Single-cell Transcriptome Analysis Identifies Senescent Osteocytes as Contributors to Bone Destruction in Breast Cancer Metastasis.

Breast cancer bone metastases increase fracture risk and are a major cause of morbidity and mortality among women. Upon colonization by tumor cells, the bone microenvironment undergoes profound reprogramming to support cancer progression that disrupts the balance between osteoclasts and osteoblasts, leading to bone lesions. Whether such reprogramming affects matrix-embedded osteocytes remains poorly understood. Here, we demonstrate that osteocytes in breast cancer bone metastasis develop premature senescence and a distinctive senescence-associated secretory phenotype (SASP) that favors bone destruction. Single-cell RNA sequencing identified osteocytes from mice with breast cancer bone metastasis enriched in senescence and SASP markers and pro-osteoclastogenic genes. Using multiplex in situ hybridization and AI-assisted analysis, we detected osteocytes with senescence-associated distension of satellites, telomere dysfunction, and p16Ink4a expression in mice and patients with breast cancer bone metastasis. In vitro and ex vivo organ cultures showed that breast cancer cells promote osteocyte senescence and enhance their osteoclastogenic potential. Clearance of senescent cells with senolytics suppressed bone resorption and preserved bone mass in mice with breast cancer bone metastasis. These results demonstrate that osteocytes undergo pathological reprogramming by breast cancer cells and identify osteocyte senescence as an initiating event triggering bone destruction in breast cancer metastases.

Case report: Death caused by multi-organ metastatic calcifications as a result of intramuscular injections with paraffin oil.

In this forensic case report, we present autopsy findings from a young male in his thirties who had been self-injecting paraffin oil into his upper extremities 8 years prior to death. The injections induced an inflammatory response, leading to granuloma formation. This, in turn, resulted in severe hypercalcemia. The external autopsy examination revealed gross macroscopic ulcerations and enlargement of upper extremities, while calcifications of ligaments, heart, kidneys and dura mater was revealed on postmortem CT-scans. Histopathological examination showed extensive multiorgan metastatic calcifications in several tissues including the lungs, heart and kidney. Cause of death was estimated to be the extensive calcific deposits in the heart likely resulting in cardiac arrest. To our knowledge this is the first case reporting findings from an autopsy in which the cause of death was linked to cosmetic oil injections.

Bone marrow adipocytes provide early sign for progression from MGUS to multiple myeloma.

Multiple Myeloma (MM) is the second most common hematological malignancy and is characterized by clonal expansion of malignant plasma cells in the bone marrow. In spite of recent advances in the field of MM, the disease has remained incurable. MM is preceded by a premalignant state known as monoclonal gammopathy of undetermined significance (MGUS), with a risk of progression to MM of 1% per year. Establishing a scalable approach that refines the identification of MGUS patients at high risk of progression to MM can transform the clinical management of the disease, improve the patient's quality of life, and will have significant socioeconomic implications. Here, we provide evidence that changes in the bone marrow adipose tissue (BMAT) provide an early sign for progression from MGUS to MM. We employed AI-assisted histological analysis of unstained bone marrow biopsies from MGUS subjects with or without progression to MM within 10 years (n = 24, n = 17 respectively). Although the BMAT fraction was not different between the two groups, bone marrow adipocyte (BMAd) density was decreased in MGUS patients who developed MM, compared to non-progressing MGUS patients. Importantly, the distribution profile for BMAd size and roundness was significantly different between the two groups, indicating a shift toward increased BMAd size and roundness in MGUS patients who developed MM. These early changes in the BMAT could serve as valuable early indicators for the transition from MGUS to MM, potentially enabling timely interventions and personalized treatment strategies. Finally, the AI-based approach for histological characterization of unstained bone marrow biopsies is cost-effective and fast, rendering its clinical implementation feasible.

Novel Developments in the Treatment of Multiple Myeloma-Associated Bone Disease.

Osteolytic bone disease is present in about 80% of patients with multiple myeloma at the time of diagnosis. Managing bone disease in patients with multiple myeloma is a challenge and requires a multi-faceted treatment approach with medication, surgery, and radiation. The established treatments with intravenous or subcutaneous antiresorptives can cause debilitating adverse events for patients, mainly osteonecrosis of the jaw, which, traditionally, has been difficult to manage. Now, oral surgery is recommended and proven successful in 60-85% of patients. Patients with spinal involvement may benefit from surgery in the form of vertebroplasty and kyphoplasty for pain relief, improved mobility, and reestablished sagittal balance, as well as the restoration of vertebral height. These procedures are considered safe, but the full therapeutic impact needs to be investigated further. Ixazomib, the first oral proteasome inhibitor, increases osteoblast differentiation, and recently published preliminary results in patients treated with Ixazomib maintenance have promisingly shown increased trabecular volume caused by prolonged bone formation activity. Other novel potential treatment strategies are discussed as well.

Disturbed bone marrow adiposity in patients with Cushing's syndrome and glucocorticoid- and postmenopausal- induced osteoporosis.

Background: Skeletal stem/progenitor cells (SSPCs) in the bone marrow can differentiate into osteoblasts or adipocytes in response to microenvironmental signalling input, including hormonal signalling. Glucocorticoids (GC) are corticosteroid hormones that promote adipogenic differentiation and are endogenously increased in patients with Cushing´s syndrome (CS). Here, we investigate bone marrow adiposity changes in response to endogenous or exogenous GC increases. For that, we characterize bone biopsies from patients with CS and post-menopausal women with glucocorticoid-induced osteoporosis (GC-O), compared to age-matched controls, including postmenopausal osteoporotic patients (PM-O). Methods: Transiliac crest bone biopsies from CS patients and healthy controls, and from postmenopausal women with GC-O and matched controls were analysed; an additional cohort included biopsies from women with PM-O. Plastic-embedded biopsies were sectioned for histomorphometric characterization and quantification of adipocytes. The fraction of adipocyte area per tissue (Ad.Ar/T.Ar) and marrow area (Ad.Ar/Ma.Ar), mean adipocyte profile area (Ad.Pf.Ar) and adipocyte profile density (N.Ad.Pf/Ma.Ar) were determined and correlated to steroid levels. Furthermore, the spatial distribution of adipocytes in relation to trabecular bone was characterized and correlations between bone marrow adiposity and bone remodeling parameters investigated. Results: Biopsies from patients with CS and GC-O presented increased Ad.Ar/Ma.Ar, along with adipocyte hypertrophy and hyperplasia. In patients with CS, both Ad.Ar/Ma.Ar and Ad.Pf.Ar significantly correlated with serum cortisol levels. Spatial distribution analyses revealed that, in CS, the increase in Ad.Ar/Ma.Ar near to trabecular bone (<100 µm) was mediated by both adipocyte hypertrophy and hyperplasia, while N.Ad.Pf/Ma.Ar further into the marrow (>100 µm) remained unchanged. In contrast, patients with GC-O only presented increased Ad.Ar/Ma.Ar and mean Ad.Pf.Ar>100 µm from trabecular bone surface, highlighting the differential effect of increased endogenous steroid accumulation. Finally, the Ad.Ar/Ma.Ar and Ad.Ar/T.Ar correlated with the canopy coverage above remodeling events. Conclusion: Increased cortisol production in patients with CS induces increased bone marrow adiposity, primarily mediated by adipocyte hypertrophy. This adiposity is particularly evident near trabecular bone surfaces, where hyperplasia also occurs. The differential pattern of adiposity in patients with CS and GC-O highlights that bone marrow adipocytes and their progenitors may respond differently in these two GC-mediated bone diseases.

Metastatic Infiltration of Nervous Tissue and Periosteal Nerve Sprouting in Multiple Myeloma-Induced Bone Pain in Mice and Human.

Multiple myeloma (MM) is a neoplasia of B plasma cells that often induces bone pain. However, the mechanisms underlying myeloma-induced bone pain (MIBP) are mostly unknown. Using a syngeneic MM mouse model, we show that periosteal nerve sprouting of calcitonin gene-related peptide (CGRP+) and growth associated protein 43 (GAP43+) fibers occurs concurrent to the onset of nociception and its blockade provides transient pain relief. MM patient samples also showed increased periosteal innervation. Mechanistically, we investigated MM induced gene expression changes in the dorsal root ganglia (DRG) innervating the MM-bearing bone of male mice and found alterations in pathways associated with cell cycle, immune response and neuronal signaling. The MM transcriptional signature was consistent with metastatic MM infiltration to the DRG, a never-before described feature of the disease that we further demonstrated histologically. In the DRG, MM cells caused loss of vascularization and neuronal injury, which may contribute to late-stage MIBP. Interestingly, the transcriptional signature of a MM patient was consistent with MM cell infiltration to the DRG. Overall, our results suggest that MM induces a plethora of peripheral nervous system alterations that may contribute to the failure of current analgesics and suggest neuroprotective drugs as appropriate strategies to treat early onset MIBP.SIGNIFICANCE STATEMENT Multiple myeloma (MM) is a painful bone marrow cancer that significantly impairs the quality of life of the patients. Analgesic therapies for myeloma-induced bone pain (MIBP) are limited and often ineffective, and the mechanisms of MIBP remain unknown. In this manuscript, we describe cancer-induced periosteal nerve sprouting in a mouse model of MIBP, where we also encounter metastasis to the dorsal root ganglia (DRG), a never-before described feature of the disease. Concomitant to myeloma infiltration, the lumbar DRGs presented blood vessel damage and transcriptional alterations, which may mediate MIBP. Explorative studies on human tissue support our preclinical findings. Understanding the mechanisms of MIBP is crucial to develop targeted analgesic with better efficacy and fewer side effects for this patient population.

Increased Bone Volume by Ixazomib in Multiple Myeloma: 3-Month Results from an Open Label Phase 2 Study.

Multiple myeloma (MM) is an incurable bone marrow cancer characterized by the development of osteolytic lesions due to the myeloma-induced increase in osteoclastogenesis and decrease in osteoblastic activity. The standard treatment of MM often involves proteasome inhibitors (PIs), which can also have a beneficial off-target bone anabolic effect. However, long-term treatment with PIs is unadvised due to their high side-effect burden and inconvenient route of administration. Ixazomib is a new-generation, oral PI that is generally well tolerated; however, its bone effect remains unknown. Here, we describe the 3-month results of a single-center phase II clinical trial investigating the effect of ixazomib treatment on bone formation and bone microstructure. Thirty patients with MM in stable disease not receiving antimyeloma treatment for ≥3 months and presenting ≥2 osteolytic lesions received monthly ixazomib treatment cycles. Serum and plasma samples were collected at baseline and monthly thereafter. Sodium 18 F-Fluoride positron emission tomography (NaF-PET) whole-body scans and trephine iliac crest bone biopsies were collected before and after three treatment cycles. The serum levels of bone remodeling biomarkers suggested an early ixazomib-induced decrease in bone resorption. NaF-PET scans indicated unchanged bone formation ratios; however, histological analyses of bone biopsies revealed a significant increase in bone volume per total volume after treatment. Further analyses of bone biopsies showed unchanged osteoclast number and COLL1A1High -expressing osteoblasts on bone surfaces. Next, we analyzed the superficial bone structural units (BSUs), which represent each recent microscopic bone remodeling event. Osteopontin staining revealed that following treatment, significantly more BSUs were enlarged (>200,000 µm2 ), and the distribution frequency of their shape was significantly different from baseline. Overall, our data suggest that ixazomib induces overflow remodeling-based bone formation by decreasing the level of bone resorption and promoting longer bone formation events, making it a potentially valuable candidate for future maintenance treatment. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Bone Pain in Multiple Myeloma (BPMM)-A Protocol for a Prospective, Longitudinal, Observational Study.

Multiple myeloma (MM) is a bone marrow neoplasia that causes bone pain in 70% patients. While preclinical models of MM have suggested that both nerve sprouting and nerve injury may be causative for the pain, there is a lack of clinical data. Thus, the primary aims of this clinical study are: (1) to provide a deep characterization of the subjective experience of pain and quality of life in MM patients; (2) to investigate disturbances in the bone innervation of MM patients. Secondary aims include exploring correlations between pain and serum inflammatory and bone turnover biomarkers. In a prospective, observational study (clinicaltrials.gov: NCT04273425), patients with suspected MM requiring a diagnostic iliac crest biopsy at Sheffield Teaching Hospital (UK) are invited to participate. Consenting patients answer seven standardized questionnaires assessing pain, quality of life and catastrophizing. Bone turnover biomarkers and inflammatory cytokines are measured in fasting serum samples, and bone innervation is evaluated in diagnostic biopsies. MM patients are invited to a follow-up upon completion of first line treatment. This will be the first deep characterization of pain in MM patients and its correlation with disturbances in bone innervation. Understanding how bone turnover and inflammation correlate to pain in MM is crucial to identify novel analgesic targets for this condition.

The nociceptin/orphanin FQ receptor system as a target to alleviate cancer-induced bone pain in rats: Model validation and pharmacological evaluation.

BACKGROUND AND PURPOSE: Cancer-induced bone pain remains inadequately controlled, and current standard of care analgesics is accompanied by several side effects. Nociceptin/orphanin FQ peptide (NOP) receptor agonists have demonstrated broad analgesic properties in rodent neuropathic and inflammatory pain models. Here, we investigate the analgesic potential of NOP receptor activation in a rodent cancer-induced bone pain model. EXPERIMENTAL APPROACH: Model validation by intratibial inoculation in male Sprague Dawley rats was performed with varying MRMT-1/Luc2 cell quantities (0.5-1.5 × 106 ·ml-1 ) and a behavioural battery (>14 days post-surgery) including evoked and non-evoked readouts: paw pressure test, cold plate, von Frey, open field, and weight distribution. Anti-allodynic potential of the endogenous NOP receptor ligand nociceptin (i.t.) and NOP receptor agonist Ro65-6570 ( i.p.) was tested using von Frey filaments, followed by a combination experiment with Ro65-6570 and the NOP receptor antagonist J-113397 (i.p.). Plasma cytokine levels and NOP receptor gene expression in dorsal root ganglion (DRG, L4-L6) and bone marrow were examined. KEY RESULTS: Inoculation with 1.5 × 106 ·ml-1 of MRMT-1/Luc2 cells resulted in a robust and progressive pain-related phenotype. Nociceptin and Ro65-6570 treatment inhibited cancer-induced mechanical allodynia. J-113397 selectively antagonized the effect of Ro65-6570. MRMT-1/Luc2-bearing animals demonstrated elevated plasma cytokine levels of IL-4, IL-5, IL-6 and IL-10 plus unaltered NOP-r gene expression in DRG and reduced expression in bone marrow. CONCLUSION AND IMPLICATIONS: Nociceptin and Ro65-6570 selectively and dose-dependently reversed cancer-induced bone pain-like behaviour. The NOP receptor system may be a potential target for cancer-induced bone pain treatment. LINKED ARTICLES: This article is part of a themed issue on The molecular pharmacology of bone and cancer-elated bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc.

Multiple myeloma-A painful disease of the bone marrow.

Multiple myeloma is a bone marrow neoplasia with an incidence of 6/100,000/year in Europe. While the disease remains incurable, the development of novel treatments such as autologous stem cell transplantation, proteasome inhibitors and monoclonal antibodies has led to an increasing subset of patients living with long-term myeloma. However, more than two thirds of patients suffer from bone pain, often described as severe, and knowledge on the pain mechanisms and its effect on their health-related quality of life (HRQoL) is limited. In this review, we discuss the mechanisms of myeloma bone disease, the currently available anti-myeloma treatments and the lessons learnt from clinical studies regarding HRQoL in myeloma patients. Moreover, we discuss the mechanisms of cancer-induced bone pain and the knowledge that animal models of myeloma-induced bone pain can provide to identify novel analgesic targets. To date, information regarding bone pain and HRQoL in myeloma patients is still scarce and an effort should be made to use standardised questionnaires to assess patient-reported outcomes that allow inter-study comparisons of the available clinical data.

Modulation of Rat Cancer-Induced Bone Pain is Independent of Spinal Microglia Activity.

The dissemination of cancer to bone can cause significant cancer-induced bone pain (CIBP), severely impairing the patient's quality of life. Several rodent models have been developed to explore the nociceptive mechanisms of CIBP, including intratibial inoculation of breast carcinoma cells in syngeneic Sprague Dawley rats. Using this model, we investigated whether resident spinal microglial cells are involved in the transmission and modulation of CIBP, a long-debated disease feature. Immunohistochemical staining of ionizing calcium-binding adaptor molecule 1 (Iba-1) and phosphorylated p38-mitogen-activated protein kinase (P-p38 MAPK) showed no spinal microglial reaction in cancer-bearing rats, independently of disease stage, sex, or carcinoma cell line. As a positive control, significant upregulation of both Iba-1 and P-p38 was observed in a rat model of neuropathic pain. Additionally, intrathecal administration of the microglial inhibitor minocycline did not ameliorate pain-like behaviors in cancer-bearing rats, in contrast to spinal morphine administration. Our results indicate that microglial reaction is not a main player in CIBP, adding to the debate that even within the same models of CIBP, significant variations are seen in disease features considered potential drug targets. We suggest that this heterogeneity may reflect the clinical landscape, underscoring the need for understanding the translational value of CIBP models.

Hypophosphatemic Hypovitaminosis D Induces Osteomalacia in the Adult Female Rat.

Osteomalacia is a bone-demineralizing disease of adulthood, often caused by hypovitaminosis D. Current animal models of the disease mimic osteomalacia as a consequence of gastric bypass or toxic exposure to metals, but a relevant model of diet-induced osteomalacia is lacking. For that purpose, 7-month-old female Sprague Dawley rats were randomly assigned into 2 weight-stratified groups and maintained for 4 months on synthetic diets containing negligible or normal levels of vitamin D. The dietary regimen resulted in vitamin D deficiency as measured by 25-hydroxyvitamin D serum levels; however, hypovitaminosis D per se did not affect biomarkers of calcium metabolism and bone turnover, nor did it result in increased osteoid. Thus, vitamin D depletion through the diet was found to be insufficient to induce an osteomalacia-like phenotype in the adult rat. After 4 months, the phosphate content of the vitamin D-depleted diet had decreased to 0.16% (calcium:phosphorus ratio of 5.85), resulting in an osteomalacic-like condition (trabecular osteoid surface/bone surface constituted 33%; CI, 26-40). The diet change also affected both metabolic and bone turnover biomarkers, including significantly suppressing serum fibroblast growth factor 23. Furthermore, decreased dietary phosphate in a vitamin D-depleted diet led to microarchitectural changes of trabecular and cortical bone, lower bone mass density, lower bone mass content and decreased bone strength, all indicating reduced bone quality. Taken together, our results show that osteomalacia can be induced in the adult female rat by depleting vitamin D and lowering phosphate content in the diet.

Differential Pain-Related Behaviors and Bone Disease in Immunocompetent Mouse Models of Myeloma.

Bone pain is a serious and debilitating symptom of multiple myeloma (MM) that impairs the quality of life of patients. The underlying mechanisms of the pain are unknown and understudied, and there is a need for immunocompetent preclinical models of myeloma-induced bone pain. The aim of this study was to provide the first in-depth behavioral characterization of an immunocompetent mouse model of MM presenting the clinical disease features: osteolytic bone disease and bone pain. We hypothesized that a widely used syngeneic model of MM, established by systemic inoculation of green fluorescent protein-tagged myeloma cells (5TGM1-GFP) in immunocompetent C57Bl/KaLwRijHsd (BKAL) mice, would present pain-related behaviors. Disease phenotype was confirmed by splenomegaly, high serum paraprotein, and tumor infiltration in the bone marrow of the hind limbs; however, myeloma-bearing mice did not present pain-related behaviors or substantial bone disease. Thus, we investigated an alternative model in which 5TGM1-GFP cells were directly inoculated into the intrafemoral medullary cavity. This localized myeloma model presented the hallmarks of the disease, including high serum paraprotein, tumor growth, and osteolytic bone lesions. Compared with control mice, myeloma-bearing mice presented myeloma-induced pain-related behaviors, a phenotype that was reversed by systemic morphine treatment. Micro-computed tomography analyses of the myeloma-inoculated femurs showed bone disease in cortical and trabecular bone. Repeated systemic bisphosphonate treatment induced an amelioration of the nociceptive phenotype, but did not completely reverse it. Furthermore, intrafemorally injected mice presented a profound denervation of the myeloma-bearing bones, a previously unknown feature of the disease. This study reports the intrafemoral inoculation of 5TGM1-GFP cells as a robust immunocompetent model of myeloma-induced bone pain, with consistent bone loss. Moreover, the data suggest that myeloma-induced bone pain is caused by a combinatorial mechanism including osteolysis and bone marrow denervation. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Cancer-induced Bone Pain Impairs Burrowing Behaviour in Mouse and Rat.

BACKGROUND: Cancer-induced bone pain remains a serious public health concern, with a need for translational behavioural tests in order to assess nociception in preclinical models of this condition. Burrowing is an innate, ethologically relevant rodent behaviour that has been proven sensitive to chronic pain conditions. Herein, we studied for the first time whether burrowing performance is altered in preclinical models of cancer-induced bone pain. MATERIALS AND METHODS: Mice and rats were inoculated with syngeneic breast cancer cells. Bone degradation was radiographically evaluated and nociception was assessed in limb-use and burrowing tests. RESULTS: Cancer-bearing rodents showed reduced relative bone density and limb-use scores, confirming disease development. Burrowing performance decreased over time in both rodent models. CONCLUSION: Burrowing performance was reduced in both rodent models, indicating that the burrowing test is a relevant and reproducible behavioural test for assessing disease development in both mouse and rat models of cancer-induced bone pain.

Neuropeptide Y is Up-regulated and Induces Antinociception in Cancer-induced Bone Pain.

Pain remains a major concern in patients suffering from metastatic cancer to the bone and more knowledge of the condition, as well as novel treatment avenues, are called for. Neuropeptide Y (NPY) is a highly conserved peptide that appears to play a central role in nociceptive signaling in inflammatory and neuropathic pain. However, little is known about the peptide in cancer-induced bone pain. Here, we evaluate the role of spinal NPY in the MRMT-1 rat model of cancer-induced bone pain. Our studies revealed an up-regulation of NPY-immunoreactivity in the dorsal horn of cancer-bearing rats 17 days after inoculation, which could be a compensatory antinociceptive response. Consistent with this interpretation, intrathecal administration of NPY to rats with cancer-induced bone pain caused a reduction in nociceptive behaviors that lasted up to 150 min. This effect was diminished by both Y1 (BIBO3304) and Y2 (BIIE0246) receptor antagonists, indicating that both receptors participate in mediating the antinociceptive effect of NPY. Y1 and Y2 receptor binding in the spinal cord was unchanged in the cancer state as compared to sham-operated rats, consistent with the notion that increased NPY results in a net antinociceptive effect in the MRMT-1 model. In conclusion, the data indicate that NPY is involved in the spinal nociceptive signaling of cancer-induced bone pain and could be a new therapeutic target for patients with this condition.