Intravital bone imaging by two-photon excitation microscopy to identify osteocytic osteolysis in vivo

Sano, Hideto; Kikuta, Junichi; Furuya, Masayuki; Kondo, Naoki; Endo, Naoto; Ishii, Masaru

doi:10.1016/j.bone.2015.01.013

Cited by 41 publications

(45 citation statements)

References 31 publications

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“…OCLs express a3V-ATPase on their plasma membrane in the ruffled border (15,47) through which H + are secreted during bone resorption, creating an acidic extracellular microenvironment (15–17). Histological examination of bone of JJN3-injected mice showed that the pits formed by TRAP + OCLs on endosteal bone surfaces (Figure 7A, top) were acidic (Figure 7A, bottom, arrowheads), and that CGRP + SNs innervated in close proximity to TRAP + OCLs (Figure 7B).…”

Section: Resultsmentioning

confidence: 99%

“…TRPV1 on SNs is activated at pH < 6.0 (27). The pH e of the acidic microenvironment created by JJN3 cells and bone-resorbing OCLs is 6.5–6.9 (Figure 3) and 4.5–6.0 (15–17), respectively. These results suggest that the pH e of specific areas in JJN3-colonized bone may be < 6.0, a pH at which TRPV1 is activated.…”

Section: Discussionmentioning

confidence: 99%

“…Osteoclasts (OCLs) 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) (16,17). H + are a well-known potent pain-inducing mediator (18,19), suggesting that the acidic microenvironment created by increased H + release from bone-resorbing OCLs via the a3V-ATPase activates pH-sensitive SNs to elicit bone pain.…”

Section: Introductionmentioning

confidence: 99%

“…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) (18). TRPV1 is activated by pH values < 6.0 (27), equivalent to the pH under the OCL ruffled border (15–17). Previous studies reported that TRPV1 plays a critical role in cancer-associated bone pain (28,29).…”

Section: Introductionmentioning

confidence: 99%

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Bone Pain Induced by Multiple Myeloma Is Reduced by Targeting V-ATPase and ASIC3

et al. 2017

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Multiple myeloma (MM) patients experience severe bone pain (MMBP) that is undertreated and poorly understood. In this study, we studied MMBP in an intratibial mouse xenograft model which employs JJN3 human MM 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+ SN 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 co-cultured with JJN3 cells showed increased neurite outgrowth and excitation inhibited by bafilomycin A1 or APETx2. Further, combining APETx2 with bafilomycin A1 reduced MMBP to a greater extent than either agent alone. Lastly, 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 MM cooperate to induce an acidic bone microenvironment that evokes MMBP as a result of the excitation of ASIC3-activated sensory neurons. Further, they present a mechanistic rationale for targeting ASIC3 on neurons along with the MM-induced acidic bone microenvironment as a strategy to relieve MMBP in patients.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Bone Pain Induced by Multiple Myeloma Is Reduced by Targeting V-ATPase and ASIC3

et al. 2017

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“…As bone structures heavily scatter lights and the high collagen content generates second-harmonic signals (SHG), these advantages won two-photon microscopy increasing popularity in research of cellular activities and interactions within bone and marrow, particularly in identifying the haematopoietic stem cell niche and detecting bone metastasis-initiating cancer cells in bone. [17][18][19][20][21][22][23] In this article, using the detection of breast cancer cell bone colonisation by confocal and two-photon microscopy as a representative example, we will describe a step-by-step methodology, from sample preparation to data analyses, used to investigate cellular events in frozen and fixed/decalcified mouse bone samples ex vivo (see schematic outline, Figure 2). Advantages and limitations of this technology is also discussed to guide the reader as to which is the most appropriate for their research question.…”

Section: Introductionmentioning

confidence: 99%

Confocal/two-photon microscopy in studying colonisation of cancer cells in bone using xenograft mouse models

Allocca¹,

Kusumbe²,

Ramasamy³

et al. 2016

BoneKEy Reports

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Confocal and two-photon microscopy has been widely used in bone research to not only produce high quality, three-dimensional images but also to provide valuable structural and quantitative information. In this article, we describe step-by-step protocols for confocal and two-photon microscopy to investigate earlier cellular events during colonisation of cancer cells in bone using xenograft mouse models. This includes confocal/two-photon microscopy imaging of paraformaldehyde fixed thick bone sections and frozen bone samples.

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An Acid‐Activatable Fluorescence Probe for Imaging Osteocytic Bone Resorption Activity in Deep Bone Cavities

et al. 2020

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A rationally designed pH‐activatable fluorescent probe (pHocas‐RIS) has been used to measure localised pH levels in osteocytic lacunae in bone tissue. Conjugation of the moderate bone‐binding drug risedronate to a pH‐activatable BODIPY fluorophore enables the probe to penetrate osteocytic lacunae cavities that are embedded deep within the bone matrix. After injection of pHocas‐RIS, any osteocytic lacunae caused by bone‐resorbing osteocytes cause the probe to fluoresce in vivo, thus allowing imaging by intravital two‐photon excitation microscopy. This pH responsive probe enabled the visualization of the bone mineralizing activities of acid producing osteocytes in real time, thus allowing the study of their central role in remodeling the bone‐matrix in healthy and disease states.

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Intravital bone imaging by two-photon excitation microscopy to identify osteocytic osteolysis in vivo

Cited by 41 publications

References 31 publications

Bone Pain Induced by Multiple Myeloma Is Reduced by Targeting V-ATPase and ASIC3

Bone Pain Induced by Multiple Myeloma Is Reduced by Targeting V-ATPase and ASIC3

Confocal/two-photon microscopy in studying colonisation of cancer cells in bone using xenograft mouse models

An Acid‐Activatable Fluorescence Probe for Imaging Osteocytic Bone Resorption Activity in Deep Bone Cavities

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