Bone Marrow Microenvironment as a Regulator and Therapeutic Target for Prostate Cancer Bone Metastasis.

Bone is the most common site of prostate cancer metastasis. Once prostate cancer cells metastasize to bone, the mortality rate of prostate cancer patients increases significantly. Furthermore, bone metastases produce multiple skeletal complications, including bone pain that impairs the patients' quality of life. Effective therapies for bone metastatic disease are underdeveloped with most current therapies being primarily palliative with modest survival benefit. Although the exact mechanisms through which prostate cancer metastasizes to bone are unclear, growing evidence suggests that the bone marrow microenvironment, particularly its hematopoietic activity, is a significant mediator of prostate cancer bone tropism. Moreover, the bone microenvironment may regulate metastatic prostate cancer cells between dormant and proliferative states. In this review, we discuss (1) how prostate cancer cells interact with the bone microenvironment to establish bone metastases and (2) current and future potential treatments for prostate cancer patients with bone metastases.

Interactions Between Disseminated Tumor Cells and Bone Marrow Stromal Cells Regulate Tumor Dormancy.

PURPOSE OF REVIEW: To succinctly summarize recent findings concerning dormancy regulating interactions between bone marrow stromal cells and disseminated tumor cells. RECENT FINDINGS: Recent studies have highlighted roles of the bone marrow microenviroment, including osteoblasts, mesenchymal stem cells (MSCs), and endothelial cells, in inducing or maintaining cancer cell dormancy. Key pathways of interest include: osteoblast-induced transforming growth factor (TGF)-ß2 signaling, transfer of MSC-derived exosomes containing dormancy inducing microRNA, cancer cell cannibalism of MSCs, and endothelial cell secretion of thrombospondin 1 (TSP1). The bone marrow is a common site of metastatic disease recurrence following a period of cancer cell dormancy. Understanding why disseminated tumor cells enter into dormancy and later resume cell proliferation and growth is vital to developing effective therapeutics against these cells. The bone marrow stroma and the various pathways through which it participates in crosstalk with cancer cells are essential to furthering understanding of how dormancy is regulated.

Efficacy, safety and albuminuria-reducing effect of gemigliptin in Korean type 2 diabetes patients with moderate to severe renal impairment: A 12-week, double-blind randomized study (the GUARD Study).

AIMS: This multicentre, randomized, double-blind study investigated the efficacy and safety of gemigliptin in Korean type 2 diabetes mellitus (T2DM) patients with moderate to severe renal impairment (RI). METHODS: The study comprised a 12-week main part and a 40-week extension. We report here the results from the main part. In total, 132 patients were randomized to receive gemigliptin (n = 66) or placebo (n = 66). Changes in glycated haemoglobin (HbA1c; primary endpoint), other glycaemic control parameters (fasting plasma glucose, glycated albumin and fructosamine), lipid profiles, renal function parameters and safety profiles were evaluated. RESULTS: Baseline characteristics were comparable between the groups (mean HbA1c, 8.4% [68 mmol/mol]; age, 62.0 years; duration of type 2 diabetes, 16.3 years; estimated glomerular filtration rate, 33.3 mL/min/1.73 m2 ). At Week 12, the adjusted mean change ± standard error in HbA1c with gemigliptin was -0.82% ± 0.14% (-8.9 ± 1.5 mmol/mol), whereas it was 0.38% ± 0.14% (4.2 ± 1.5 mmol/mol) with placebo (significant between-group difference, P < .001). Other glycaemic control parameters showed beneficial changes as well. Body weight change (gemigliptin, -0.3 kg; placebo, -0.2 kg) was not significant. In the gemigliptin group, the mean decrease in urinary albumin creatinine ratio (UACR) was significant, both in patients with microalbuminuria (-41.9 mg/g creatinine, P = .03) and macroalbuminuria (-528.9 mg/g creatinine, P < .001). Drug-related adverse events were similar with gemigliptin and placebo (15% and 12%, respectively). CONCLUSIONS: A 12-week treatment with gemigliptin improved glycaemic control and provided UACR reduction in T2DM patients with moderate to severe RI. Gemigliptin was well tolerated, with no additional risk of hypoglycaemia and change in body weight.

Secretory factors released from high dose radiation-activated osteoclasts increase the expression level of pain-associated neuropeptides in sensory neuronal cultures.

Stereotactic Body Radiation Therapy for lung tumors near the chest wall often causes significant chest wall pain (CWP), negatively impacting patients' quality of life. The mechanisms behind SBRT-induced CWP remain unclear and may involve multiple factors. We investigated the potential crosstalk between radiation-activated osteoclasts and sensory neurons, focusing on osteoclast-derived factors in CWP. Using the murine pre-osteoclast cell line Raw264.7, we induced differentiation with RANKL, followed by 10Gy gamma-irradiation. Conditioned media from these irradiated osteoclasts was used to treat sensory neuronal cultures from mouse dorsal root ganglia. Neuronal cultures were also directly exposed to 10Gy radiation, with and without osteoclast co-culture. Analysis of osteoclast markers and pain-associated neuropeptides was conducted using RT-qPCR and histochemical staining. Osteoclast differentiation and activity were inhibited using Osteoprotegerin and risedronate. Results showed that high-dose radiation significantly increased osteoclast size, resorption pit size, and activity biomarkers. Neurons treated with CM from irradiated osteoclasts showed increased expression of pain-associated neuropeptides CGRP and Substance P, which was mitigated by osteoprotegerin and risedronate. This study suggests that high-dose radiation enhances osteoclast activity, upregulating pain-associated neuropeptides in sensory neurons, and that inhibitors like osteoprotegerin and risedronate may offer therapeutic strategies for managing radiation-induced pain.

Crosstalk between bone metastatic cancer cells and sensory nerves in bone metastatic progression.

Although the role of peripheral nerves in cancer progression has been appreciated, little is known regarding cancer/sensory nerve crosstalk and its contribution to bone metastasis and associated pain. In this study, we revealed that the cancer/sensory nerve crosstalk plays a crucial role in bone metastatic progression. We found that (i) periosteal sensory nerves expressing calcitonin gene-related peptide (CGRP) are enriched in mice with bone metastasis; (ii) cancer patients with bone metastasis have elevated CGRP serum levels; (iii) bone metastatic patient tumor samples express elevated calcitonin receptor-like receptor (CRLR, a CGRP receptor component); (iv) higher CRLR levels in cancer patients are negatively correlated with recurrence-free survival; (v) CGRP induces cancer cell proliferation through the CRLR/p38/HSP27 pathway; and (vi) blocking sensory neuron-derived CGRP reduces cancer cell proliferation in vitro and bone metastatic progression in vivo. This suggests that CGRP-expressing sensory nerves are involved in bone metastatic progression and that the CGRP/CRLR axis may serve as a potential therapeutic target for bone metastasis.

Clinical characteristics of defecation and micturition syncope compared with common vasovagal syncope.

BACKGROUND: Little is known about the clinical characteristics of patients with situational syncope such as defecation syncope (DS) or micturition syncope (MS) compared with those with common vasovagal syncope (VVS). METHODS: Among 680 consecutive patients, who underwent a head-up tilt test between January 2006 and November 2010, 282 patients (40.4±16.7 years; 48.6% men) diagnosed as DS (n = 38), MS (n = 38), or common VVS (n = 208) were included. RESULTS: Ages at diagnosis (38.7±17.3 vs 48.3±14.1 vs 42.0±13.8, P = 0.004) and the first syncope (33.7±18.4 vs 44.5±15.3 vs 37.5±14.6, P = 0.002) were significantly less in patients with common VVS than those with DS or MS, respectively. The patients with MS were more likely to be men (73.7%, P = 0.036), whereas patients with DS were more commonly women (73.7%). No sexual preference was observed in patients with common VVS. Body mass index was significantly lower (P = 0.047) and syncopal episodes were more recurrent (P = 0.049) in patients with common VVS than those with DS or MS. The frequency of drinking alcoholbefore syncope was significantly higher in patients with MS (39.5%, P < 0.001). CONCLUSIONS: DS tended to occur in older women, whereas MS tended to occur in middle-aged men and drinking alcohol was an important precipitating factor for MS. However, common VVS was observed more in a thin and young population, which was more recurrent compared with those situational syncopes.

Functional roles of neuromedin B and gastrin-releasing peptide in regulating itch and pain in the spinal cord of non-human primates.

Despite accumulating evidence in rodents, the functional role of neuromedin B (NMB) in regulating somatosensory systems in primate spinal cord is unknown. We aimed to compare the expression patterns of NMB and its receptor (NMBR) and the behavioral effects of intrathecal (i.t.) NMB with gastrin-releasing peptide (GRP) on itch or pain in non-human primates (NHPs). We used six adult rhesus monkeys. The mRNA or protein expressions of NMB, GRP, and their receptors were evaluated by quantitative reverse transcription polymerase chain reaction, immunohistochemistry, or in situ hybridization. We determined the behavioral effects of NMB or GRP via acute thermal nociception, capsaicin-induced thermal allodynia, and itch scratching response assays. NMB expression levels were greater than those of GRP in the dorsal root ganglia and spinal dorsal horn. Conversely, NMBR expression was significantly lower than GRP receptor (GRPR). I.t. NMB elicited only mild scratching responses, whereas GRP caused robust scratching responses. GRP- and NMB-elicited scratching responses were attenuated by GRPR (RC-3095) and NMBR (PD168368) antagonists, respectively. Moreover, i.t. NMB and GRP did not induce thermal hypersensitivity and GRPR and NMBR antagonists did not affect peripherally elicited thermal allodynia. Consistently, NMBR expression was low in both itch- and pain-responsive neurons in the spinal dorsal horn. Spinal NMB-NMBR system plays a minimal functional role in the neurotransmission of itch and pain in primates. Unlike the functional significance of the GRP-GRPR system in itch, drugs targeting the spinal NMB-NMBR system may not effectively alleviate non-NMBR-mediated itch.

Osteoblasts derived from mouse mandible enhance tumor growth of prostate cancer more than osteoblasts derived from long bone.

Prostate cancer (PCa) metastasizes to bone, where the bone marrow microenvironment controls disease progression. However, the cellular interactions that result in active bone marrow metastases are poorly understood. A better understanding of these interactions is critical to success in the pursuit of effective treatments for this life ending disease. Anecdotally, we observe that after intracardiac injection of PCa cells, one of the greatest tools to investigate the mechanisms of bone-metastatic disease, animals frequently present with mandible metastasis before hind limb metastasis. Therefore, in this study, we investigated whether the bone cells derived from the mouse mandible influence PCa progression differently than those from the hind limb. Interestingly, we found that osteoblasts harvested from mouse mandibles grew faster, expressed more vascular endothelial growth factor (VEGF), increased vascularity and formed more bone, and stimulated faster growth of PCa cells when cultured together than osteoblasts harvested from mouse hind limbs. Additionally, these findings were confirmed in vivo when mouse mandible osteoblasts were co-implanted into mice with PCa cells. Importantly, the enhancement of PCa growth mediated by mandible osteoblasts was not shown to be due to their differentiation or proliferation activities, but may be partly due to increased vascularization and expression of VEGF.

Activated mast cells in skeletal muscle can be a potential mediator for cancer-associated cachexia.

BACKGROUND: Eighty per cent of United States advanced cancer patients faces a worsened prognosis due to cancer-associated cachexia. Inflammation is one driver of muscle atrophy in cachexia, and skeletal muscle-resident immune cells could be a source of inflammation. This study explores the efficacy of cancer activated skeletal muscle-resident mast cells as a biomarker and mediator of cachexia. METHODS: Individual gene markers for immune cells were assessed in a publicly available colon carcinoma cohort of normal (n = 3), moderate cachexia (n = 3), and severe cachexia (n = 4) mice. Lewis lung carcinoma (LL/2) cells induced cachexia in C57BL/6 mice, and a combination of toluidine blue staining, immunofluorescence, quantitative polymerase chain reaction, and western blots measured innate immune cell expression in hind limb muscles. In vitro measurements included C2C12 myotube diameter before and after treatment with media from primary murine mast cells activated with LL/2 conditioned media. To assess translational potential in human samples, innate immune cell signatures were assessed for correlation with skeletal muscle atrophy and apoptosis, dietary excess, and cachexia signatures in normal skeletal muscle tissue. Gene set enrichment analysis was performed with innate immune cell signatures in publicly available cohorts for upper gastrointestinal (GI) cancer and pancreatic ductal adenocarcinoma (PDAC) patients (accession: GSE34111 and GSE130563, respectively). RESULTS: Individual innate immunity genes (TPSAB1 and CD68) showed significant increases in severe cachexia (weight loss > 15%) mice in a C26 cohort (GSE24112). Induction of cachexia in C57BL/6 mice with LL/2 subcutaneous injection significantly increased the number of activated skeletal muscle-resident degranulating mast cells. Murine mast cells activated with LL/2 conditioned media decreased C2C12 myotube diameter (P ≤ 0.05). Normal human skeletal muscle showed significant positive correlations between innate immune cell signatures and muscle apoptosis and atrophy, dietary excess, and cachexia signatures. The mast cell signature was up-regulated (positive normalized enrichment score and false discovery rate ≤ 0.1) in upper GI cachectic patients (n = 12) compared with control (n = 6), as well as in cachectic PDAC patients (n = 17) compared with control patients (n = 16). CONCLUSIONS: Activated skeletal muscle-resident mast cells are enriched in cachectic muscles, suggesting skeletal-muscle resident mast cells may serve as a biomarker and mediator for cachexia development to improve patient diagnosis and prognosis.

Usefulness of the measurement of neurite outgrowth of primary sensory neurons to study cancer-related painful complications.

Abnormal outgrowth of sensory nerves is one of the important contributors to pain associated with cancer and its treatments. Primary neuronal cultures derived from dorsal root ganglia (DRG) have been widely used to study pain-associated signal transduction and electrical activity of sensory nerves. However, there are only a few studies using primary DRG neuronal culture to investigate neurite outgrowth alterations due to underlying cancer-related factors and chemotherapeutic agents. In this study, primary DRG sensory neurons derived from mouse, non-human primate, and human were established in serum and growth factor-free conditions. A bovine serum albumin gradient centrifugation method improved the separation of sensory neurons from satellite cells. The purified DRG neurons were able to maintain their heterogeneous subpopulations, and displayed an increase in neurite growth when exposed to cancer-derived conditioned medium, while they showed a reduction in neurite length when treated with a neurotoxic chemotherapeutic agent. Additionally, a semi-automated quantification method was developed to measure neurite length in an accurate and time-efficient manner. Finally, these exogenous factors altered the gene expression patterns of murine primary sensory neurons, which are related to nerve growth, and neuro-inflammatory pain and nociceptor development. Together, the primary DRG neuronal culture in combination with a semi-automated quantification method can be a useful tool for further understanding the impact of exogenous factors on the growth of sensory nerve fibers and gene expression changes in sensory neurons.

Determining Competitive Potential of Bone Metastatic Cancer Cells in the Murine Hematopoietic Stem Cell Niche.

The ability of cancer cells to compete with hematopoietic stem cells (HSCs) to target the bone marrow microenvironment, or the HSC niche, during the dissemination process is critical for the development of bone metastasis. Here, we describe the methods for testing the relative potential of cancer cells to compete with HSCs for occupancy of the HSC niche by measuring the peripheral blood level of engrafted HSCs by flow cytometry in mice after bone marrow transplantation and tandem cancer cell inoculation. This method is useful for determining the molecular mechanisms for the roles of HSCs in the regulation of bone metastases.

Role of the Bone Microenvironment in the Development of Painful Complications of Skeletal Metastases.

Cancer-induced bone pain (CIBP) is the most common and painful complication in patients with bone metastases. It causes a significant reduction in patient quality of life. Available analgesic treatments for CIBP, such as opioids that target the central nervous system, come with severe side effects as well as the risk of abuse and addiction. Therefore, alternative treatments for CIBP are desperately needed. Although the exact mechanisms of CIBP have not been fully elucidated, recent studies using preclinical models have demonstrated the role of the bone marrow microenvironment (e.g., osteoclasts, osteoblasts, macrophages, mast cells, mesenchymal stem cells, and fibroblasts) in CIBP development. Several clinical trials have been performed based on these findings. CIBP is a complex and challenging condition that currently has no standard effective treatments other than opioids. Further studies are clearly warranted to better understand this painful condition and develop more effective and safer targeted therapies.

Adeno-associated virus serotype rh10 is a useful gene transfer vector for sensory nerves that innervate bone in immunodeficient mice.

Adeno-associated virus (AAV) is frequently used to manipulate gene expression in the sensory nervous system for the study of pain mechanisms. Although some serotypes of AAV are known to have nerve tropism, whether AAV can distribute to sensory nerves that innervate the bone or skeletal tissue has not been shown. This information is crucial, since bone pain, including cancer-induced bone pain, is an area of high importance in pain biology. In this study, we found that AAVrh10 transduces neurons in the spinal cord and dorsal root ganglia of immunodeficient mice with higher efficacy than AAV2, 5, 6, 8, and 9 when injected intrathecally. Additionally, AAVrh10 has tropism towards sensory neurons in skeletal tissue, such as bone marrow and periosteum, while it occasionally reaches the sensory nerve fibers in the mouse footpad. Moreover, AAVrh10 has higher tropic affinity to large myelinated and small peptidergic sensory neurons that innervate bone, compared to small non-peptidergic sensory neurons that rarely innervate bone. Taken together, these results suggest that AAVrh10 is a useful gene delivery vector to target the sensory nerves innervating bone. This finding may lead to a greater understanding of the molecular mechanisms of chronic bone pain and cancer-induced bone pain.

Elevated plasma osteopontin levels in patients with hepatocellular carcinoma.

OBJECTIVES: Osteopontin (OPN) is a secreted glycoprotein, frequently associated with various tumors. We investigated the usefulness of plasma OPN level as a biomarker for hepatocellular carcinoma (HCC). METHODS: We determined plasma levels of OPN, alpha-fetoprotein (AFP), and prothrombin induced by vitamin K absence II (PIVKA II) in a group of 62 HCC patients, in 60 patients with chronic liver diseases, and in 60 healthy control individuals using a standardized ELISA kit. To determine the source of elevated plasma level of OPN, immunohistochemical analysis of 285 HCC samples on tissue microarray was performed. RESULTS: Plasma OPN levels in the HCC patients (median 954 ng/mL, range 168-5,742) were significantly higher (p-value < 0.001) than those patients with chronic liver diseases (381 ng/mL, 29-1,688) or of a healthy control group (155 ng/mL, 10-766). Within the HCC patient group, plasma OPN level increased significantly with advancing degree of Child-Pugh class and of tumor stage. Diagnostic sensitivity and specificity of OPN for HCC was 87% and 82%, respectively (cut-off value: 617.6 ng/mL). OPN had a greater area under curve value (0.898) than AFP (0.745) or PIVKA II (0.578), suggesting superior diagnostic accuracy of OPN. Immunohistochemistry of 285 samples of HCC showed that OPN was expressed in 92 of 285 tumors (32.3%). OPN expression was found in the malignant hepatocytes and cancer-infiltrating macrophages, not in the noncancerous hepatocytes or Kupffer cells. CONCLUSIONS: These data propose elevated plasma OPN levels as a potential biomarker for HCC.