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.

PDF file - 115K, Cyclophosphamide treatment reduced body weight but did not cause systemic inflammation

PDF file - 129K, Cyclophosphamide suppressed PC-3 prostate tumor growth in the tibiae

Cancer-induced bone pain (CIBP) is the most common and devastating symptom of bone metastatic cancer that substantially disrupts patients’ quality of life. Currently, there are few effective analgesic treatments for CIBP other than opioids which come with severe side effects. In order to better understand the factors and mechanisms responsible for CIBP it is essential to have clinically relevant animal models that mirror pain-related symptoms and disease progression observed in patients with bone metastatic cancer.In the current study, we characterize a syngeneic mouse model of prostate cancer induced bone pain. We transfected a prostate cancer cell line (RM1) with green fluorescent protein (GFP) and luciferase reporters in order to visualize tumor growth longitudinally in vivo and to assess the relationship between sensory neurons and tumor cells within the bone microenvironment. Following intra-femoral injection of the RM1 prostate cancer cell line into male C57BL/6 mice, we observed a progressive increase in spontaneous guarding of the inoculated limb between 12 and 21 days post inoculation in tumor bearing compared to sham operated mice. Daily running wheel performance was evaluated as a measure of functional impairment and potentially movement evoked pain. We observed a progressive reduction in the distance traveled and percentage of time at optimal velocity between 12 and 21 days post inoculation in tumor bearing compared to sham operated mice. We utilized histological, radiographic and μCT analysis to examine tumor induced bone remodeling and observed osteolytic lesions as well as extra-periosteal aberrant bone formation in the tumor bearing femur, similar to clinical findings in patients with bone metastatic prostate cancer. Within the tumor bearing femur, we observed reorganization of blood vessels, macrophage and nerve fibers within the intramedullary space and periosteum adjacent to tumor cells. Tumor bearing mice displayed significant increases in the injury marker ATF3 and upregulation of the neuropeptides SP and CGRP in the ipsilateral DRG as well as increased measures of central sensitization and glial activation in the ipsilateral spinal cord. This immunocompetent mouse model will be useful when combined with cell type selective transgenic mice to examine tumor, immune cell and sensory neuron interactions in the bone microenvironment and their role in pain and disease progression associated with bone metastatic prostate cancer.

<p>PDF file - 157K, A single dose of cyclophosphamide did not induce vascular disruption in lung, liver and kidney</p>

BACKGROUND AND AIMS:Deficiency in the thiol transferase glutaredoxin 1 (Grx1) in aging mice promotes, in a sexually dimorphic manner, dysregulation of macrophages and atherogenesis. However, the underlying mechanisms are not known. Here we tested the hypothesis that macrophage-restricted overexpression of Grx1 protects atherosclerosis-prone mice against macrophage reprogramming and dysfunction induced by a high-calorie diet (HCD) and thereby reduces the severity of atherosclerosis.METHODS:We generated lentiviral vectors carrying cluster of differentiation 68 (CD68) promoter-driven enhanced green fluorescent protein (EGFP) or Grx1 constructs and conducted bone marrow (BM) transplantation studies to overexpress Grx1 in a macrophage-specific manner in male and female atherosclerosis-prone LDLR-/- mice, and fed these mice a HCD to induce atherogenesis. Atherosclerotic lesion size was determined in both the aortic root and the aorta. We isolated BM-derived macrophages (BMDM) to assess protein S-glutathionylation levels and loss of mitogen-activated protein kinase phosphatase 1 (MKP-1) activity as measures of HCD-induced thiol oxidative stress. We also conducted gene profiling on these BMDM to determine the impact of Grx1 activity on HCD-induced macrophage reprogramming.RESULTS:Overexpression of Grx1 protected macrophages against HCD-induced protein S-glutathionylation, reduced monocyte chemotaxis in vivo, limited macrophage recruitment into atherosclerotic lesions, and was sufficient to reduce the severity of atherogenesis in both male and female mice. Gene profiling revealed major sex differences in the transcriptional reprogramming of macrophages induced by HCD feeding, but Grx1 overexpression only partially reversed HCD-induced transcriptional reprogramming of macrophages.CONCLUSIONS:Macrophage Grx1 plays a major role in protecting mice atherosclerosis mainly by maintaining the thiol redox state of the macrophage proteome and preventing macrophage dysfunction.

Research indicates gender differences in functional performance at advanced ages, but little is known about their impact on longevity for men and women.To derive a set of motor function factors from a battery of functional performance measures and examine their associations with mortality, incorporating possible gender interactions.Analyses were performed on the longitudinal Swedish Adoption/Twin Study of Aging (SATSA) including twenty-four assessments of motor function up to six times over a 19-year period. Three motor factors were derived from several factor analyses; fine motor, balance/upper strength, and flexibility. A latent growth curve model was used to capture longitudinal age changes in the motor factors and generated estimates of intercept at age 70 (I), rates of change before (S1) and after age 70 (S2) for each factor. Cox regression models were used to determine how gender in interaction with the motor factors was related to mortality.Females demonstrated lower functional performance in all motor functions relative to men. Cox regression survival analyses demonstrated that both balance/upper strength, and fine motor function were significantly related to mortality. Gender specific analyses revealed that this was true for women only. For men, none of the motor factors were related to mortality.Women demonstrated more difficulties in all functioning facets, and only among women were motor functioning (balance/upper strength and fine motor function) associated with mortality. These results provide evidence for the importance of considering motor functioning, and foremost observed gender differences when planning for individualized treatment and rehabilitation.

Many types of solid tumors metastasize to the bone, where it causes significant morbidity and mortality in patients with advanced disease. Bone metastases are not only incurable but also affect bone health which impairs patients' quality of life. In order to understand the mechanisms and develop effective treatments for bone-metastatic disease, it is first necessary to develop animal models that permit the assessment of tumor growth in the bone and progressive structural changes of the bone simultaneously. Longitudinal analysis of bone tumor progression is generally performed by bioluminescent imaging; however, this method is not able to assess progressive structural changes of the bone. Here, we describe a simple method for assessment of bone lesions using a scoring system that takes into account disease burden and bone destruction using longitudinal radiographs.

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.

Abstract 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.

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.

More than 80% of patients with advanced prostate cancer (PCa) experience bone metastasis, which negatively impacts overall survival and patient quality of life. Various mouse models have been used to study the mechanisms of bone metastasis over the years; however, there is currently no model that fully recapitulates what happens in humans because bone metastasis rarely occurs in spontaneous PCa mouse models. Nevertheless, animal models of bone metastasis using several different tumor inoculation routes have been developed to help study bone metastatic progression, which occurs particularly in late-stage PCa patients. This chapter describes the protocols commonly used to develop models of bone metastatic cancer in mice using different percutaneous injection methods (Intracardiac and Intraosseous). These models are useful for understanding the molecular mechanisms of bone metastatic progression, including tumor tissue tropism and tumor growth within the bone marrow microenvironment. Better understanding of the mechanisms involved in these processes will clearly lead to the development of new therapeutic strategies for PCa patients with bone 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.

To succinctly summarize recent findings concerning dormancy regulating interactions between bone marrow stromal cells and disseminated tumor cells.

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.

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.