De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis.

Osteoclasts play a central role in cancer-cell-induced osteolysis, but the molecular mechanisms of osteoclast activation during bone metastasis formation are incompletely understood. By performing RNA sequencing on a mouse breast carcinoma cell line with higher bone-metastatic potential, here we identify the enzyme CYP11A1 strongly upregulated in osteotropic tumor cells. Genetic deletion of Cyp11a1 in tumor cells leads to a decreased number of bone metastases but does not alter primary tumor growth and lung metastasis formation in mice. The product of CYP11A1 activity, pregnenolone, increases the number and function of mouse and human osteoclasts in vitro but does not alter osteoclast-specific gene expression. Instead, tumor-derived pregnenolone strongly enhances the fusion of pre-osteoclasts via prolyl 4-hydroxylase subunit beta (P4HB), identified as a potential interaction partner of pregnenolone. Taken together, our results demonstrate that Cyp11a1-expressing tumor cells produce pregnenolone, which is capable of promoting bone metastasis formation and osteoclast development via P4HB.

Immunotherapy targeting PD­1/PD­L1: A potential approach for the treatment of cancer bone metastases (Review).

Immune checkpoint molecules, such as programmed cell death 1 (PD­1) and programmed cell death ligand 1 (PD­L1), have a critical role in regulating immune responses, including in tumor tissues. Monoclonal antibodies against these molecules, known as immune checkpoint inhibitors (ICIs), have been shown to be effective against a variety of cancers; however, significant patient populations are resistant to such treatment. Clinical studies to date have shown that ICIs are less effective in cancer patients with bone metastases. The effect of anti­PD­1/PD­L1 antibodies on bone metastases, as assessed by the bone metastasis­specific response classification criteria, was relatively low. In addition, the presence of bone metastases showed a trend toward worse progression­free survival and overall survival in cancer patients treated with ICIs. To improve the efficacy of ICIs in bone metastases, several combination therapies are under investigation and certain studies have reported better responses. The present review summarizes the current understanding of the effects of anti­PD­1/PD­L1 antibodies on bone metastases based on the reported clinical and preclinical studies.

Primary tumor-induced immunity suppresses bone metastases of breast cancer in syngeneic immunocompetent mouse models.

The immune system plays a crucial role in cancer development and progression. More than a century ago, mouse models showed that primary tumors suppressed the growth of newly implanted secondary tumors. This phenomenon, in which tumor-primed T cells mediate the rejection of tumor growth at a distant site, is known as concomitant tumor immunity. Here, we investigated the role of concomitant immunity in the development of breast cancer bone metastases using newly developed syngeneic immunocompetent mouse models. The presence of primary breast tumors developed by tumor cell injection into the mammary fat pads (MFPs) significantly reduced bone metastases of mouse breast cancer 4T1 and EMT6 cells induced by cell injection through the caudal artery (CA). Similar results were obtained when primary tumors were surgically resected prior to CA injection of tumor cells. In contrast, no inhibition was found when MFP and CA injections were performed using different cell combinations. Immunohistochemical studies revealed that the number of CD8+ T cells in bone metastases of 4T1 and EMT6 cells was significantly increased in the presence of primary tumors. The primary tumor-induced inhibition of bone metastases was not reproduced in T cell-deficient athymic nude mice. Furthermore, depletion of CD8+ T cells using an anti-CD8α antibody also abolished the primary tumor-induced inhibition of bone metastases. Taken together, these results suggest that immune cell priming by orthotopic breast tumors inhibits the development of breast cancer bone metastases, which is predominantly mediated by CD8+ cytotoxic T lymphocytes.

Sclerostin blockade promotes bone metastases of Wnt-responsive breast cancer cells.

The secreted protein sclerostin is primarily produced by osteocytes and suppresses osteoblast differentiation and function by inhibiting the canonical Wnt signaling pathway. Genetic and pharmacological inhibition of sclerostin has been shown to increase bone formation and an anti-sclerostin antibody has been clinically approved for the treatment of osteoporosis. Canonical Wnt signaling is also involved in the progression of several types of cancers including breast cancer. Here, we studied the effects of sclerostin inhibition on the development of bone metastases of breast cancer using mouse models. TOPFLASH assay and real-time PCR analysis of AXIN2, a target of canonical Wnt signaling, revealed that, among four cell lines tested, MDA-MB-231 human breast cancer cells responded highly to the canonical Wnt ligand Wnt3a, whereas other cell lines exhibited marginal responses. Consistent with these results, treatment with an anti-sclerostin antibody significantly increased the bone metastases of MDA-MB-231 but not those of other breast cancer cells. Immunohistochemical studies demonstrated that an anti-sclerostin antibody induced intracellular accumulation of ß-catenin in bone-colonized MDA-MB-231 cells. Suspension culture assays showed that Wnt3a accelerated the tumorsphere formation of MDA-MB-231 cells, whereas monolayer cell proliferation and migration were not affected. Furthermore, the numbers of osteoclasts and their precursor cells in bone metastases of MDA-MB-231 were significantly increased in mice treated with an anti-sclerostin antibody. These results collectively suggest that sclerostin blockade activates canonical Wnt signaling in ligand-responsive breast cancer cells metastasized to bone, thereby increasing bone metastases, likely to have been mediated at least in part by enhancing stem cell-like properties of cancer cells and osteoclastogenesis.

Opposing Effects of Granulocyte Colony-Stimulating Factor on the Initiation and Progression of Breast Cancer Bone Metastases.

Granulocyte colony stimulating factor (G-CSF), an essential cytokine regulating granulopoiesis, is expressed in a substantial proportion of breast cancers, and it has been implicated in cancer progression. Here, we examined effects of G-CSF on the development of bone metastases of breast cancer using immunocompetent mouse models. The expression of CXC chemokine ligand 12 (CXCL12) in bone marrow stromal cells, which plays a critical role in the maintenance of hematopoietic stem cells and also in cancer cell homing to bone, was markedly decreased in mice treated with G-CSF. Flow cytometric analysis revealed that pretreatment of mice with G-CSF reduced the number of bone-homing cancer cells. G-CSF also increased the population of myeloid-derived suppressor cells (MDSCs) in bone marrow. Depletion of MDSCs using anti-Gr-1 antibody treatment significantly decreased the metastatic tumor burden in bone. The overall effects of G-CSF on bone metastases were finally examined using two different treatment protocols. When mice were treated with G-CSF prior to the tumor cell inoculation, G-CSF did not change bone metastatic-tumor burden. In contrast, when G-CSF treatment was started after the tumor cells had homed to bone, G-CSF significantly accelerated bone metastases formation. These results suggest that G-CSF suppressed cancer cell homing to bone by downregulating CXCL12 expression in bone marrow stromal cells, whereas G-CSF stimulated the progression of bone metastases at least in part by MDSC-mediated mechanisms. IMPLICATIONS: G-CSF had opposing effects on the initiation and progression of bone metastases of breast cancer and the balance may regulate the metastatic tumor burden.

Altered immunolocalization of FGF23 in murine femora metastasized with human breast carcinoma MDA-MB-231 cells.

INTRODUCTION: After the onset of bone metastasis, tumor cells appear to modify surrounding microenvironments for their benefit, and particularly, the levels of circulating fibroblast growth factor (FGF) 23 in patients with tumors have been highlighted. MATERIALS AND METHODS: We have attempted to verify if human breast carcinoma MDA-MB-231 cells metastasized in the long bone of nu/nu mice would synthesize FGF23. Serum concentrations of calcium, phosphate (Pi) and FGF23 were measured in control nu/nu mice, bone-metastasized mice, and mice with mammary gland injected with MDA-MB-231 cells mimicking primary mammary tumors. RESULTS AND CONCLUSIONS: MDA-MB-231 cells revealed intense FGF23 reactivity in metastasized lesions, whereas MDA-MB-231 cells cultured in vitro or when injected into the mammary glands (without bone metastasis) showed weak FGF23 immunoreactivity. Although the bone-metastasized MDA-MB-231 cells abundantly synthesized FGF23, osteocytes adjacent to the FGF23-immunopositive tumors, unlike intact osteocytes, showed no FGF23. Despite significantly elevated serum FGF23 levels in bone-metastasized mice, there was no significant decrease in the serum Pi concentration when compared with the intact mice and mice with a mass of MDA-MB-231 cells in mammary glands. The metastasized femora showed increased expression and FGFR1 immunoreactivity in fibroblastic stromal cells, whereas femora of control mice showed no obvious FGFR1 immunoreactivity. Taken together, it seems likely that MDA-MB-231 cells synthesize FGF23 when metastasized to a bone, and thus affect FGFR1-positive stromal cells in the metastasized tumor nest in a paracrine manner.

Bone-targeting phospholipid polymers to solubilize the lipophilic anticancer drug.

Current chemotherapy methods have limited effectiveness in eliminating bone metastasis, which leads to a poor prognosis associated with severe bone disorders. To provide regional chemotherapy for this metastatic tumor, a bone-targeting drug carrier was produced by introducing the osteotropic bisphosphonate alendronate (ALN) units into an amphiphilic phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate). The polymer can form nanoparticles with a diameter of less than 30 nm; ALN units were exposed to the outer layer of the particle. A simple mixing procedure was used to encapsulate a hydrophobic anticancer drug, known as docetaxel (DTX), in the polymer nanoparticle, providing a uniform solution of a polymer-DTX complex in the aqueous phase. The complex showed anticancer activities against several breast cancer cell lines, and the complex formation did not hamper the pharmacological effect of DTX. The fluorescence observations evaluated by an in vivo imaging system and fluorescence microscopy showed that the addition of ALN to the polymer-DTX complex enhanced bone accumulation. Bone-targeting phospholipid polymers are potential solubilizing excipients used to formulate DTX and deliver the hydrophobic drug to bone tissues by blood administration.

Stromal fibroblasts induce metastatic tumor cell clusters via epithelial-mesenchymal plasticity.

Emerging evidence supports the hypothesis that multicellular tumor clusters invade and seed metastasis. However, whether tumor-associated stroma induces epithelial-mesenchymal plasticity in tumor cell clusters, to promote invasion and metastasis, remains unknown. We demonstrate herein that carcinoma-associated fibroblasts (CAFs) frequently present in tumor stroma drive the formation of tumor cell clusters composed of two distinct cancer cell populations, one in a highly epithelial (E-cadherinhiZEB1lo/neg: Ehi) state and another in a hybrid epithelial/mesenchymal (E-cadherinloZEB1hi: E/M) state. The Ehi cells highly express oncogenic cell-cell adhesion molecules, such as carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and CEACAM6 that associate with E-cadherin, resulting in increased tumor cell cluster formation and metastatic seeding. The E/M cells also retain associations with Ehi cells, which follow the E/M cells leading to collective invasion. CAF-produced stromal cell-derived factor 1 and transforming growth factor-ß confer the Ehi and E/M states as well as invasive and metastatic traits via Src activation in apposed human breast tumor cells. Taken together, these findings indicate that invasive and metastatic tumor cell clusters are induced by CAFs via epithelial-mesenchymal plasticity.

Parathyroid Hormone Shifts Cell Fate of a Leptin Receptor-Marked Stromal Population from Adipogenic to Osteoblastic Lineage.

Intermittent parathyroid hormone (iPTH) treatment induces bone anabolic effects that result in the recovery of osteoporotic bone loss. Human PTH is usually given to osteoporotic patients because it induces osteoblastogenesis. However, the mechanism by which PTH stimulates the expansion of stromal cell populations and their maturation toward the osteoblastic cell lineage has not be elucidated. Mouse genetic lineage tracing revealed that iPTH treatment induced osteoblastic differentiation of bone marrow (BM) mesenchymal stem and progenitor cells (MSPCs), which carried the leptin receptor (LepR)-Cre. Although these findings suggested that part of the PTH-induced bone anabolic action is exerted because of osteoblastic commitment of MSPCs, little is known about the in vivo mechanistic details of these processes. Here, we showed that LepR+ MSPCs differentiated into type I collagen (Col1)+ mature osteoblasts in response to iPTH treatment. Along with osteoblastogenesis, the number of Col1+ mature osteoblasts increased around the bone surface, although most of them were characterized as quiescent cells. However, the number of LepR-Cre-marked lineage cells in a proliferative state also increased in the vicinity of bone tissue after iPTH treatment. The expression levels of SP7/osterix (Osx) and Col1, which are markers for osteoblasts, were also increased in the LepR+ MSPCs population in response to iPTH treatment. In contrast, the expression levels of Cebpb, Pparg, and Zfp467, which are adipocyte markers, decreased in this population. Consistent with these results, iPTH treatment inhibited 5-fluorouracil- or ovariectomy (OVX)-induced LepR+ MSPC-derived adipogenesis in BM and increased LepR+ MSPC-derived osteoblasts, even under the adipocyte-induced conditions. Treatment of OVX rats with iPTH significantly affected the osteoporotic bone tissue and expansion of the BM adipose tissue. These results indicated that iPTH treatment induced transient proliferation of the LepR+ MSPCs and skewed their lineage differentiation from adipocytes toward osteoblasts, resulting in an expanded, quiescent, and mature osteoblast population. © 2019 American Society for Bone and Mineral Research.

Bone metastasis: Interaction between cancer cells and bone microenvironment.

BACKGROUND: Bone is one of the most common target organs for cancer metastasis, especially in patients with advanced breast and prostate cancers. Despite recent advances in therapeutic approaches, bone metastases remain incurable and produce multiple complications called skeletal-related events, including hypercalcemia, pathological fractures, spinal compression, and bone pain, which are associated with poor prognosis. HIGHLIGHT: Although the precise mechanisms are yet to be fully elucidated, accumulating evidence suggests that bone provides a favorable microenvironment that enables circulating cancer cells to home, proliferate, and colonize, resulting in the formation of metastasis. Cancer cells that metastasize to bone also possess unique features, enabling them to utilize the bone microenvironment. Thus, communication between cancer cells and bone is believed to be critical for the development and progression of bone metastases. CONCLUSION: Continued studies are warranted to understand the molecular mechanisms underlying bone metastases and to develop mechanism-based and effective therapeutic interventions.

Establishment and characterization of a C57BL/6 mouse model of bone metastasis of breast cancer.

Bone is one of the most common sites of metastasis in patients with advanced breast cancer; however, the mechanisms of bone metastasis remain to be fully elucidated. Animal models are essential research tools for investigating the mechanisms of diseases and drug actions. To date, there have only been a few reports in which C57BL/6 mice were used for the study of bone metastases of breast cancer. In the current study, we found that intracardiac inoculation of C57BL/6 mouse-derived parental E0771 breast cancer cells (E0771/Pa) frequently lead to bone metastases in C57BL/6 mice within 2 weeks. The bone-metastatic clone of E0771 (E0771/Bone) established by sequential in vivo selection demonstrated a higher bone-metastatic potential. Although there were no apparent differences in cell morphology or proliferation in monolayer cultures, E0771/Bone showed increased tumorsphere formation in suspension cultures and tumor formation in the orthotopic mammary fat pad in C57BL/6 mice compared with E0771/Pa. Furthermore, E0771/Bone expressed breast cancer stem-like cell surface markers CD24-/CD44+. These findings suggest that E0771/Bone possesses cancer stem-like properties. Quantitative PCR analysis revealed that mRNA expression of parathyroid hormone-related protein (PTHrP), the most common mediator of osteolytic bone metastases of breast cancer, was significantly upregulated in E0771/Bone. Thus, cancer stem-like properties and elevated PTHrP expression likely contribute to the enhanced metastatic potential of E0771/Bone. We believe that this new mouse model is a useful tool for in vivo studies of bone metastases of breast cancer, especially for those using genetically engineered mice with a C57BL/6 background.

Hypoxic Microenvironment and Metastatic Bone Disease.

Hypoxia is a common feature of solid tumors and is associated with an increased risk of metastasis and a poor prognosis. Recent imaging techniques revealed that bone marrow contains a quite hypoxic microenvironment. Low oxygen levels activate hypoxia signaling pathways such as hypoxia-inducible factors, which play critical roles in the key stages of metastatic dissemination including angiogenesis, epithelial-mesenchymal transition, invasion, maintenance of cancer stem cells, tumor cell dormancy, release of extracellular vesicles, and generation of pre-metastatic niches. Hypoxia also affects bone cells, such as osteoblasts and osteoclasts, and immune cells, which also act to support the development and progression of bone metastases. Paradoxically, hypoxia and related signaling molecules are recognized as high-priority therapeutic targets and many candidate drugs are currently under preclinical and clinical investigation. The present review focuses on our current knowledge of the potential roles of hypoxia in cancer metastasis to bone by considering the interaction between metastatic cancer cells and the bone microenvironment. Current therapeutic approaches targeting hypoxia are also described.

Expression and localization of CRAMP in rat tooth germ and during reparative dentin formation.

OBJECTIVES: Cathelicidin-related antimicrobial peptide (CRAMP) is an antimicrobial peptide in mice and rats homologous to LL-37 in humans. In addition to its antibacterial activity, CRAMP has various physiological functions by binding to formyl peptide receptor 2 (FPR2). However, the role of these peptides in teeth is unknown. Therefore, we investigated the role of CRAMP and FPR2 in tooth development, reparative dentin formation, and defense response. MATERIAL AND METHODS: First, we examined the localization of CRAMP and FPR2 during tooth development by immunohistochemical analysis. Next, we investigated the localization of CRAMP, FPR2, and CD68-positive macrophages by immunohistochemical analysis during pulp inflammation and reparative dentin formation after cavity preparation. Finally, we analyzed the effect of lipopolysaccharide (LPS) on the expression of CRAMP and FPR2 in dental pulp cells by real-time reverse transcription PCR. RESULTS: At the late bell stage in tooth development, CRAMP was detected in odontoblasts, and FPR2 was observed in the sub-odontoblastic layer. In mature teeth, CRAMP was not detected, but FPR2 continued to be localized in the sub-odontoblastic layer. After cavity preparation, CRAMP-positive cells and macrophages were found in dental pulp tissues below the cavity at an early stage of repair. At subsequent stages of reparative dentin formation, CRAMP was observed in odontoblast-like cells that contacted reparative dentin. FPR2 immunoreactivity was also detected in odontoblast-like cells and neighboring cells. LPS stimulated the expression of CRAMP mRNA in dental pulp cells in vitro. CONCLUSIONS: Localization of CRAMP and its receptor FPR2-positive cells were observed during physiological and reparative dentin formation. CLINICAL RELEVANCE: CRAMP/LL-37 has a possibility that induce reparative dentin formation.

Decreased sensory nerve excitation and bone pain associated with mouse Lewis lung cancer in TRPV1-deficient mice.

Bone pain is one of the most common and life-limiting complications of cancer metastasis to bone. Although the mechanism of bone pain still remains poorly understood, bone pain is evoked as a consequence of sensitization and excitation of sensory nerves (SNs) innervating bone by noxious stimuli produced in the microenvironment of bone metastases. We showed that bone is innervated by calcitonin gene-related protein (CGRP)+ SNs extending from dorsal root ganglia (DRG), the cell body of SNs, in mice. Mice intratibially injected with Lewis lung cancer (LLC) cells showed progressive bone pain evaluated by mechanical allodynia and flinching with increased CGRP+ SNs in bone and augmented SN excitation in DRG as indicated by elevated numbers of pERK- and pCREB-immunoreactive neurons. Immunohistochemical examination of LLC-injected bone revealed that the tumor microenvironment is acidic. Bafilomycin A1, a selective inhibitor of H+ secretion from vacuolar proton pump, significantly alleviated bone pain, indicating that the acidic microenvironment contributes to bone pain. We then determined whether the transient receptor potential vanilloid 1 (TRPV1), a major acid-sensing nociceptor predominantly expressed on SNs, plays a role in bone pain by intratibially injecting LLC cells in TRPV1-deficient mice. Bone pain and SN excitation in the DRG and spinal dorsal horn were significantly decreased in TRPV1 -/- mice compared with wild-type mice. Our results suggest that TRPV1 activation on SNs innervating bone by the acidic cancer microenvironment in bone contributes to SN activation and bone pain. Targeting acid-activated TRPV1 is a potential therapeutic approach to cancer-induced bone pain.

Osteogenic Factor Runx2 Marks a Subset of Leptin Receptor-Positive Cells that Sit Atop the Bone Marrow Stromal Cell Hierarchy.

Bone marrow mesenchymal stem and progenitor cells (BM-MSPCs) maintain homeostasis of bone tissue by providing osteoblasts. Although several markers have been identified for labeling of MSPCs, these labeled cells still contain non-BM-MSPC populations. Studies have suggested that MSPCs are observed as leptin receptor (LepR)-positive cells, whereas osteoblasts can be classified as positive for Runx2, a master regulator for osteoblastogenesis. Here, we demonstrate, using Runx2-GFP reporter mice, that the LepR-labeled population contains Runx2-GFPlow sub-population, which possesses higher fibroblastic colony-forming units (CFUs) and mesensphere capacity, criteria for assessing stem cell activity, than the Runx2-GFP- population. In response to parathyroid hormone (PTH), a bone anabolic hormone, LepR+Runx2-GFPlow cells increase Runx2 expression and form multilayered structures near the bone surface. Subsequently, the multilayered cells express Osterix and Type I collagen α, resulting in generation of mature osteoblasts. Therefore, our results indicate that Runx2 is weakly expressed in the LepR+ population without osteoblastic commitment, and the LepR+Runx2-GFPlow stromal cells sit atop the BM stromal hierarchy.

Comparison of Different Disease-Specific Health-Related Quality of Life Measurements in Patients with Long-Term Noninvasive Ventilation.

BACKGROUND: Two disease-specific questionnaires have been developed to assess health-related quality of life (HRQL) in patients with chronic respiratory failure: the Severe Respiratory Insufficiency (SRI) Questionnaire and the Maugeri Respiratory Failure (MRF) Questionnaire. We aimed to compare the characteristics of the SRI, MRF-26, and St. George's Respiratory Questionnaire (SGRQ) for use in patients with home noninvasive ventilation (NIV). METHODS: Fifty-six outpatients receiving long-term NIV were recruited and underwent assessments of pulmonary function, arterial blood gas, HRQL, dyspnea, and psychological status. RESULTS: Correlations of the SRI and MRF-26 with the SGRQ were modest. While pulmonary function was weakly related to only some domains of the SRI and MRF-26, the modified Medical Research Council (mMRC) dyspnea scale and Hospital Anxiety and Depression Scale (HADS) were significantly related to all domains of the SRI and MRF-26. Multiple regression analyses showed that HADS depression and mMRC accounted for 34% and 27% of the variance in the SRI, 24% and 37% in the MRF-26, and 17% and 46% in the SGRQ, respectively. CONCLUSIONS: The SRI and MRF-26 were reliable questionnaires for patients receiving long-term NIV. Dyspnea and psychological status were their main common determinants. The SRI covers more psychological health impairments than the MRF. This trial is registered with ClinicalTrials.gov Identifier: NCT00905476.

Differentiation by NK cells is a prerequisite for effective targeting of cancer stem cells/poorly differentiated tumors by chemopreventive and chemotherapeutic drugs.

Natural Killer (NK) cells target oral, pancreatic, lung, breast, glioblastoma and melanoma stem-like/poorly differentiated tumors. Differentiation of the abovementioned tumors with supernatants from split-anergized NK cells decreases their susceptibility to NK cells, but increases their sensitivity to cisplatin (CDDP)-mediated cell death. Breast and melanoma tumor cells with CD44 knockdown display enhanced susceptibility to NK cell-mediated lysis, potentially due to decreased differentiation. We also demonstrate that sulindac, a non-steroidal anti-inflammatory drug and a chemopreventive agent, not only limits the growth of oral tumor cells, but also aids in cancer cell elimination by NK cells. Treatment of oral tumors with sulindac, but not adriamycin inversely modulates the expression and function of NFκB and JNK, resulting in a significant down-regulation of IL-6, and VEGF secretion by oral tumor cells. In addition, increased secretion of IL-6 and VEGF is blocked by sulindac during interaction of oral tumors with NK cells. Sulindac treatment prevents synergistic induction of VEGF secretion by the tumor cells after their co-culture with untreated NK cells since non-activated NK cells lack the ability to efficiently kill tumor cells. Moreover, sulindac is able to profoundly reduce VEGF secretion by tumor cells cultured with IL-2 activated NK cells, which are able to significantly lyse the tumor cells. Based on the data presented in this study, we propose the following combinatorial approach for the treatment of stem-like/ poorly differentiated tumors in cancer patients with metastatic disease. Stem-like/ poorly differentiated tumor cells may in part undergo lysis or differentiation after NK cell immunotherapy, followed by treatment of differentiated tumors with chemotherapy and chemopreventive agents to eliminate the bulk of the tumor. This dual approach should limit tumor growth and prevent metastasis.

Validation of the Japanese Severe Respiratory Insufficiency Questionnaire in hypercapnic patients with noninvasive ventilation.

BACKGROUND: The Severe Respiratory Insufficiency (SRI) Questionnaire was originally developed in German to assess health-related quality of life (HRQL) and was validated as a multidimensional instrument with high psychometric properties in chronic hypercapnic respiratory failure (CHRF) patients receiving noninvasive ventilation (NIV). We aimed to investigate the intercultural adaptation of the Japanese SRI Questionnaire and whether it is a reliable and valid HRQL questionnaire to administer to those patients. METHODS: The SRI Questionnaire was adapted to Japanese using a translation and back-translation procedure, followed by equivalency assessment. It was validated in 56 stable outpatients receiving NIV for CHRF, primarily due to chronic obstructive pulmonary disease (COPD) and/or pulmonary tuberculosis sequelae. RESULTS: Examination of the frequency distribution of the Japanese SRI Questionnaire showed that the subscales and summary were approximately normally distributed and well balanced. There were no significant differences in SRI scores between patients with COPD and pulmonary tuberculosis sequelae. Cronbach׳s α values representing internal consistency of seven SRI subscales ranged from 0.56 to 0.80; attendant symptoms and sleep had the lowest values. Cronbach׳s α value was 0.92 for the SRI summary. The SRI summary score was significantly related to all eight subscales of the Medical Outcomes Study 36-item short form, with correlation coefficients of 0.41-0.66. CONCLUSIONS: The Japanese SRI Questionnaire was produced using a standardized procedure and an equivalency study. It has high psychometric properties with internal consistency and concurrent validity. The Japanese SRI Questionnaire can be used to assess HRQL in patients on NIV for CHRF.

Analysis of the relationship between health status and mortality in hypercapnic patients with noninvasive ventilation.

BACKGROUND: Health status and mortality are important outcomes in patients with advanced pulmonary diseases receiving noninvasive ventilation (NIV). However, their relationship has not been thoroughly investigated. METHODS: The present study prospectively recruited 56 stable outpatients treated with NIV for chronic hypercapnic respiratory failure caused by chronic obstructive pulmonary disease and/or pulmonary tuberculosis sequelae. At baseline, health status was measured by the Medical Outcomes Study 36-item short form, a generic questionnaire; the St. George's Respiratory Questionnaire (SGRQ), a respiratory-specific questionnaire; and two respiratory failure-specific questionnaires, the Maugeri Respiratory Failure questionnaire and the Severe Respiratory Insufficiency (SRI) questionnaire. Arterial blood gas, pulmonary function, dyspnea and psychological status were also measured. RESULTS: In cross-sectional comparisons of the four health status questionnaires, the SGRQ and SRI questionnaire had lower floor and ceiling effects. During the 3-year follow-up, 16 patients (29%) died. Health status shown by the SGRQ and SRI was significantly predictive of mortality, independently of the physiological measures of low body mass index (BMI), hypercapnia, and low pulmonary function. Stepwise multivariate analyses indicated that the SRI summary score was the most significant predictor of mortality (P = 0.0006) followed by BMI (P = 0.012). CONCLUSION: There was a significant relationship between health status and 3-year mortality in patients with NIV, independently of under-nutrition, hypercapnia and low pulmonary function. Health status measurement is important not only to comprehensively evaluate disease severity in relation to its close association with mortality, but also to elucidate factors that improve the survival of patients with advanced respiratory diseases.

Comparable roles of CD44v8-10 and CD44s in the development of bone metastases in a mouse model.

Cluster of differentiation (CD)44 has been implicated in cancer metastasis to bone. Clinical and experimental studies have suggested that the standard isoform of CD44 (CD44s) and the variant isoform of CD44 (CD44v) enhance metastasis. The present study examined the differential roles of CD44s and CD44v, particularly CD44v8-10, in the development of bone metastases. For this purpose, MDA-MB-231 human breast cancer cells and A549 human lung cancer cells were stably transduced with epithelial splicing regulatory protein 1 (ESRP1), which regulates the alternative splicing of several genes, including CD44. The introduction of ESRP1 induced a splicing switch from CD44s to CD44v, particularly to CD44v8-10, while the total amount of CD44 was rarely affected. However, ESRP1 did not significantly affect cell proliferation, migration, invasion or tumor sphere formation in vitro. Furthermore, ESRP1 did not cause significant differences in the development of bone metastases in a mouse model. As an alternative approach, cancer cells transduced with the CD44v8-10 gene were also established. The overexpression of CD44v8-10 in MCF-7 human breast cancer cells, which rarely express any isoform of CD44, promoted cell migration and sphere formation, whereas the overexpression of CD44v8-10 in MDA-MB-231 cells, which endogenously express high levels of CD44s, did not exert these effects. The results of the present study collectively suggest that the ability of CD44v8-10 to promote tumor aggressiveness and bone metastases is similar to that of CD44s. CD44v8-10 and CD44s may represent potential therapeutic targets for the treatment of bone metastases.