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Liver fibrosis is the static and main (70%-80%) component of portal hypertension (PH). We investigated dynamic components of PH by a three-dimensional analysis based on correlation of hepatic collagen proportionate area (CPA) with portal pressure (PP) in animals or HVPG in patients.Different animal models (bile duct ligation: n = 31, carbon tetrachloride: n = 12, thioacetamide: n = 12, choline-deficient high-fat diet: n = 12) and patients with a confirmed single etiology of cholestatic (primary biliary cholangitis/primary sclerosing cholangitis: n = 16), alcohol-associated (n = 22), and metabolic (NASH: n = 19) liver disease underwent CPA quantification on liver specimens/biopsies. Based on CPA-to-PP/HVPG correlation, potential dynamic components were identified in subgroups of animals/patients with lower-than-expected and higher-than-expected PP/HVPG. Dynamic PH components were validated in a patient cohort (n = 245) using liver stiffness measurement (LSM) instead of CPA. CPA significantly correlated with PP in animal models (Rho = 0.531; p0.001) and HVPG in patients (Rho = 0.439; p0.001). Correlation of CPA with PP/HVPG varied across different animal models and etiologies in patients. In models, severity of hyperdynamic circulation and specific fibrosis pattern (portal fibrosis: p = 0.02; septa width: p = 0.03) were associated with PH severity. In patients, hyperdynamic circulation (p = 0.04), vascular dysfunction/angiogenesis (VWF-Ag: p = 0.03; soluble vascular endothelial growth factor receptor 1: p = 0.03), and bile acids (p = 0.04) were dynamic modulators of PH. The LSM-HVPG validation cohort confirmed these and also indicated IL-6 (p = 0.008) and hyaluronic acid (HA: p0.001) as dynamic PH components.The relative contribution of "static" fibrosis on PH severity varies by type of liver injury. Next to hyperdynamic circulation, increased bile acids, VWF-Ag, IL-6, and HA seem to indicate a pronounced dynamic component of PH in patients.

Graphical Abstract, Background Calcification propensity is associated with the risk for cardiovascular events and death in end-stage renal disease patients. Here we investigated the effect of lowering serum phosphate with oral phosphate binder therapy on calcification propensity. Methods We performed an open-label, randomized, controlled, crossover study in chronic haemodialysis patients with hyperphosphataemia. Patients (n = 39) were randomized in a 1:1 ratio to either low-dose (250 mg/day) sucroferric oxyhydroxide (SO) followed by high-dose (2000 mg/day) SO or vice versa, with washout phases before and after SO treatment. The primary endpoint was changed in calcification propensity as measured by calciprotein particle formation time (T50 test) between washout and high-dose SO treatment in patients with ≥85% adherence to the prescribed SO dose (per-protocol analysis). Results In the primary per-protocol analysis (n = 28), 2000 mg/day SO treatment resulted in a mean increase in T50 of 66 min (95% CI 49–84 min, P

The targeted delivery of tumor necrosis factor-α (TNF-α) with antibodies specific to splice isoforms of fibronectin [e.g., F8-TNF, specific to the extra-domain A (EDA) domain of fibronectin] has already shown efficacy against experimental sarcomas but has not yet been investigated in orthotopic sarcomas. Here, we investigated F8-TNF in a syngeneic K7 M2–derived orthotopic model of osteosarcoma as a treatment against pulmonary metastases, the most frequent cause of osteosarcoma-related death. Immunofluorescence on human osteosarcoma tissue confirmed the presence of EDA in primary tumors (PTs) as well as metastases. In mice, the efficacy of F8-TNF against PTs and early pulmonary metastases was evaluated. Intratibial PT growth was not affected by F8-TNF, yet early micrometastases were reduced possibly due to an F8-TNF–dependent attraction of pulmonary CD4+, CD8+, and natural killer cells. Furthermore, immunofluorescence revealed stronger expression of EDA in early pulmonary metastases compared with PT tissue. To study progressing pulmonary metastases, a hind limb amputation model was established, and the efficacy of F8-TNF, alone or combined with doxorubicin, was investigated. Despite the presence of EDA in metastases, no inhibition of progressive metastatic growth was detected. No significant differences in numbers of CD4+ or CD8+ cells or F4/80+ and Ly6G+ myeloid-derived cells were observed, although a strong association between metastatic growth and presence of pulmonary Ly6G+ myeloid-derived cells was detected. In summary, these findings demonstrate the potential of F8-TNF in activating the immune system and reducing early metastatic growth yet suggest a lack of efficacy of F8-TNF alone or combined with doxorubicin against progressing osteosarcoma metastases.

Current osteosarcoma therapies cause severe treatment-related side effects and chemoresistance, and have low success rates. Consequently, alternative treatment options are urgently needed. Photodynamic therapy (PDT) is a minimally invasive, local therapy with proven clinical efficacy for a variety of tumor types. PDT is cytotoxic, provokes anti-vascular effects and stimulates tumor cell targeting mechanisms of the immune system and, consequently, has potential as a novel therapy for osteosarcoma patients. This study investigated the uptake and the dark- and phototoxicity and cytotoxic mechanisms of the photosensitizer (PS) 5,10,15,20-tetrakis(meta-hydroxyphenyl) chlorine (mTHPC, Foscan) and a liposomal mTHPC formulation (Foslip) in the human 143B and a mouse K7M2-derived osteosaroma cell line (K7M2L2) in vitro. Secondly the tumor- and metastasis-suppressive efficacies of mTHPC formulations based PDT and associated mechanisms in intratibial, metastasizing osteosarcoma mouse models (143B/SCID and syngeneic K7M2L2/BALB/c) were studied. The uptake of Foscan and Foslip in vitro was time- and dose-dependent and resulted in mTHPC and light dose-dependent phototoxicity associated with apoptosis. In vivo, the uptake of both i.v. administered mTHPC formulations was higher in tumor than in healthy control tissue. PDT caused significant (Foscan P

Current osteosarcoma therapies cause severe treatment-related side effects and chemoresistance, and have low success rates. Consequently, alternative treatment options are urgently needed. Photodynamic therapy (PDT) is a minimally invasive, local therapy with proven clinical efficacy for a variety of tumor types. PDT is cytotoxic, provokes anti-vascular effects and stimulates tumor cell targeting mechanisms of the immune system and, consequently, has potential as a novel therapy for osteosarcoma patients. This study investigated the uptake and the dark- and phototoxicity and cytotoxic mechanisms of the photosensitizer (PS) 5,10,15,20-tetrakis(meta-hydroxyphenyl) chlorine (mTHPC, Foscan) and a liposomal mTHPC formulation (Foslip) in the human 143B and a mouse K7M2-derived osteosaroma cell line (K7M2L2) in vitro. Second, the tumor- and metastasis-suppressive efficacies of mTHPC formulations based PDT and associated mechanisms in intratibial, metastasizing osteosarcoma mouse models (143B/SCID and syngeneic K7M2L2/BALB/c) were studied. The uptake of Foscan and Foslip in vitro was time- and dose-dependent and resulted in mTHPC and light dose-dependent phototoxicity associated with apoptosis. In vivo, the uptake of both i.v. administered mTHPC formulations was higher in tumor than in healthy control tissue. PDT caused significant (Foscan p 0.05, Foslip p 0.001) tumor growth inhibition in both models. A significant (Foscan p 0.001, Foslip p 0.001) immune system-dependent suppression of lung metastasis was only observed in the K7M2L2/BALB/c model and was associated with a marked infiltration of T-lymphocytes at the primary tumor site. In conclusion, mTHPC-based PDT is effective in clinically relevant experimental osteosarcoma and suppresses lung metastasis in immunocompetent mice with beneficial effects of the liposomal mTHPC formulation Foslip.

Reliable prognostic factors for the outcome of patients with osteosarcoma (OS) remain elusive. We analyzed the relationship between immunohistochemical expression of deoxyribonucleic acid (DNA) mismatch repair (MMR) proteins, MutS protein homolog 2 (MSH2) and MSH6 using a tissue microarray (TMA) with respect to OS patient demographics and survival time.We retrieved tumor tissue specimens from bone tissue originating from surgical primary tumor specimens of OS patients to generate a TMA and stained sections with antibodies against MSH2 and MSH6.Tumor resections of 67 patients with a mean follow-up of 98 months were evaluated. MSH2 was expressed in nine (13%), MSH6 in ten (15%) and combined MSH2 and MSH6 (MSH2/6) in six (9%) patients. Significantly shorter survival times were associated with expression of MSH6, MSH2/6, as well as simultaneous non-response to chemotherapy and presence of metastasis.The survival time of patients with OS may be predicted by local expression of MSH6 and MSH2/6 in surgical primary tumor resections. This study shows the prognostic value of the local expression of DNA MMR proteins, as markers for poor prognosis of OS patients.

Neoadjuvant chemotherapy in osteosarcoma increased the long-term survival of patients with localized disease considerably but metastasizing osteosarcoma remained largely treatment resistant. Neuropilins, transmembrane glycoproteins, are important receptors for VEGF dependent hyper-vascularization in tumor angiogenesis and their aberrant expression promotes tumorigenesis and metastasis in many solid tumors. Our analysis of Neuropilin-1 (NRP1) and Neuropilin-2 (NRP2) immunostaining in a tissue microarray of 66 osteosarcoma patients identified NRP2 as an indicator of poor overall, metastasis-free and progression free survival while NRP1 had no predictive value. Patients with tumors that expressed NRP2 in the absence of NRP1 had a significantly worse prognosis than NRP1(-)/NRP2(-), NRP1(+) or NRP1(+)/NRP2(+) tumors. Moreover, patients with overt metastases and with NRP2-positive primary tumors had a significantly shorter survival rate than patients with metastases but NRP2-negative tumors. Furthermore, the expression of both NRP1 and NRP2 in osteosarcoma cell lines correlated to a variable degree with the metastatic potential of the respective cell line. To address the functional relevance of Neuropilins for VEGF signaling we used shRNA mediated down-regulation and blocking antibodies of NRP1 and NRP2 in the metastatic 143B and HuO9-M132 cell lines. In 143B cells, VEGFA signaling monitored by AKT phosphorylation was more inhibited by blocking of NRP1, whereas in HuO9-M132 cells NRP2 blocking was more effective indicating that NRP1 and NRP2 can substitute each other in the functional interaction with VEGFR1. Altogether, these data point to NRP2 as a powerful prognostic marker in osteosarcoma and together with NRP1 as a novel target for tumor-suppressive therapy.

It is hardly possible to define osteosarcoma (OS) patients at greatest risk for non-response to chemotherapy, metastasis and short survival times. Our goal was the investigation of local expression of insulin-like growth factor (IGF-1) with regard to survival time of OS patients using a tissue microarray (TMA).Tumor tissue specimens from surgical primary tumor resections were collected from patients with OS. A TMA was composed, sections were stained with rabbit anti-IGF-1 and grading was performed. Statistics involved Kaplan-Meier curves and the log-rank test.We analyzed immunohistochemical expression of local IGF-1 on a TMA based on surgical primary tumor resections of 67 OS patients. The mean clinical follow-up time was 98 months. Twenty-two (33%) OS patients stained negatively and 44 (66%) OS patients stained positively for IGF-1. Significantly shorter survival was detected with expression of IGF-1 (p=0.007). The 5-year survival rate for patients expressing IGF-1 was 63% compared to 92% in patients without expression of IGF-1. Non-responders to chemotherapy and patients with metastasis, who also stained positively for IGF-1 manifested a significantly (p=0.002 and p0.0001, respectively) shorter survival.Expression of local IGF-1 in primary tumor tissue appears to significantly affect the aggressiveness of OS, may predict survival time and, above all, may discriminate patients with non-response to chemotherapy and metastasis. This represents the basis for successful patient selection with regard to the decision process for or against chemotherapy and the choice of the most effective therapeutic drug. It may be a more important marker of tumor progression and indicator of prognosis than serum IGF-1. Novel tumor markers and therapeutic agents targeting the local IGF-1 pathway may increase the likelihood of therapeutic success.

Osteosarcoma (OS) is a malignancy of bone and five-year survival rates are still at a low 20% if metastatic disease is present. Metastatic OSs most frequently metastasize to the lung. TNF-α is a powerful cytokine already shown to be active against many cancers. Nevertheless, TNF-α is highly toxic at clinically relevant concentrations. To overcome this limitation, targeted forms of TNF-α can be used. In this study, targeting is achieved through linking TNF-α to the antibody fragment F8, targeting the EDA-domain of fibronectin. To test the efficacy of F8-TNF-α against OS, we sought to investigate the effects of F8-TNF-α in an orthotopic syngeneic OS model. In addition to evaluating compound efficacy, we also tested whether the route of administration (systemic versus local application) can even further increase treatment efficacy. Ultimately, we aimed at measuring treatment success by assessing different stages of OS progression, especially metastatic disease. To show clinical relevance, we performed immunofluorescence (IF) of EDA on primary human OS samples. In a second step, we evaluated drug efficacy in a syngeneic OS mouse model. K7M2L2 OS cells, stably infected to express the marker proteins lacZ and mCherry, were orthotopically injected into the tibia of mice. Once primary tumors were detectable, the mice were treated with either PBS or F8-TNF-α. The compound was given either systemically (intravenously (i.v.), tail vein) or locally (intraarterial (i.a.), femoral artery). Four days after the last treatment, all mice were sacrificed, terminal blood sampling was performed and blood samples were analyzed for the presence of circulating tumor cells (CTCs) via FACS/mCherry fluorescence. Following X-gal staining, metastases on the surface of lungs were counted. EDA and infiltration of immune cells (CD4+, CD8+, natural killer (NK) cells, CD19+) were assessed histologically. Using IF, we detected strong expression of EDA in primary tumors as well as bone and lung metastases from human OS patients. Preliminary results from our treatment study showed a significant decrease in the number of lung metastases as well as the number of CTCs in mice treated with i.a. F8-TNF-α while the treatment was well tolerated by the mice. The reduction of lung metastases could, at least in part, be explained by a strong expression of EDA in the lung metastases. In addition, significant increases in lung-infiltrating immune cells were observed after administration of F8-TNF-α (i.v. F8- TNF-α: increased CD4+ T-cells; i.a. F8-TNF-α: increased NK cells). Although significant reductions in numbers of metastases and CTCs were detected, F8-TNF-α did not significantly impair the growth of the primary tumors. In conclusion, our study demonstrates a reduction of CTCs as well as lung metastases after administration of F8-TNF-α and thus, provides a rationale for further studying the anti-metastatic effects of this compound in a clinically relevant model of OS. Citation Format: Bernhard Robl, Sander Martijn Botter, Aleksandar Boro, Dario Neri, Bruno Fuchs. Targeting of metastatic osteosarcoma with F8-TNF-alpha in an orthotopic mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 589.

We show that after tail amputation in Ambystoma mexicanum (Axolotl) the correct number and spacing of dorsal root ganglia are regenerated. By transplantation of spinal cord tissue and nonclonal neurospheres, we show that the central spinal cord represents a source of peripheral nervous system cells. Interestingly, melanophores migrate from preexisting precursors in the skin. Finally, we demonstrate that implantation of a clonally derived spinal cord neurosphere can result in reconstitution of all examined cell types in the regenerating central spinal cord, suggesting derivation of a cell with spinal cord stem cell properties.

Background Primary bone cancers are among the deadliest cancer types in adolescents, with osteosarcomas being the most prevalent form. Osteosarcomas are commonly treated with multi-drug neoadjuvant chemotherapy and therapy success as well as patient survival is affected by the presence of tumor suppressors. In order to assess the prognostic value of tumor-suppressive biomarkers, primary osteosarcoma tissues were analyzed prior to and after neoadjuvant chemotherapy. Methods We constructed a tissue microarray from high grade osteosarcoma samples, consisting of 48 chemotherapy naïve biopsies (BXs) and 47 tumor resections (RXs) after neoadjuvant chemotherapy. We performed immunohistochemical stainings of P53, P16, maspin, PTEN, BMI1 and Ki67, characterized the subcellular localization and related staining outcome with chemotherapy response and overall survival. Binary logistic regression analysis was used to analyze chemotherapy response and Kaplan-Meier-analysis as well as the Cox proportional hazards model was applied for analysis of patient survival. Results No significant associations between biomarker expression in BXs and patient survival or chemotherapy response were detected. In univariate analysis, positive immunohistochemistry of P53 (P = 0.008) and P16 (P16; P = 0.033) in RXs was significantly associated with poor survival prognosis. In addition, presence of P16 in RXs was associated with poor survival in multivariate regression analysis (P = 0.003; HR = 0.067) while absence of P16 was associated with good chemotherapy response (P = 0.004; OR = 74.076). Presence of PTEN on tumor RXs was significantly associated with an improved survival prognosis (P = 0.022). Conclusions Positive immunohistochemistry (IHC) of P16 and P53 in RXs was indicative for poor overall patient survival whereas positive IHC of PTEN was prognostic for good overall patient survival. In addition, we found that P16 might be a marker of osteosarcoma chemotherapy resistance. Therefore, our study supports the use of tumor RXs to assess the prognostic value of biomarkers. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1397-4) contains supplementary material, which is available to authorized users.