Multiple myeloma patients experience severe bone pain (MMBP) that is undertreated and poorly understood. In this study, we studied MMBP in an intratibial mouse xenograft model that employs JJN3 human multiple myeloma cells. In this model, mice develop MMBP associated in bone with increased sprouting of calcitonin gene-related peptide-positive (CGRP+) 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 single injection of the selective V-ATPase inhibitor, bafilomycin A1, decreased MMBP, CGRP+ sensory neuron sprouting, and pERK1/2 and pCREB expression in DRG. CGRP+ sensory nerves also expressed increased levels of the acid-sensing nociceptor ASIC3. Notably, a single injection of the selective ASIC3 antagonist APETx2 dramatically reduced MMBP in the model. Mechanistic investigations in primary DRG neurons cocultured with JJN3 cells showed increased neurite outgrowth and excitation inhibited by bafilomycin A1 or APETx2. Furthermore, combining APETx2 with bafilomycin A1 reduced MMBP to a greater extent than either agent alone. Finally, combining bafilomycin A1 with the osteoclast inhibitor zoledronic acid was sufficient to ameliorate MMBP, which was refractory to zoledronic acid. Overall, our results show that osteoclasts and multiple myeloma cooperate to induce an acidic bone microenvironment that evokes MMBP as a result of the excitation of ASIC3-activated sensory neurons. Furthermore, they present a mechanistic rationale for targeting ASIC3 on neurons along with the multiple myeloma-induced acidic bone microenvironment as a strategy to relieve MMBP in patients. Cancer Res; 77(6); 1283–95. ©2017 AACR.
Multiple myeloma is a malignant plasma cell disorder accounting for approximately 10% of all hematologic cancers (1). Seventy percent of multiple myeloma patients present with severe bone pain associated with osteolytic bone lesions that result from increased osteoclastic bone resorption and reduced osteoblastic bone formation (2). Bone pain contributes to increased mortality and morbidity (3). The degree of bone pain in multiple myeloma is often greater than pain experienced with solid tumors (4–6), and bone pain is the most common presenting complaint of multiple myeloma patients (7). Control of bone pain is thus a major goal in the management of multiple myeloma patients. Despite this, the pathophysiology of bone pain associated with multiple myeloma (MMBP) is poorly understood, and MMBP is frequently inadequately treated (3).
Bone is densely innervated by sensory neurons (SN), especially the calcitonin gene-related peptide-positive (CGRP+) SN (8–12), which show pathologic sprouting in the presence of tumors (13, 14). Osteoclasts are also increased in cancer-colonized bone and actively secrete protons (H+) to destroy mineralized bone via the plasma membrane a3 isoform vacuolar-H+-ATPase (a3V-ATPase) localized in the ruffled borders (15), creating an acidic microenvironment (pH range = 4.0–6.0; refs. 16, 17). H+ is a well-known potent pain-inducing mediator (18, 19), suggesting that the acidic microenvironment created by increased H+ release from bone-resorbing osteoclasts via the a3V-ATPase activates pH-sensitive SNs to elicit bone pain. Consistent with this notion, specific inhibitors of osteoclastic bone resorption, including bisphosphonates and denosumab, significantly reduce bone pain in patients with multiple myeloma (2, 20) and bone metastases of solid cancers (21–23), indicating that osteoclast bone resorption contributes to the activation of nociceptive SNs in the bone to induce bone pain.
However, bisphosphonate or denosumab treatment does not prevent the progression of bone pain in bone metastasis at advanced stages in patients with solid tumors (22, 23), suggesting that tumor cells also contribute to bone pain. Solid tumor cells release increased the levels of H+ via various types of plasma membrane pH regulators, thereby inducing an acidic extracellular microenvironment (pH range = 6.5–7.0; refs. 24, 25) that can also excite pH-sensitive nociceptive SNs innervating bone to evoke bone pain. Similarly, it has been clinically well recognized that MMBP in patients at advanced stages is unresponsive to bisphosphonates or denosumab. Thus, multiple myeloma cells may also contribute to induce MMBP through the creation of acidic extracellular microenvironment via plasma membrane pH regulators as do solid tumors.
A subpopulation of peripheral nerve fibers, termed nociceptors (18), recognizes and transduces local nociceptive stimuli into electrochemical signals. The nociceptors in turn transmit these noxious signals to the central nervous system (CNS) and brain via the dorsal root ganglion (DRG), which is the cell body of primary afferent SN fibers innervating peripheral tissues and serves as the gateway for peripheral noxious signals to the CNS (26). The specific acid-sensing nociceptors related to acid-induced bone pain are the transient receptor potential channel-vanilloid subfamily 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 in cancer-associated bone pain (28, 29). In contrast, little is known about the contributions of ASIC3 to bone pain, despite that ASIC3 senses milder pH between 6.5 and 7.0 (30), equivalent to the pH of the bone microenvironment of cancer-involved bone (24, 25).
In this study, we show that H+ released via V-ATPase by bone-resorbing osteoclasts and JJN3 multiple myeloma cells create an acidic bone microenvironment that excites SNs innervating bone via activation of ASIC3 to induce MMBP. Furthermore, blocking the generation of the acidic bone microenvironment and the activation of ASIC3 on SNs significantly reduced MMBP and could be a mechanism-based therapeutic approach for MMBP.
Materials and Methods
The sources for the reagents were as follows: APETx2 and SB366791 (Tocris); zoledronic acid monohydrate (Sigma); rabbit mAbs to ERK1/2, CREB, pERK1/2, pCREB, horseradish peroxidase (HRP)-conjugated horse anti-mouse IgG and goat anti-rabbit IgG antibody (Cell Signaling Technology); bafilomycin A1 (Baf A1), rabbit polyclonal antibodies to ASIC3, TRPV1, the goat polyclonal antibody against CGRP, and HRP-conjugated mouse mAb against β-actin (Abcam); Alexa Fluor 488-conjugated donkey anti-rabbit IgG and donkey anti-chicken IgY, and rhodamine red-X (RRX)-conjugated donkey anti-goat IgG antibody (Jackson); rhodamine phalloidin (Life Technologies); and rabbit polyclonal antibodies to RANKL, IL6, mouse mAb to MIP-1α, and HRP-conjugated goat anti-chicken antibody (Santa Cruz Biotechnology).
The JJN3 human multiple myeloma cell line was provided by Dr. Nicola Giuliani (Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy; ref. 31). JJN3, H929, and U266 multiple myeloma cell lines and primary CD138+ multiple myeloma cells and CD138− cells (isolated from bone marrow aspirates of multiple myeloma patients using MACS MicroBead) were cultured in RPMI1640 medium with 5% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. The mouse osteocytic MLO-A5 and MLO-Y4 (Dr. T. Bellido, Department of Anatomy and Cell Biology, School of Medicine, Indiana University, Indianapolis, IN), mouse osteoblastic MC3T3-E1 and F11 SN, human breast cancer MCF-7, and MDA-MB-231 cells were cultured in α-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 growth medium (Lonza) with 2% FBS. All cell lines were analyzed and authenticated by targeted genomic and RNA sequencing. All procedures involving patients were performed with written informed consent according to the Declaration of Helsinki and under a protocol approved by the Indiana University (IU) Institutional Review Board.
Animal model of multiple myeloma
All animal studies were approved by the Institutional Animal Care and Use Committee at IU School of Medicine (Indianapolis, IN). JJN3 cells (5 × 105) or PBS (sham) were injected into the right tibia marrow of female C57BL/6 SCID mice (4–6 weeks old) under anesthesia (32).
Behavioral pain tests
Mechanical allodynia and thermal hyperalgesia were evaluated by von Frey and plantar tests using the Dynamic Plantar Aesthesiometer and Plantar Test Instrument (Ugo Basile), respectively (33, 34). These tests are widely used for bone pain assessment in rodents (35). Tests were performed prior to multiple myeloma cell injection to determine baseline behaviors and every 5 days following cell injection. Throughout the experiment, behavioral tests were performed under blind conditions by a single researcher. JJN3 cell injections were performed by a researcher blinded to the experiment. The investigator performing the behavioral tests was blinded as to the experimental condition of the animals.
Acridine orange accumulation
Acridine orange (Molecular Probes), which selectively deposits 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 detected using an EVOS fluorescence microscope (Advanced Microscopy).
Tartrate-resistant acid phosphatase staining
Bone sections were stained for tartrate-resistant acid phosphatase (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 measurement
Sera were collected from mice at the indicated time, and C-terminal telopeptide of type I collagen (CTx) was measured by RatLaps (CTX-I) EIA Kit (Immunodiagnostic Systems) according to the manufacturer's instructions.
Extracellular pH measurement
Cells (105/96-well) were cultured for 48 hours in the presence of adriamycin (30 nmol/L) to prevent cell proliferation and extracellular pH (pHe) was measured by FiveEasy pH meter (Mettler Toledo) immediately after removal from the CO2 incubator.
Knockdown of a3V-ATPase in JJN3 cells
JJN3 cells were infected with 20 μL of control (sc-108080) or TCIRG1 (sc-96928-V) shRNA lentiviral particles (Santa Cruz Biotechnology) in the presence of 5 μg/mL polybrene. One day later, cells were cultured in RPMI1640 plus 5% FBS for 7 days in the presence of 2 μg/mL puromycin (Gibco) to select cells stably expressing the shRNAs.
Neurite outgrowth determination
1° N cells (1 × 103/6-well) were cocultured with JJN3 cells (5 × 103) or alone for 120 hours. Neurite outgrowth was visualized by staining the cultures with calcein AM (Life Technologies), and the length of neurites was quantified with a fluorescent microscope using Neuron J (37).
Imaging of intracellular Ca2+ influx
1° N or F11 SN cells were seeded in FluoroDish (World Precision) and loaded with fura-2 AM (3 μmol/L, Molecular Probes) for 25 minutes (38). Intracellular calcium was measured by digital video microfluorimetry, using an intensified CCD camera coupled to a microscope and MetaFluor software (Molecular Devices). Cells were illuminated, and the excitation wavelengths for fura-2 (340/380 nm) were selected by a filter changer. Inhibitors were added directly into the bathing solution 2 hours before stimulation with 0.1 N hydrochloric acid (HCl).
Cells or tissues were lysed in lysis buffer (Invitrogen), electrophoresed on a 10% SDS-PAGE, and blotted onto PVDF membranes (Bio-Rad; ref. 32). After blocking, the membranes were incubated with primary antibodies overnight at 4°C, and then with HRP-conjugated secondary antibodies for 1 hour. Protein bands were visualized with a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).
Bones were fixed, embedded in OCT compound, and sectioned at 30-μm thickness using a cryostat. After blocking, sections were incubated with the primary antibodies overnight at 4°C and secondary antibodies for 60 minutes, mounted with coverslips in VECTASHIELD anti-fade mounting medium (Vector), and observed under the Leica TCS SP8 confocal laser scanning microscope.
For TRAP and CGRP double-staining, sections were first stained with TRAP and incubated with anti-CGRP antibody (1/200) overnight at 4°C, and then with HRP-conjugated secondary antibodies and diaminobenzidine substrate (Vector).
JJN3 cells (1 × 103/96-well) were fixed with 10% neutral-buffered formalin, incubated with 3% BSA-PBS blocking solution, and then with a3V-ATPase antibody (1/200; ref. 39) at 4°C overnight, followed by Alexa Fluor 594 anti-chicken secondary antibody (1/1,000) and Alexa Fluor 488 phalloidin (1/2,000). Nuclei were counterstained with DAPI.
Data are presented as the mean ± SEM. Statistical differences were determined by using one-way, two-way, two-way repeated measures ANOVA followed by Bonferroni or Dunnet post hoc test, as indicated. All statistical analyses were performed by using Statcell (OMS). P < 0.05 was considered statistically significant.
Animal model of MMBP
Mice intratibially injected with JJN3 cells developed characteristic osteolytic lesions on X-ray (Fig. 1A) and μCT (Fig. 1B). These lesions showed aggressive proliferation of JJN3 cells in the bone marrow (Fig. 1C) with increased TRAP+ osteoclast bone resorption (Fig. 1D and E) and elevated serum CTx (Fig. 1F). We found that JJN3 cells produced osteoclastogenic cytokines, including RANKL, IL6, and MIP-1α (Fig. 1G). Interestingly, MDA-MB-231 human breast cancer cells (25), used as a representative aggressive solid cancer cells that develop osteolytic bone lesions to compare with multiple myeloma cells, showed undetectable production of MIP-1α. PBS-injected sham mice exhibited no osteolytic lesions.
The right leg of mice harboring JJN3 cells initially displayed mechanical allodynia (Fig. 2A) and thermal hyperalgesia (Fig. 2B) at day 20 of postinjection, and these pain behaviors progressed in parallel with bone destruction. Sham mice showed no evidence of MMBP (Fig. 2A and B). Left legs of JJN3-injected mice had no evidence of MMBP, demonstrating that MMBP is associated with local JJN3 colonization rather than a systemic effect of JJN3 cells. Sprouting of CGRP+ SNs in JJN3-injected bone was increased in parallel with the progression of MMBP (Fig. 2C and D).
5TGM1 multiple myeloma cells intratibially injected into immunocompetent C57BL/KaLwRij mice also developed osteolytic lesions and induced progressive MMBP (Supplementary Fig. S1A and S1B).
Acidification and MMBP are associated with JJN3-colonized bone
We next determined whether the JJN3-colonized bone is acidic using the pH sensor fluorescent dye, acridine orange. No fluorescence was detected in the right tibiae of sham mice (Fig. 2E, left). In contrast, the entire marrow of right tibiae injected with JJN3 cells was fluorescent (Fig. 2E, middle), demonstrating that JJN3-colonized bone is acidic. The bone marrow injected with 5TGM1 cells was also acidic (Supplementary Fig. S1D, right). We then assessed whether the selective V-ATPase inhibitor, Baf A1, blocked established MMBP in JJN3-injected mice. Baf A1 inhibited acidification by osteoclast bone resorption (40), and we reported reduced bone pain in breast cancer bone metastases (41). Repeated injections of Baf A1 inhibited tumor growth (42) and bone resorption (40) that alter the status of bone pain. To examine whether Baf A1 directly inhibits SN activation and reduces MMBP independent of inhibition of tumor growth and bone resorption, JJN3-injected mice manifesting MMBP and sham mice were given a single intraperitoneal injection of Baf A1 at day 25 (arrow in Fig. 2A and B) and evaluated for changes in MMBP as a function of time. Baf A1 treatment blocked acidification in JJN3-colonized bone (Fig. 2E, right). Importantly, Baf A1 blockade of the acidification of JJN3-colonized bone inhibited the progression of mechanical allodynia (Fig. 2F) and thermal hyperalgesia (Fig. 2G) as early as 12 hours after injection. The effects lasted up to 24 hours and were lost by 48 hours after injection.
In parallel with noxious behaviors, JJN3-injected mice demonstrated increased expression of pERK1/2 and pCREB, which are molecular indicators for neuronal excitation (43), in their DRG SNs compared with sham mice (Fig. 2H). Similarly, 5TGM1-injected mice also exhibited elevated expression of pERK and pCREB 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 noxious behaviors, is inhibited by blocking the acidification of JJN3-colonized bone. These results suggest that H+ protons released via V-ATPase directly excite SNs in bone to evoke MMBP. They also suggest that Baf A1 ameliorates MMBP by inhibiting SN excitation in a time period too short to be due to inhibition of JJN3 tumor growth and bone resorption.
Expression of V-ATPase and pHe in JJN3 cells
We next examined whether JJN3 cells contribute to acidification of the bone microenvironment by releasing H+ via V-ATPase. Human multiple myeloma cell lines, JJN3, H929, and U266, and MDA-MB-231 human breast cancer cell line (serve as a positive control) expressed a3V-ATPase (Fig. 3A). Importantly, CD138+, but not CD138−, cells derived from three multiple myeloma patients expressed a3V-ATPase (Fig. 3B). JJN3 expressed the a3V-ATPase on their plasma membrane (Fig. 3C), as did highly invasive MDA-MB-231 cells (44). The pHe of JJN3, H929, and U266 and MDA-MB-231 and CD138+ cells was acidic (Fig. 3D). The human multiple myeloma cells MM.1S expressed undetectable a3V-ATPase (Fig. 3A), and the pHe of MM.1S was not acidic (Fig. 3D). The expression of a3V-ATPase in MC3T3-E1 osteoblastic and MLO-A5 and MLO-Y4 osteocytic cells was undetectable (Fig. 3A), and their pHe was not acidic (Fig. 3D). Thus, osteoblasts and osteocytes unlikely contribute to extracellular acidification of multiple myeloma–colonized bone. Baf A1 significantly blocked extracellular acidification in JJN3 cultures (Fig. 3E) with no inhibition of cell proliferation (Fig. 3F). In contrast, Baf A1 showed no effects on pHe in other cells. Furthermore, knockdown of a3V-ATPase in JJN3 cells significantly blocked extracellular acidification (Fig. 3G).
Thus, the a3V-ATPase contributes to the acidification of JJN3 cell extracellular microenvironment.
5TGM1 cells also expressed plasma membrane a3V-ATPase, and the pHe of 5TGM1 cells was acidic, which was neutralized by Baf A1 (Supplementary Fig. S1E–S1G).
Effects of acidic microenvironment of JJN3-colonized bone on SNs
Tumors in the bone induce SN sprouting in the bone, thereby enhancing bone pain (13, 14). We therefore determined the extent of CGRP+ SN sprouting with or without treatment with Baf A1 in JJN3-colonized bone. Baf A1 inhibited the progression of mechanical allodynia in mice injected intratibially with JJN3 cells (Fig. 4A). IHC showed increased CGRP+ SN sprouting within the CD138+ 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 CGRP+ SNs within the JJN3 tumor in bone (Fig. 4B, bottom and C).
Thus, the acidic microenvironment of JJN3-colonized bone resulting from H+ release via V-ATPase increases sprouting of CGRP+ SNs in vivo.
To further analyze the effects of the acidic microenvironment created by JJN3 cells on SN sprouting, 1° N cells were cocultured with JJN3 cells and assessed for neurite outgrowth, an in vitro indicator for sprouting (45). Neurite outgrowth of 1° N cells was increased in the cocultures (Fig. 4D and E). Importantly, Baf A1 significantly decreased neurite outgrowth of primary DRG neuron cells in the cocultures (Fig. 4D and E). Furthermore, neurite outgrowth of 1° N cells cultured in media at pH 6.5, which is equivalent to the pHe of JJN3 cultures, was also increased compared with that cultured in media at pH 7.4 (Fig. 4F).
Thus, H+ protons released via V-ATPase from JJN3 cells increase neurite outgrowth of SNs in vitro.
Activation of acid-sensing nociceptors by the acidic microenvironment of JJN3-colonized bone
To study the mechanism underlying increased sprouting of CGRP+ SNs in bone occurring in the acidic microenvironment of JJN3 cells, we determined the expression of the acid-sensing nociceptors, ASIC3, on CGRP+ SNs in DRG of JJN3-injected mice. We found increased expression of ASIC3 on CGRP+ SNs of DRG in JJN3-injected mice by double immunofluorescent (Fig. 5A) and Western blot analysis (Fig. 5B) compared with sham mice. Expression of the acid-sensing nociceptor, TRPV1, was also increased on CGRP+ SNs of DRG in JJN3-injected mice compared with sham mice (Supplementary Fig. S2A and S2B).
We then examined the role of ASIC3 in SN excitation in response to JJN3-induced acidic microenvironment by assessing intracellular Ca2+ influx, an early indicator of SN excitation in vitro (38), in 1° N cells. Coculture of 1° N cells with JJN3 cells rapidly induced a transient Ca2+ influx (Fig. 5D, iii), which was blocked by APETx2 (Fig. 5D, iv). Addition of culture medium caused no Ca2+ influx (Fig. 5D, i). Addition of HCl at a final pH 6.5 also induced a Ca2+ influx (Fig. 5E), whereas PBS at pH 7.4 had no effect. Importantly, no Ca2+ influx was induced by the addition of JJN3 cells (Fig. 5D, iv) or pH 6.5 medium (Fig. 5F) when 1° N cells were pretreated with APETx2 for 2 hours.
Thus, the acidic extracellular microenvironment of JJN3 cells promotes neurite outgrowth and excites 1° N SNs via ASIC3 activation.
Role of ASIC3 in MMBP
We then determined the role of ASIC3 in MMBP by testing APETx2 in vivo. JJN3-bearing and sham mice were treated with a single intraplantar injection of APETx2 at day 25 (arrow in Fig. 6A) and evaluated for changes in MMBP. APETx2 reduced mechanical allodynia as early as 1 hour after injection, which lasted until 12 hours and disappeared after 24 hours (Fig. 6B, open square). As shown in Fig. 2G, Baf A1 exhibited its analgesic 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 was decreased in APETx2- and Baf A1–treated JJN3-injected mice compared with vehicle-treated JJN3-injected mice at 6 hours after APETx2 injection (Fig. 6C).
Thus, inhibition of ASIC3 or V-ATPase by APETx2 or Baf A1 administered in an “as-needed manner” alleviates MMBP, respectively. These results indicate that ASIC3 plays an important role in MMBP induced by acidic bone microenvironment.
The selective TRPV1 antagonist, SB366791, also alleviated mechanical allodynia and decreased pERK1/2 and pCREB in DRG in JJN3-injected mice following a single intraplantar injection (Supplementary Fig. S2C and S2D).
Effects of inhibition of both V-ATPase and ASIC3 on SNs on MMBP
The analgesic effects of APETx2 were observed 1 hour after injection and lost after 18 hours (Fig. 6B, open square), whereas Baf A1 exhibited its analgesic effects 12 hours after injection until 24 hours (Fig. 6B, open triangle). We therefore examined whether treatment of JJN3-injected mice with a combination of APETx2 and Baf A1 produces rapid and sustained analgesic effects on MMBP. As expected, combined treatment with a single intraplantar injection of APETx2 and a single intraperitoneal injection of Baf A1 decreased mechanical allodynia (Fig. 6B, closed square) as early as 1 hour after injection to a greater extent and for a longer period than did each agent alone.
Expression of pERK1/2 and pCREB in DRG in JJN3-injected mice was also more profoundly downregulated by the combined treatment than each agent alone (Fig. 6C).
Contributions of osteoclasts to MMBP
Osteoclasts express a3V-ATPase on their plasma membrane in the ruffled border (15, 47) through which H+ protons are secreted during bone resorption, creating an acidic extracellular microenvironment (15–17). Histologic examination of bone of JJN3-injected mice showed that the pits formed by TRAP+ osteoclasts on endosteal bone surfaces (Fig. 7A, top) were acidic (Fig. 7A, bottom, arrowheads) and that CGRP+ SNs innervated in close proximity to TRAP+ osteoclasts (Fig. 7B). These findings suggest that the osteoclast-generated acidic microenvironments can directly activate pH-sensitive SNs in the bone and elicit MMBP. To determine the role of osteoclasts in SN activation and 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 with ZOL at days 10, 14, and 18 (arrows in Fig. 7C) using an established protocol (48). ZOL significantly reduced mechanical allodynia (Fig. 7C, closed triangle). Of note, however, the analgesic effects of ZOL were lost at day 35. These results are consistent with the clinical observations that ZOL does not prevent the progression of bone pain in multiple myeloma patients at advanced stages (2, 3, 22) and suggest that suppression of osteoclast bone resorption no longer ameliorates MMBP and that multiple myeloma cells predominantly contribute to MMBP at advanced stages.
Effects of inhibition of both osteoclasts and V-ATPase on MMBP
We therefore examined whether inhibition of H+ release via V-ATPase from JJN3 cells by Baf A1 alleviates MMBP that became refractory to analgesic effects of ZOL. As already shown in Figs. 2G and 6B, a single intraperitoneal injection of Baf A1 to untreated JJN3-bearing mice reduced MMBP (Fig. 7D, closed diamond). Of note, a single intraperitoneal injection of Baf A1 to ZOL-treated JJN3-bearing mice that lost responsiveness to analgesic effects of ZOL at day 35 (arrowhead in Fig. 7C) decreased mechanical allodynia (Fig. 7D, closed square) as early as 1 hour until 24 hours. Furthermore, the combination of Baf A1 and ZOL decreased pERK1/2 and pCREB expression in DRG more profoundly than did ZOL or Baf A1 alone (Fig. 7E). These results indicate that V-ATPase on multiple myeloma cells as well as osteoclasts contribute to SN excitation and MMBP at advanced stages of multiple myeloma.
We investigated the contributions of the acidic bone microenvironment associated with multiple myeloma colonization in bone to MMBP. We showed that bone-colonizing JJN3 cells release H+ via V-ATPase, creating an acidic bone microenvironment. This pathologic acidic microenvironment then stimulates the sprouting of CGRP+ SNs innervating bone and activates the acid-sensing nociceptors, ASIC3, on the CGRP+ SNs, which in turn excites the SNs to evoke MMBP. Blocking H+ release and development of the acidic bone microenvironment by the selective V-ATPase inhibitor, Baf A1, decreased the sprouting of CGRP+ SNs in bone, inhibited the excitation of the SNs, and reduced MMBP in JJN3-injected mice. Inhibiting H+-induced activation of ASIC3 by the specific ASIC3 antagonist, APETx2, inhibited SN excitation and alleviated MMBP. Furthermore, a combination of a single injection of Baf A1 and APETx2 showed greater and longer analgesic effects on MMBP than each agent alone. Taken together, these results indicate that secretion of H+ via V-ATPase on multiple myeloma cells and subsequent activation of SNs by H+ via ASCI3 are key steps responsible for inducing MMBP, and that these steps could be mechanism-based targets for the treatment of MMBP in patients.
Our results for the first time show that not only osteoclasts but also multiple myeloma cells directly contribute to SN activation to evoke MMBP through releasing H+ via V-ATPase. The result in part explains the observations that osteoclast-specific inhibitors alone reduce MMBP at early to advanced stages, however, fail to prevent the progression of MMBP at advanced to terminal stages of the disease (2, 20). Consistent with these observations, ZOL reduced MMBP at early stages but lost its analgesic effects at terminal stages in our animal model. Of importance, amelioration of MMBP was restored by additional administration of a single injection of Baf A1 to ZOL-treated mice, demonstrating that blockade of H+ release from multiple myeloma cells via suppression of V-ATPase mitigates MMBP refractory to the analgesic action of ZOL. These results suggest that administration of V-ATPase inhibitor combined with osteoclast inhibitor may be an effective approach for management of MMBP in patients with advanced multiple myeloma.
ZOL was shown to decrease tumor burden in preclinical models of multiple myeloma (48). Thus, alleviation of MMBP by ZOL in JJN3-injected mice could be secondary to decreased multiple myeloma tumor burden rather than inhibition of osteoclast bone resorption. However, clinical observations that ZOL fails to prevent the progression of MMBP at advanced stages of the disease (2, 20) together with our results suggest that the analgesic effects of ZOL are unlikely accounted by the inhibition of multiple myeloma growth.
Expression of the acid-sensing nociceptor TRPV1 was also increased on CGRP+ SNs in JJN3-injected mice. Previous studies reported that TRPV1 antagonists or disruption of the TRPV1 gene attenuated cancer-induced bone pain (28, 29). We also showed that a single intraplantar injection of the selective TRPV1 antagonist, SB366791, significantly decreased SN excitation and MMBP in JJN3-injected mice. TRPV1 on SNs is activated at pH <6.0 (27). The pHe of the acidic microenvironment created by JJN3 cells and bone-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 specific areas in JJN3-colonized bone may be <6.0, a pH at which TRPV1 is activated. We propose that the pHe of multiple myeloma–colonized bone varies spatially and temporally and induces activation of either ASIC3, TRPV1, or both on pH-sensitive SNs to induce MMBP, depending on the status of multiple myeloma progression and osteoclast bone resorption.
Although our results demonstrate significant contribution of bone-resorbing osteoclast to MMBP in JJN3-injected mice, osteoclast bone resorption that occurs under physiologic conditions rarely induces bone pain. As an explanation for this, recent studies reported that TRPV1 and ASIC3 dramatically decrease their threshold for sensing noxious stimuli in the presence of tissue damage and inflammation (18). Thus, it is speculated that SNs are not sensitive enough to be excited to evoke bone pain in response to acidic microenvironments created by physiologic osteoclast bone resorption. Furthermore, episodic musculoskeletal pain termed “growing pain” that often occurs in children in early childhood (49) suggests that bone turnover rate may be critical.
Solid tumors express various plasma membrane pH regulators, including V-ATPase, monocarboxylate transporter 1/4, and carbonic anhydrases (24) through which H+ are extruded to avoid intracellular acidification due to elevated aerobic glycolysis (Warburg effect; ref. 50) and acidify the extracellular microenvironment. Among these pH regulators, a3V-ATPase has 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 cells express V-ATPase was unknown. Here, we show that JJN3 cells express plasma membrane a3V-ATPase that acidifies extracellular environments by releasing H+ and directly contributes to SN activation to induce MMBP. However, whether a3V-ATPase expression is associated with multiple myeloma progression as is the case of solid tumors is unknown. Our results showed that a single injection of Baf A1 had no effects on JJN3 progression in bone and that Baf A1 did not inhibit JJN3 proliferation in culture, although long-term effects of Baf A1 on multiple myeloma progression were not examined in this study. Interestingly, however, CD138+ primary cells derived from the bone marrow 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 relationship between a3V-ATPase expression and multiple myeloma progression.
In conclusion, this study shows that the creation of acidic extracellular bone microenvironments by osteoclasts and multiple myeloma cells via H+ secretion through plasma membrane V-ATPase and responses of SNs innervating bone to the acidic microenvironment via ASIC3 are critical contributors to the pathophysiology of MMBP. Targeting these pathways may provide mechanism-based effective therapies for control of MMBP, which is currently undertreated.
Disclosure of Potential Conflicts of Interest
G.D. Roodman is a consultant/advisory board member for Amgen. No potential conflicts of interest were disclosed by the other authors.
Conception and design: M. Hiasa, F.A. White, T. Yoneda
Development of methodology: M. Hiasa, T. Okui, F.A. White, T. Yoneda
Acquisition 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. Yoneda
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Hiasa, T. Okui, Y.M. Allette, F.A. White, T. Yoneda
Writing, review, and/or revision of the manuscript: M. Hiasa, G.D. Roodman, T. Yoneda
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H. Wakabayashi
Study supervision: T. Yoneda
This study was supported by the Project Development Team within the ICTSI NIH/NCRR (#TR000006), the Indiana University Health Strategic Research Initiative in Oncology, and start-up fund of Indiana University School of Medicine (T. Yoneda) and Merit Review Funds from the Veterans Administration (G.D. Roodman), the NIH (#DK100905 to F.A. White), MERIT Review Award (#BX002209) from the U.S. Department of Veterans Affairs, Biomedical Laboratory Research and Development Service (F.A. White), grants from St. Vincent Indianapolis Hospital and the St. Vincent Foundation to F.A. White. Support for Y.M. Allette as an Indiana CTSI Predoctoral trainee was provided by UL1 (#TR001108), NIH/NCATS (A. Shekhar, principal investigator), and Japan Society for the Promotion of Science Grants-in-aid for Research Activity Start-up, and Postdoctoral Fellowship for Research Abroad (M. Hiasa).
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