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INTRODUCTION: Neurogenic lower urinary tract dysfunction (NLUTD), including neurogenic detrusor overactivity (NDO) and detrusor sphincter dyssynergia, is one of the most frequent and devastating sequelae of spinal cord injury (SCI), as it can lead to urinary incontinence and secondary damage such as renal failure. Transcutaneous tibial nerve stimulation (TTNS) is a promising, non-invasive neuromodulatory intervention that may prevent the emergence of the C-fibre evoked bladder reflexes that are thought to cause NDO. This paper presents the protocol for TTNS in acute SCI (TASCI), which will evaluate the efficacy of TTNS in preventing NDO. Furthermore, TASCI will provide insight into the mechanisms underlying TTNS, and the course of NLUTD development after SCI. METHODS AND ANALYSIS: TASCI is a nationwide, randomised, sham-controlled, double-blind clinical trial, conducted at all four SCI centres in Switzerland. The longitudinal design includes a baseline assessment period 5-39 days after acute SCI and follow-up assessments occurring 3, 6 and 12 months after SCI. A planned 114 participants will be randomised into verum or sham TTNS groups (1:1 ratio), stratified on study centre and lower extremity motor score. TTNS is performed for 30 min/day, 5 days/week, for 6-9 weeks starting within 40 days after SCI. The primary outcome is the occurrence of NDO jeopardising the upper urinary tract at 1 year after SCI, assessed by urodynamic investigation. Secondary outcome measures assess bladder and bowel function and symptoms, sexual function, neurological structure and function, functional independence, quality of life, as well as changes in biomarkers in the urine, blood, stool and bladder tissue. Safety of TTNS is the tertiary outcome. ETHICS AND DISSEMINATION: TASCI is approved by the Swiss Ethics Committee for Northwest/Central Switzerland, the Swiss Ethics Committee Vaud and the Swiss Ethics Committee Zürich (#2019-00074). Findings will be disseminated through peer-reviewed

Current combined surgical and neo-adjuvant chemotherapy of primary metastatic osteosarcoma (OS) is ineffective, reflected by a 5-year survival rate of affected patients of less than 20%. Studies in experimental OS metastasis models pointed to the CXCR4/CXCL12 homing axis as a novel target for OS metastasis-suppressive treatment. The present study investigated for the first time the CXCR4-blocking principle in a spontaneously metastasizing human 143B OS cell line-derived orthotopic xenograft mouse model. The highly metastatic 143B cells, unlike the parental non-metastatic HOS cells, express functional CXCR4 receptors at the cell surface, as revealed in this study by RT/PCR of gene transcripts, by FACS analysis with the monoclonal anti CXCR4 antibody 12G5 (mAb 12G5) and by CXCL12 time- and dose-dependent stimulation of AKT and ERK phosphorylation. A significantly (p<0.05) higher CXCL12 dose-dependent chemotactic response of 143B compared to HOS cells in a Boyden chamber trans-well migration assay suggested a crucial role of the CXCL12/CXCR4 homing axis in 143B cell lung metastasis. Repetitive treatment of mice with 143B cell-derived intratibial tumors given intravenous bolus injections of mAb12G5 indeed inhibited significantly (p<0.01) the number of X-gal-stainable lung micrometastases of lacZ-transduced 143B cells. Antibody treatment had also a mild inhibitory effect on primary tumor growth associated with remarkably less osteolysis, but it did not affect the number of developing lung macrometastases. In conclusion, these results demonstrate considerable potential of high-affinity CXCR4-blocking agents for OS tumor cell homing suppressive treatment in metastasizing OS complementary to current (neo)-adjuvant chemotherapy.

BACKGROUND Osteosarcoma is a rare but highly malignant cancer of the bone. As a consequence, the number of established cell lines used for experimental in vitro and in vivo osteosarcoma research is limited and the value of these cell lines relies on their stability during culture. Here we investigated the stability in gene expression by microarray analysis and array genomic hybridization of three low metastatic cell lines and derivatives thereof with increased metastatic potential using cells of different passages. PRINCIPAL FINDINGS The osteosarcoma cell lines showed altered gene expression during in vitro culture, and it was more pronounced in two metastatic cell lines compared to the respective parental cells. Chromosomal instability contributed in part to the altered gene expression in SAOS and LM5 cells with low and high metastatic potential. To identify metastasis-relevant genes in a background of passage-dependent altered gene expression, genes involved in "Pathways in cancer" that were consistently regulated under all passage comparisons were evaluated. Genes belonging to "Hedgehog signaling pathway" and "Wnt signaling pathway" were significantly up-regulated, and IHH, WNT10B and TCF7 were found up-regulated in all three metastatic compared to the parental cell lines. CONCLUSIONS Considerable instability during culture in terms of gene expression and chromosomal aberrations was observed in osteosarcoma cell lines. The use of cells from different passages and a search for genes consistently regulated in early and late passages allows the analysis of metastasis-relevant genes despite the observed instability in gene expression in osteosarcoma cell lines during culture.

Osteosarcoma (OS) is the most common primary bone malignancy in children and adolescents. More than 30% of patients develop lung metastasis, which is the leading cause of mortality. Recently, the extracellular matrix protein Cyr61 has been recognized as a malignancy promoting protein in OS mouse model with prognostic potential in human OS. In this study, we aimed at the identification of novel Cyr61-interacting proteins. Here we report that Cyr61 associates with Caprin-1, and confocal microscopy showed that stable ectopic expression of Caprin-1 leads to the formation of stress granules containing Caprin-1 and Cyr61, confers resistance to cisplatin-induced apoptosis, and resulted in constitutive phosphorylation of Akt and ERK1/2. Importantly, ectopic expression of Caprin-1 dramatically enhanced primary tumor growth, remarkably increased lung metastatic load in a SCID intratibial OS mouse model, and decreased significantly (p<0.0018) the survival of the mice. Although Caprin-1 expression, evaluated with a tissue microarray including samples from 59 OS patients, failed to be an independent predictor for the patients' outcome in this limited cohort of patients, increased Caprin-1 expression indicated a tendency to shortened overall survival, and more strikingly, Cyr61/Caprin-1 co-expression was associated with worse survival than that observed for patients with tumors expressing either Cyr61 or Caprin-1 alone or none of these proteins. The findings imply that Caprin-1 may have a metastasis promoting role in OS and show that through resistance to apoptosis and via the activation of Akt and ERK1/2 pathways, Caprin-1 is significantly involved in the development of OS metastasis.

Osteosarcoma (OS) is the most frequent primary malignant bone cancer in children and adolescents with a high propensity for lung metastasis. Therefore, it is of great importance to identify molecular markers leading to increased metastatic potential in order to devise more effective therapeutic strategies that suppress metastasis, the major cause of death in OS. CD44, the principal receptor for the extracellular matrix component hyaluronan (HA), is frequently found overexpressed in tumor cells and has been implicated in metastatic spread in various cancer types. Here, we investigated the effects of stable shRNA-mediated silencing of CD44 gene products on in vitro and in vivo metastatic properties of the highly metastatic human 143-B OS cell line. In vitro, CD44 knockdown resulted in a 73% decrease in the adhesion to HA, a 57% decrease in the migration rate in a trans-filter migration assay, and a 28% decrease in the cells' capacity for anchorage-independent growth in soft agar compared to the control cells, implicating that CD44 expression contributes to the metastatic activity of 143-B cells. However, making use of an orthotopic xenograft OS mouse model, we demonstrated that reduced CD44 expression facilitated primary tumor growth and formation of pulmonary metastases. The enhanced malignant phenotype was associated with decreased adhesion to HA and reduced expression of the tumor suppressor merlin in vivo. In conclusion, our study identified CD44 as a metastasis suppressor in this particular experimental OS model.

More effective treatment of metastasizing osteosarcoma with a current mean 5-year survival rate of less than 20% requires more detailed knowledge on mechanisms and key regulatory molecules of the complex metastatic process. CXCR4, the receptor of the chemokine CXCL12, has been reported to promote tumor progression and metastasis in osteosarcoma. CXCR7 is a recently deorphanized CXCL12-scavenging receptor with so far not well-defined functions in tumor biology. The present study focused on a potential malignancy enhancing function of CXCR7 in interaction with CXCR4 in osteosarcoma, which was investigated in an intratibial osteosarcoma model in SCID mice, making use of the human 143B osteosarcoma cell line that spontaneously metastasizes to the lung and expresses endogenous CXCR4. 143B osteosarcoma cells stably expressing LacZ (143B-LacZ cells) were retrovirally transduced with a gene encoding HA-tagged CXCR7 (143B-LacZ-X7-HA cells). 143B-LacZ-X7-HA cells coexpressing CXCR7 and CXCR4 exhibited CXCL12 scavenging and enhanced adhesion to IL-1β-activated HUVEC cells compared to 143B-LacZ cells expressing CXCR4 alone. SCID mice intratibially injected with 143B-LacZ-X7-HA cells had significantly (p<0.05) smaller primary tumors, but significantly (p<0.05) higher numbers of lung metastases than mice injected with 143B-LacZ cells. Unexpectedly, 143B-LacZ-X7-HA cells, unlike 143B-LacZ cells, also metastasized with high incidence to the auriculum cordis. In conclusion, expression of the CXCL12 scavenging receptor CXCR7 in the CXCR4-expressing human 143B osteosarcoma cell line enhances its metastatic activity in intratibial primary tumors in SCID mice that predominantly metastasize to the lung and thereby closely mimic the human disease. These findings point to CXCR7 as a target, complementary to previously proposed CXCR4, for more effective metastasis-suppressive treatment in osteosarcoma.

Current combined surgical and neo-adjuvant chemotherapy of primary metastatic osteosarcoma (OS) is ineffective, reflected by a 5-year survival rate of affected patients of less than 20%. Studies in experimental OS metastasis models pointed to the CXCR4/CXCL12 homing axis as a novel target for OS metastasis-suppressive treatment. The present study investigated for the first time the CXCR4-blocking principle in a spontaneously metastasizing human 143B OS cell line-derived orthotopic xenograft mouse model. The highly metastatic 143B cells, unlike the parental non-metastatic HOS cells, express functional CXCR4 receptors at the cell surface, as revealed in this study by RT/PCR of gene transcripts, by FACS analysis with the monoclonal anti CXCR4 antibody 12G5 (mAb 12G5) and by CXCL12 time- and dose-dependent stimulation of AKT and ERK phosphorylation. A significantly (p<0.05) higher CXCL12 dose-dependent chemotactic response of 143B compared to HOS cells in a Boyden chamber trans-well migration assay suggested a crucial role of the CXCL12/CXCR4 homing axis in 143B cell lung metastasis. Repetitive treatment of mice with 143B cell-derived intratibial tumors given intravenous bolus injections of mAb12G5 indeed inhibited significantly (p<0.01) the number of X-gal-stainable lung micrometastases of lacZ-transduced 143B cells. Antibody treatment had also a mild inhibitory effect on primary tumor growth associated with remarkably less osteolysis, but it did not affect the number of developing lung macrometastases. In conclusion, these results demonstrate considerable potential of high-affinity CXCR4-blocking agents for OS tumor cell homing suppressive treatment in metastasizing OS complementary to current (neo)-adjuvant chemotherapy.