensionalmCT images. C, JJN3 cells in bone marrowwith osteoclast bone resorption(arrowheads, hematoxylin and eosin,�400). D, TRAP staining of bone (�40).E, Number of TRAPþ osteoclasts in bone.�� , P < 0.01 versus sham mice (one-wayANOVA). F, Serum CTx levels in mice.� , P < 0.05 versus sham mice (two-wayANOVA).G,Production of osteoclastogeniccytokines in JJN3 and MDA-MB-231 cells.

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Effects of acidic microenvironment of JJN3-colonizedbone on SNs

Tumors in the bone induce SN sprouting in the bone,thereby enhancing bone pain (13, 14). We therefore deter-mined the extent of CGRPþ SN sprouting with or withouttreatment with Baf A1 in JJN3-colonized bone. Baf A1 inhib-ited the progression of mechanical allodynia in mice injectedintratibially with JJN3 cells (Fig. 4A). IHC showed increasedCGRPþ SN sprouting within the CD138þ JJN3 tumor in tibiae(Fig. 4B, middle and C) compared with tibiae of sham mice(Fig. 4B, top and C). Treatment with Baf A1 decreased the

sprouting of CGRPþ SNs within the JJN3 tumor in bone (Fig.4B, bottom and C).

Thus, the acidic microenvironment of JJN3-colonized boneresulting from Hþ release via V-ATPase increases sprouting ofCGRPþ SNs in vivo.

To further analyze the effects of the acidic microenvironmentcreated by JJN3 cells on SN sprouting, 1� N cells were coculturedwith JJN3 cells and assessed for neurite outgrowth, an in vitroindicator for sprouting (45). Neurite outgrowth of 1� N cells wasincreased in the cocultures (Fig. 4D and E). Importantly, Baf A1significantly decreased neurite outgrowth of primaryDRGneuron

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Figure 2.

Bone pain and SN activation in JJN3-injectedmice. A, Mechanical allodynia. � , P < 0.05;�� , P < 0.01 vs. sham mice (mean � SE, n ¼ 16,two-way repeated measures ANOVA). Arrow,a single intraperitoneal injection of Baf A1 (25mg/kg) or vehicle (0.1% DMSO in PBS).B, Thermal hyperalgesia. � , P < 0.05 versusshammice (mean� SE, n¼ 16). Arrow, a singleintraperitoneal injection of BafA1 (25mg/kg) orvehicle (0.1% DMSO in PBS, two-way repeatedmeasuresANOVA).C,Sprouting ofCGRPþSNsin tibiae. D, Quantitative data of C. � , P < 0.05versus shammice (mean� SE, n¼ 16, two-wayANOVA). E, Acidification of bone marrowshown by acridine orange accumulation.F, Time course of inhibition of mechanicalallodynia, following a single intraperitonealinjection of Baf A1 (25 mg/kg) or vehicle (0.1%DMSO in PBS; arrow in A and B). � , P < 0.05versus vehicle-treated JJN3-injected mice(mean � SE, n ¼ 8, two-way repeatedmeasures ANOVA). G, Thermal hyperalgesia,following a single intraperitoneal injection ofBaf A1 (25 mg/kg) or vehicle (0.1% DMSO inPBS; arrow in A and B). � , P < 0.05 versusvehicle-treated JJN3-injected mice(mean � SE, n ¼ 8, two-way repeatedmeasures ANOVA). H, Excitation of sensorynerves determined by pERK1/2 andpCREB expression in DRG in sham andJJN3-injected mice.






cells in the cocultures (Fig. 4D and E). Furthermore, neuriteoutgrowth of 1� N cells cultured in media at pH 6.5, which isequivalent to the pHe of JJN3 cultures, was also increased com-pared with that cultured in media at pH 7.4 (Fig. 4F).

Thus,Hþprotons released via V-ATPase from JJN3 cells increaseneurite outgrowth of SNs in vitro.

Activation of acid-sensing nociceptors by the acidicmicroenvironment of JJN3-colonized bone

To study the mechanism underlying increased sprouting ofCGRPþ SNs in bone occurring in the acidic microenvironment of

JJN3 cells, we determined the expression of the acid-sensingnociceptors, ASIC3, on CGRPþ SNs inDRGof JJN3-injectedmice.We found increased expression of ASIC3 on CGRPþ SNs of DRGin JJN3-injectedmice by double immunofluorescent (Fig. 5A) andWestern blot analysis (Fig. 5B) compared with sham mice.Expression of the acid-sensing nociceptor, TRPV1, was alsoincreased on CGRPþ SNs of DRG in JJN3-injectedmice comparedwith sham mice (Supplementary Fig. S2A and S2B).

Increased neurite outgrowth of 1� N cells in cocultures withJJN3 cells was reduced by treatment with the specific ASIC3antagonist, APETx2 (46) or Baf A1 (Fig. 5C).

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Expression of plasma membrane a3V-ATPase. A, a3V-ATPase expression inhuman multiple myeloma cell lines(MM.1S, H929, U266, and JJN3),MC3T3-E1 mouse osteoblastic cells,MLO-A5 and MLO-Y4 mouseosteocytic cells, and MDA-MB-231human breast cancer cells. B, a3V-ATPase expression in primary CD138þ

and CD138� cells derived from threemultiple myeloma patients. C,Immunocytochemistry of a3V-ATPaseexpression in JJN3 and MDA-MB-231cells. D, pHe in cultures of humanmultiple myeloma cell lines, CD138þ

and CD138� cells, and MC3T3-E1,MLO-A5, MLO-Y4, and MDA-MB-231cells. � , P < 0.05; �� , P < 0.01 versuscontrol (mean � SE, n ¼ 6, one-wayANOVA with Dunnet post hoc test).E, Inhibition of acidification of pHe byBaf A1 (50 ng/mL, 48 hours). � ,P <0.01versus Baf A1-treated JJN3 cells(mean� SE, n¼ 6, one-way ANOVA).F, Effects of Baf A1 (50 ng/mL, 48hours) on cell proliferation (mean �SE, n¼ 6, one-way ANOVA). G, pHe incultures of JJN3/sh a3V-ATPase or/shcontrol cells. � , P < 0.01 versus control.��, P < 0.05 versus JJN3/sh controlcells (mean � SE, n ¼ 6, one-wayANOVA with Bonferroni post hoctest).

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We then examined the role of ASIC3 in SN excitation inresponse to JJN3-induced acidic microenvironment by asses-sing intracellular Ca2þ influx, an early indicator of SN excita-tion in vitro (38), in 1� N cells. Coculture of 1� N cells with JJN3cells rapidly induced a transient Ca2þ influx (Fig. 5D, iii), whichwas blocked by APETx2 (Fig. 5D, iv). Addition of culturemedium caused no Ca2þ influx (Fig. 5D, i). Addition of HClat a final pH 6.5 also induced a Ca2þ influx (Fig. 5E), whereasPBS at pH 7.4 had no effect. Importantly, no Ca2þ influx wasinduced by the addition of JJN3 cells (Fig. 5D, iv) or pH 6.5medium (Fig. 5F) when 1� N cells were pretreated with APETx2for 2 hours.

Thus, the acidic extracellular microenvironment of JJN3 cellspromotes neurite outgrowth and excites 1� N SNs via ASIC3activation.

Role of ASIC3 in MMBPWe then determined the role of ASIC3 in MMBP by testing

APETx2 in vivo. JJN3-bearing and shammice were treated with asingle intraplantar injection of APETx2 at day 25 (arrow in Fig.6A) and evaluated for changes in MMBP. APETx2 reducedmechanical allodynia as early as 1 hour after injection, whichlasted until 12 hours and disappeared after 24 hours (Fig. 6B,open square). As shown in Fig. 2G, Baf A1 exhibited itsanalgesic effects 12 hours after injection until 24 hours (Fig.6B, open triangle) following a single intraperitoneal injection(arrow in Fig. 6A).

Furthermore, pERK1/2 and pCREB expression in DRG wasdecreased in APETx2- and Baf A1–treated JJN3-injected micecompared with vehicle-treated JJN3-injected mice at 6 hours afterAPETx2 injection (Fig. 6C).

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Sprouting and neurite outgrowth ofSNs. A, Inhibition of mechanicalallodynia by Baf A1 in JJN3-injectedmice. Arrows, Baf A1 or vehicleinjection. � , P <0.05; ��, P <0.01 versussham mice. #, P < 0.05 versus vehicle-treated JJN3-injected mice (mean �SE, n¼ 8, two-way repeatedmeasuresANOVA with Dunnet post hoc test). B,Sprouting of CGRPþ SNs in tibiae. Left,CD138 (green); middle, CGRP (red);right, merged image. Tibiae injectedwith PBS (top; sham); JJN3 cells andtreated with vehicle (middle); andJJN3 cells and treated with Baf A1(bottom). Sections were incubatedwith rabbit anti-CD138 (1:100) andgoat anti-CGRP (1:200), then withdonkey anti-rabbit IgG (1:100) andanti-goat IgG (1:100), respectively.C,Quantitative analysis ofB. � ,P <0.01versus sham. �� , P < 0.01 versusvehicle-treated JJN3-injected tibiae(mean � SE, n ¼ 6, one-way ANOVAwith Dunnet post hoc test). D, Neuriteoutgrowth from 1� N cells in coculturewith JJN3 cells. Cocultures weretreated with Baf A1 (50 ng/mL) orvehicle (0.1% DMSO in PBS) for 5 days,labeled with calcein AM, andquantitated. E, Quantitative dataof D. � , P < 0.01 versus 1� N cells alone.�� , P < 0.01 versus vehicle-treatedcocultures (mean � SE, n ¼ 6, one-way ANOVA with Dunnet post hoctest). F, Neurite outgrowth of 1�

N cells. � , P < 0.05 versus pH 7.4(mean� SE, n¼ 6, one-way ANOVA).






Thus, inhibition of ASIC3 or V-ATPase by APETx2 or Baf A1administered in an "as-neededmanner" alleviates MMBP, respec-tively. These results indicate that ASIC3 plays an important role inMMBP induced by acidic bone microenvironment.

The selective TRPV1 antagonist, SB366791, also alleviatedmechanical allodynia and decreased pERK1/2 and pCREB inDRGin JJN3-injected mice following a single intraplantar injection(Supplementary Fig. S2C and S2D).

Effects of inhibition of both V-ATPase and ASIC3 on SNs onMMBP

The analgesic effects of APETx2 were observed 1 hour afterinjection and lost after 18 hours (Fig. 6B, open square), whereasBaf A1 exhibited its analgesic effects 12 hours after injection until24 hours (Fig. 6B, open triangle). We therefore examinedwhether

treatment of JJN3-injected mice with a combination of APETx2and Baf A1 produces rapid and sustained analgesic effects onMMBP. As expected, combined treatment with a single intraplan-tar injection of APETx2 and a single intraperitoneal injection ofBaf A1 decreasedmechanical allodynia (Fig. 6B, closed square) asearly as 1 hour after injection to a greater extent and for a longerperiod than did each agent alone.

Expression of pERK1/2 and pCREB in DRG in JJN3-injectedmice was also more profoundly downregulated by the combinedtreatment than each agent alone (Fig. 6C).

Contributions of osteoclasts to MMBPOsteoclasts express a3V-ATPase on their plasma membrane

in the ruffled border (15, 47) through which Hþ protons aresecreted during bone resorption, creating an acidic extracellular

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Role of ASIC3 in sprouting andexcitation of SNs. A, Expression ofASIC3 (left, green) on CGRPþ SNs(middle, red) in DRG from JJN3-injected (top) and sham mice(bottom). Sections were incubatedwith a goat anti-CGRP (1:200) andrabbit anti-ASIC3 (1:300), then adonkey anti-goat IgG (1:100) and anti-rabbit IgG (1:100), respectively.B, ASIC3 expression in DRGs isolatedfrom JJN3-injected and sham mice.C, Neurite outgrowth from 1� N cells incocultures with JJN3 cells. Cocultureswere treated with Baf A1 (50 ng/mL),APETx2 (0.5 mmol/L), or vehicle (0.1%DMSO in PBS) for 5 days. �, P < 0.05versus 1� N cells alone. �� , P < 0.05versus vehicle-treated cocultures(mean � SE, n ¼ 6, one-way ANOVAwith Dunnet post hoc test).D, Intracellular Ca2þ mobilization incocultures. 1� N cells were loaded withfura 2AM (3 mmol/L), treated withAPETx2 (0.5 mmol/L; ii and iv) orvehicle (0.1%DMSO inPBS; i and iii) for2 hours, then received JJN3 cells(3 � 105/300 mL; iii and iv, arrows) orculture medium (i and ii), incubatedand Ca2þ influx determined.E, Ca2þ influx in 1� N cells. 1� N cellswere pretreated with APETx2 (0.5mmol/L) for 2 hours, treated with PBSor HCl (arrows), and Ca2þ influxdetermined.

Hiasa et al.





microenvironment (15–17). Histologic examination of bone ofJJN3-injected mice showed that the pits formed by TRAPþ osteo-clasts on endosteal bone surfaces (Fig. 7A, top) were acidic (Fig.7A, bottom, arrowheads) and that CGRPþ SNs innervated inclose proximity to TRAPþ osteoclasts (Fig. 7B). These findingssuggest that the osteoclast-generated acidic microenvironmentscan directly activate pH-sensitive SNs in the bone and elicitMMBP. To determine the role of osteoclasts in SN activationand MMBP, we tested the potent inhibitor of osteoclasts,zoledronic acid (ZOL), which reduces MMBP in patients (2).JJN3-injected mice (Fig. 7C, closed circle) were treated withZOL at days 10, 14, and 18 (arrows in Fig. 7C) using anestablished protocol (48). ZOL significantly reduced mechan-ical allodynia (Fig. 7C, closed triangle). Of note, however, theanalgesic effects of ZOL were lost at day 35. These results areconsistent with the clinical observations that ZOL does notprevent the progression of bone pain in multiple myelomapatients at advanced stages (2, 3, 22) and suggest that suppres-sion of osteoclast bone resorption no longer ameliorates MMBPand that multiple myeloma cells predominantly contribute toMMBP at advanced stages.

Effects of inhibition of both osteoclasts andV-ATPase onMMBPWe therefore examined whether inhibition of Hþ release via V-

ATPase from JJN3 cells by Baf A1 alleviates MMBP that becamerefractory to analgesic effects of ZOL. As already shown in Figs. 2Gand 6B, a single intraperitoneal injection of Baf A1 to untreatedJJN3-bearing mice reduced MMBP (Fig. 7D, closed diamond). Ofnote, a single intraperitoneal injection of Baf A1 to ZOL-treatedJJN3-bearing mice that lost responsiveness to analgesic effects ofZOL at day 35 (arrowhead in Fig. 7C) decreased mechanicalallodynia (Fig. 7D, closed square) as early as 1 hour until 24hours. Furthermore, the combination of Baf A1 and ZOLdecreased pERK1/2 and pCREB expression in DRG more pro-foundly than did ZOL or Baf A1 alone (Fig. 7E). These resultsindicate that V-ATPase on multiple myeloma cells as well asosteoclasts contribute to SN excitation and MMBP at advancedstages of multiple myeloma.

DiscussionWe investigated the contributions of the acidic bone microen-

vironment associated with multiple myeloma colonization in

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Contribution of a3V-ATPase andASIC3 to MMBP and SN excitation.A, Mechanical allodynia. Arrow, asingle injection of either vehicle,APETx2 (intraplantar), Baf A1(intraperitoneal), or APETx2combined with Baf A1. � , P < 0.05;�� , P < 0.01 versus sham (mean � SE,n ¼ 8, two-way repeated measuresANOVA). B, Time course of inhibitionof mechanical allodynia followingtreatment with either vehicle (0.1%DMSO in PBS), APETx2 (20 mmol/L),Baf A1 (25 mg/kg), or APETx2 plus BafA1 (arrow in A) in sham and JJN3-injected mice. � , P < 0.05 versus JJN3-injected mice treated with vehicle. #,P < 0.05 versus JJN3-injected micetreated with APETx2 or Baf A1 (mean� SE, n ¼ 8, two-way repeatedmeasures ANOVA with Bonferronipost hoc test). C, Expressionof pERK1/2 and pCREB in DRGs inmice shown in B at 12 hours.






bone to MMBP. We showed that bone-colonizing JJN3 cellsrelease Hþ via V-ATPase, creating an acidic bone microenviron-ment. This pathologic acidic microenvironment then stimulatesthe sprouting of CGRPþ SNs innervating bone and activates theacid-sensing nociceptors, ASIC3, on the CGRPþ SNs, which inturn excites the SNs to evoke MMBP. Blocking Hþ release anddevelopment of the acidic bone microenvironment by the selec-tive V-ATPase inhibitor, Baf A1, decreased the sprouting ofCGRPþ

SNs in bone, inhibited the excitation of the SNs, and reducedMMBP in JJN3-injectedmice. InhibitingHþ-induced activation ofASIC3 by the specific ASIC3 antagonist, APETx2, inhibited SN

excitation and alleviatedMMBP. Furthermore, a combination of asingle injection of Baf A1 and APETx2 showed greater and longeranalgesic effects onMMBP than each agent alone. Taken together,these results indicate that secretion of Hþ via V-ATPase on mul-tiple myeloma cells and subsequent activation of SNs by Hþ viaASCI3 are key steps responsible for inducing MMBP, and thatthese steps could bemechanism-based targets for the treatment ofMMBP in patients.

Our results for the first time show that not only osteoclasts butalsomultiplemyeloma cells directly contribute to SNactivation toevokeMMBP through releasingHþ viaV-ATPase. The result in part

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Contribution of osteoclasts to MMBPand SN activation. A, Acidification ofresorption lacunae formed onendosteal bone surfaces by TRAPþ

osteoclasts. Top, TRAPþ osteoclasts(arrowheads) on endosteal bonesurface; bottom, fluorescence ofacridine orange accumulation in pitsformed under osteoclasts (green;�200). B, Innervation of CGRPþ SNs(arrowheads) in the close proximity ofTRAPþ osteoclasts (arrows). Sectionswere incubated with a goat anti-CGRP(1:200), followed by HRP-conjugateddonkey anti-goat IgG (1:100). Brown,CGRPþ SNs; red, TRAPþ osteoclasts(�400). C, Inhibition of mechanicalallodynia by ZOL. Mice intratibiallyinjected with JJN3 cells ( , ), orPBS (sham; , ) were givenvehicle (PBS; , ) or ZOL(120 mg/kg; , ) at day 10, 14, and18 after JJN3 cell injection (arrows). Atday 35, mice were given a singleintraperitoneal injection of Baf A1(arrowhead). � , P < 0.05 versusvehicle-treated JJN3-injected mice(mean� SE, n¼ 8, two-way repeatedmeasures ANOVA with Dunnnet posthoc test). D, Effects of a singleintraperitoneal injection of Baf A1 atday 35 (arrowhead inC) onMMBP thatbecame unresponsive to ZOL. At day35, vehicle-treated JJN3-injectedmicewere given a single intraperitonealinjection of vehicle ( ) or Baf A1( ), and ZOL-treated JJN3-injectedmice received a single intraperitonealinjection of vehicle ( ) or Baf A1(25 mg/kg; ). � , P < 0.05 versus ZOL-treated JJN3-injectedmice. #,P<0.05versus Baf A1-treated JJN3-injectedmice (mean � SE, n ¼ 8, two-wayrepeated measures ANOVA withDunnet post hoc test). E, Expression ofpERK1/2 and pCREB in DRG SNs inmice shown in D at 12 hours.

Hiasa et al.





explains the observations that osteoclast-specific inhibitors alonereduceMMBP at early to advanced stages, however, fail to preventthe progression of MMBP at advanced to terminal stages of thedisease (2, 20). Consistent with these observations, ZOL reducedMMBPat early stages but lost its analgesic effects at terminal stagesin our animal model. Of importance, amelioration of MMBP wasrestored by additional administration of a single injection of BafA1 to ZOL-treated mice, demonstrating that blockade of Hþ

release frommultiple myeloma cells via suppression of V-ATPasemitigates MMBP refractory to the analgesic action of ZOL. Theseresults suggest that administration of V-ATPase inhibitor com-bined with osteoclast inhibitor may be an effective approach formanagement of MMBP in patients with advanced multiplemyeloma.

ZOLwas shown todecrease tumorburden inpreclinicalmodelsof multiple myeloma (48). Thus, alleviation of MMBP by ZOL inJJN3-injected mice could be secondary to decreased multiplemyeloma tumor burden rather than inhibition of osteoclast boneresorption. However, clinical observations that ZOL fails to pre-vent the progression ofMMBPat advanced stages of thedisease (2,20) together with our results suggest that the analgesic effects ofZOL are unlikely accounted by the inhibition of multiple mye-loma growth.

Expression of the acid-sensing nociceptor TRPV1 was alsoincreased on CGRPþ SNs in JJN3-injected mice. Previous studiesreported that TRPV1 antagonists or disruption of the TRPV1 geneattenuated cancer-induced bone pain (28, 29). We also showedthat a single intraplantar injection of the selective TRPV1 antag-onist, SB366791, significantly decreased SN excitation andMMBPin JJN3-injected mice. TRPV1 on SNs is activated at pH <6.0 (27).The pHe of the acidic microenvironment created by JJN3 cells andbone-resorbing osteoclasts is 6.5 to 6.9 (Fig. 3) and 4.5 to 6.0 (15–17), respectively. These results suggest that the pHe of specificareas in JJN3-colonized bonemay be <6.0, a pH atwhich TRPV1 isactivated. We propose that the pHe of multiple myeloma–colo-nized bone varies spatially and temporally and induces activationof either ASIC3, TRPV1, or both on pH-sensitive SNs to induceMMBP, dependingon the status ofmultiplemyelomaprogressionand osteoclast bone resorption.

Although our results demonstrate significant contribution ofbone-resorbing osteoclast to MMBP in JJN3-injected mice,osteoclast bone resorption that occurs under physiologic con-ditions rarely induces bone pain. As an explanation for this,recent studies reported that TRPV1 and ASIC3 dramaticallydecrease their threshold for sensing noxious stimuli in thepresence of tissue damage and inflammation (18). Thus, it isspeculated that SNs are not sensitive enough to be excited toevoke bone pain in response to acidic microenvironmentscreated by physiologic osteoclast bone resorption. Further-more, episodic musculoskeletal pain termed "growing pain"that often occurs in children in early childhood (49) suggeststhat bone turnover rate may be critical.

Solid tumors express various plasma membrane pH regula-tors, including V-ATPase, monocarboxylate transporter 1/4,and carbonic anhydrases (24) through which Hþ are extrudedto avoid intracellular acidification due to elevated aerobicglycolysis (Warburg effect; ref. 50) and acidify the extracellularmicroenvironment. Among these pH regulators, a3V-ATPasehas been implicated in solid cancer invasiveness and metastasis(25, 44). We previously reported that suppression of a3V-ATPase in highly metastatic B16-F10 melanoma decreases bone

metastasis (51). In contrast, whether multiple myeloma cellsexpress V-ATPase was unknown. Here, we show that JJN3 cellsexpress plasma membrane a3V-ATPase that acidifies extracel-lular environments by releasing Hþ and directly contributes toSN activation to induce MMBP. However, whether a3V-ATPaseexpression is associated with multiple myeloma progression asis the case of solid tumors is unknown. Our results showed thata single injection of Baf A1 had no effects on JJN3 progressionin bone and that Baf A1 did not inhibit JJN3 proliferation inculture, although long-term effects of Baf A1 on multiplemyeloma progression were not examined in this study. Inter-estingly, however, CD138þ primary cells derived from the bonemarrow of multiple myeloma patients expressed a3V-ATPase,whereas little expression of a3V-ATPase was seen in CD138�

cells. Further studies are needed to determine the relationshipbetween a3V-ATPase expression and multiple myelomaprogression.

In conclusion, this study shows that the creation of acidicextracellular bone microenvironments by osteoclasts and multi-plemyeloma cells via Hþ secretion through plasmamembrane V-ATPase and responses of SNs innervating bone to the acidicmicroenvironment via ASIC3 are critical contributors to thepathophysiology of MMBP. Targeting these pathways may pro-vide mechanism-based effective therapies for control of MMBP,which is currently undertreated.

Disclosure of Potential Conflicts of InterestG.D. Roodman is a consultant/advisory board member for Amgen. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: M. Hiasa, F.A. White, T. YonedaDevelopment of methodology: M. Hiasa, T. Okui, F.A. White, T. YonedaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):M. Hiasa, Y.M. Allette, M.S. Ripsch, G.-H. Sun-Wada,H. Wakabayashi, F.A. White, T. YonedaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):M.Hiasa, T. Okui, Y.M. Allette, F.A. White, T. YonedaWriting, review, and/or revision of themanuscript:M. Hiasa, G.D. Roodman,T. YonedaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. WakabayashiStudy supervision: T. Yoneda

Grant SupportThis study was supported by the Project Development Team within the

ICTSI NIH/NCRR (#TR000006), the Indiana University Health StrategicResearch Initiative in Oncology, and start-up fund of Indiana UniversitySchool of Medicine (T. Yoneda) and Merit Review Funds from the VeteransAdministration (G.D. Roodman), the NIH (#DK100905 to F.A. White),MERIT Review Award (#BX002209) from the U.S. Department of VeteransAffairs, Biomedical Laboratory Research and Development Service (F.A.White), grants from St. Vincent Indianapolis Hospital and the St. VincentFoundation to F.A. White. Support for Y.M. Allette as an Indiana CTSIPredoctoral trainee was provided by UL1 (#TR001108), NIH/NCATS(A. Shekhar, principal investigator), and Japan Society for the Promotionof Science Grants-in-aid for Research Activity Start-up, and PostdoctoralFellowship for Research Abroad (M. Hiasa).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 31, 2015; revised November 23, 2016; accepted Novem-ber 29, 2016; published OnlineFirst March 2, 2017.






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2017;77:1283-1295. Published OnlineFirst March 2, 2017.Cancer Res Masahiro Hiasa, Tatsuo Okui, Yohance M. Allette, et al. V-ATPase and ASIC3Bone Pain Induced by Multiple Myeloma Is Reduced by Targeting

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Bone Pain Induced by Multiple Myeloma Is Reduced by ...Microenvironment and Immunology Bone Pain Induced by Multiple Myeloma Is Reduced by Targeting V-ATPase and ASIC3 Masahiro Hiasa1,Tatsuo