Intravenous Opioid Medication with Piritramide Reduces the Risk of Pneumothorax During CT-Guided Percutaneous Core Biopsy of the Lung.

PURPOSE: CT-guided percutaneous core biopsy of the lung is usually performed under local anesthesia, but can also be conducted under additional systemic opioid medication. The purpose of this retrospective study was to assess the effect of intravenous piritramide application on the pneumothorax rate and to identify risk factors for post-biopsy pneumothorax. MATERIALS AND METHODS: One hundred and seventy-one core biopsies of the lung were included in this retrospective single center study. The incidence of pneumothorax and chest tube placement was evaluated. Patient-, procedure- and target-related variables were analyzed by univariate and multivariable logistic regression analysis. RESULTS: The overall incidence of pneumothorax was 39.2% (67/171). The pneumothorax rate was 31.5% (29/92) in patients who received intravenous piritramide and 48.1% (38/79) in patients who did not receive piritramide. In multivariable logistic regression analysis periinterventional piritramide application proved to be the only independent factor to reduce the risk of pneumothorax (odds ratio 0.46, 95%-confidence interval 0.24, 0.88; p = 0.018). Two or more pleura passages (odds ratio 3.38, 95%-confidence interval: 1.15, 9.87; p = 0.026) and prone position of the patient (odds ratio 2.27, 95%-confidence interval: 1.04, 4.94; p = 0.039) were independent risk factors for a higher pneumothorax rate. CONCLUSION: Procedural opioid medication with piritramide proved to be a previously undisclosed factor decreasing the risk of pneumothorax associated with CT-guided percutaneous core biopsy of the lung. LEVEL OF EVIDENCE 4: small study cohort.

Postoperative Epidural Fibrosis: Challenges and Opportunities - A Review.

Postoperative epidural fibrosis (EF) is still a major limitation to the success of spine surgery. Fibrotic adhesions in the epidural space, initiated via local trauma and inflammation, can induce difficult-to-treat pain and constitute the main cause of failed back surgery syndrome, which not uncommonly requires operative revision. Manifold agents and methods have been tested for EF relief in order to mitigate this longstanding health burden and its socioeconomic consequences. Although several promising strategies could be identified, few have thus far overcome the high translational hurdle, and there has been little change in standard clinical practice. Nonetheless, notable research progress in the field has put new exciting avenues on the horizon. In this review, we outline the etiology and pathogenesis of EF, portray its clinical and surgical presentation, and critically appraise current efforts and novel approaches toward enhanced prevention and treatment.

Positive Vasoreactivity Testing in Pulmonary Arterial Hypertension: Therapeutic Consequences, Treatment Patterns, and Outcomes in the Modern Management Era.

BACKGROUND: Among patients with pulmonary arterial hypertension (PAH), acute vasoreactivity testing during right heart catheterization may identify acute vasoresponders, for whom treatment with high-dose calcium channel blockers (CCBs) is recommended. However, long-term outcomes in the current era remain largely unknown. We sought to evaluate the implications of acute vasoreactivity response for long-term response to CCBs and other outcomes. METHODS: Patients diagnosed with PAH between January 1999 and December 2018 at 15 pulmonary hypertension centers were included and analyzed retrospectively. In accordance with current guidelines, acute vasoreactivity response was defined by a decrease of mean pulmonary artery pressure by ≥10 mm Hg to reach <40 mm Hg, without a decrease in cardiac output. Long-term response to CCBs was defined as alive with unchanged initial CCB therapy with or without other initial PAH therapy and World Health Organization functional classification I/II or low European Society of Cardiology/European Respiratory Society risk status at 12 months after initiation of CCBs. Patients were followed for up to 5 years; clinical measures, outcome, and subsequent treatment patterns were captured. RESULTS: Of 3702 patients undergoing right heart catheterization for PAH diagnosis, 2051 had idiopathic, hereditary, or drug-induced PAH, of whom 1904 (92.8%) underwent acute vasoreactivity testing. A total of 162 patients fulfilled acute vasoreactivity response criteria and received an initial CCB alone (n=123) or in combination with another PAH therapy (n=39). The median follow-up time was 60.0 months (interquartile range, 30.8-60.0), during which overall survival was 86.7%. At 12 months, 53.2% remained on CCB monotherapy, 14.7% on initial CCB plus another initial PAH therapy, and the remaining patients had the CCB withdrawn or PAH therapy added. CCB long-term response was found in 54.3% of patients. Five-year survival was 98.5% in long-term responders versus 73.0% in nonresponders. In addition to established vasodilator responder criteria, pulmonary artery compliance at acute vasoreactivity testing, low risk status and NT-proBNP (N-terminal pro-B-type natriuretic peptide) levels at early follow-up correlated with long-term response and predicted survival. CONCLUSIONS: Our data display heterogeneity within the group of vasoresponders, with a large subset failing to show a sustained satisfactory clinical response to CCBs. This highlights the necessity for comprehensive reassessment during early follow-up. The use of pulmonary artery compliance in addition to current measures may better identify those likely to have a good long-term response.

Comparison of ex vivo bioluminescence imaging, Alu-qPCR and histology for the quantification of spontaneous lung and bone metastases in subcutaneous xenograft mouse models.

Bioluminescence imaging (BLI) is a non-invasive state-of-the-art-method for longitudinal tracking of tumor cells in mice. The technique is commonly used to determine bone metastatic burden in vivo and also suitable ex vivo to detect even smallest bone micro-metastases in spontaneous metastasis xenograft models. However, it is unclear to which extent ex vivo BLI correlates with alternative methods for metastasis quantification. Here, we compared ex vivo BLI, human DNA-based Alu-qPCR, and histology for the quantification of bone vs. lung metastases, which are amongst the most common sites of metastasis in prostate cancer (PCa) patients and spontaneous PCa xenograft models. Data from 93 immunodeficient mice were considered, each of which were subcutaneously injected with luciferase/RGB-labeled human PCa PC-3 cells. The primary tumors were resected at ~ 0.75 cm³ and mice were sacrificed ~ 3 weeks after surgery and immediately examined by ex vivo BLI. Afterwards, the right lungs and hind limbs with the higher BLI signal (BLIHi bone) were processed for histology, whereas the left lung lobes and hind limbs with the lower BLI signal (BLILo bone) were prepared for Alu-qPCR. Our data demonstrate remarkable differences in the correlation coefficients of the different methods for lung metastasis detection (r ~ 0.8) vs. bone metastasis detection (r ~ 0.4). However, the BLI values of the BLIHi and BLILo bones correlated very strongly (r ~ 0.9), indicating that the method per se was reliable under identical limitations; the overall level of metastasis to contralateral bones was astonishingly similar. Instead, the level of lung metastasis only weakly to moderately correlated with the level of bone metastasis formation. Summarized, we observed a considerable discrepancy between ex vivo BLI and histology/Alu-qPCR in the quantification of bone metastases, which was not observed in the case of lung metastases. Future studies using ex vivo BLI for bone metastasis quantification should combine multiple methods to accurately determine metastatic load in bone samples.

Microplastics role in cell migration and distribution during cancer cell division.

Amidst the global plastic pollution crisis, the gastrointestinal tract serves as the primary entry point for daily exposure to micro- and nanoplastics. We investigated the complex dynamics between polystyrene micro- and nanoplastics (PS-MNPs) and four distinct human colorectal cancer cell lines (HT29, HCT116, SW480, and SW620). Our findings revealed a significant size- and concentration dependent uptake of 0.25, 1, and 10 µm PS-MNPs across all cell lines, with HCT116 cells exhibiting the highest uptake rates. During cell division, particles were distributed between mother and daughter cells. Interestingly, we observed no signs of elimination from the cells. Short-term exposure to 0.25 µm particles significantly amplified cell migration, potentially leading to pro-metastatic effects. Particles demonstrated high persistence in 2D and 3D cultures, and accumulation in non-proliferating parts of spheroids, without interfering with cell proliferation or division. Our study unveils the disturbing fact of the persistence and bioaccumulation of MNPs in colorectal cancer cell lines, key toxicological traits under REACH (Regulation concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals). Our observations underscore the potential of MNPs as hidden catalysts for tumor progression, particularly through enhancing cell migration and possibly fueling metastasis - a finding that sheds light on a significant and previously underexplored area of concern.

Real-World Management of Patients with Pulmonary Arterial Hypertension: Insights from EXPOSURE.

INTRODUCTION: Further insights into real-world management and outcomes of patients with pulmonary arterial hypertension (PAH) are needed. This interim analysis of the ongoing, multicentre, prospective EXPOSURE (EUPAS19085) observational study describes characteristics, treatment patterns and outcomes of patients with PAH initiating a new PAH-specific therapy in Europe/Canada. METHODS AND RESULTS: All analyses were descriptive. In total, 1944 patients with follow-up information were included; the majority were female, with World Health Organization functional class II/III symptoms and with idiopathic PAH or connective tissue disease-associated PAH. Most incident patients (N = 1100; diagnosed for ≤ 6 months) initiated treatment as monotherapy (48%) or double therapy (43%). Of those initiating monotherapy, 38% (199/530) escalated to double therapy (median [Q1, Q3] time to escalation 3.4 [1.9, 6.6] months), and of those initiating double therapy, 17% (78/457) escalated to triple therapy (median [Q1, Q3] time to escalation 7.0 [3.4, 12.7] months) during the observation period (median [Q1, Q3]: 17.0 [7.5, 29.9] months). The majority of the 834 prevalent patients (diagnosed > 6 months) entered the study on initiation of combination therapy and most did not change treatment regimen during the observation period (median [Q1, Q3]: 19.6 [10.2, 32.2] months). One-year survival was 88% for incident patients and 90% for prevalent patients. CONCLUSIONS: Results from EXPOSURE suggest a shift towards combination therapy and the alignment of real-world treatment patterns with current guideline recommendations. While survival estimates are encouraging, the extent of monotherapy use at treatment initiation and follow-up highlight an opportunity for further improvements through optimisation of treatment strategies in line with current guidelines. A graphical abstract is also available with this article. TRIAL REGISTRATION NUMBER: EUPAS19085.

Pulmonary arterial hypertension (PAH) is a progressive disease. Clinical guidelines recommend that most patients start treatment with a combination of different PAH medications. While there is no cure for PAH, these medications help to control symptoms and slow disease worsening. To understand treatments currently used in clinical practice, we analysed data from EXPOSURE (EUPAS19085), an ongoing study collecting information from patients starting a new PAH medication in Europe and Canada. Most patients in the study were female, with World Health Organization functional class II/III symptoms, and idiopathic (unknown cause) PAH or PAH associated with connective tissue disorders. Among 1100 patients who were 'recently diagnosed' (diagnosed with PAH in the past 6 months), 88% were alive after 1 year. We found that 48% started treatment with one PAH medication, and 38% of those patients had a second medication prescribed within a median period of 3 months. Among the 457 'recently diagnosed' patients treated with two PAH medications when they entered the study, 17% had a third medication prescribed within a median period of 7 months. Among 834 patients with 'established PAH' (diagnosed more than 6 months ago), 90% were alive after 1 year. Most entered the study when they started a third medication and did not have further changes in treatment. Our findings show that patients with PAH are often treated with one medication in clinical practice as well as a combination of medications. While survival rates are encouraging, the extent to which one PAH medication is used suggests there is room for treatment improvement.

ALDH1A1 drives prostate cancer metastases and radioresistance by interplay with AR- and RAR-dependent transcription.

Rationale: Current therapies for metastatic osseous disease frequently fail to provide a durable treatment response. To date, there are only limited therapeutic options for metastatic prostate cancer, the mechanisms that drive the survival of metastasis-initiating cells are poorly characterized, and reliable prognostic markers are missing. A high aldehyde dehydrogenase (ALDH) activity has been long considered a marker of cancer stem cells (CSC). Our study characterized a differential role of ALDH1A1 and ALDH1A3 genes as regulators of prostate cancer progression and metastatic growth. Methods: By genetic silencing of ALDH1A1 and ALDH1A3 in vitro, in xenografted zebrafish and murine models, and by comparative immunohistochemical analyses of benign, primary tumor, and metastatic specimens from patients with prostate cancer, we demonstrated that ALDH1A1 and ALDH1A3 maintain the CSC phenotype and radioresistance and regulate bone metastasis-initiating cells. We have validated ALDH1A1 and ALDH1A3 as potential biomarkers of clinical outcomes in the independent cohorts of patients with PCa. Furthermore, by RNAseq, chromatin immunoprecipitation (ChIP), and biostatistics analyses, we suggested the molecular mechanisms explaining the role of ALDH1A1 in PCa progression. Results: We found that aldehyde dehydrogenase protein ALDH1A1 positively regulates tumor cell survival in circulation, extravasation, and metastatic dissemination, whereas ALDH1A3 plays the opposite role. ALDH1A1 and ALDH1A3 are differentially expressed in metastatic tumors of patients with prostate cancer, and their expression levels oppositely correlate with clinical outcomes. Prostate cancer progression is associated with the increasing interplay of ALDH1A1 with androgen receptor (AR) and retinoid receptor (RAR) transcriptional programs. Polo-like kinase 3 (PLK3) was identified as a transcriptional target oppositely regulated by ALDH1A1 and ALDH1A3 genes in RAR and AR-dependent manner. PLK3 contributes to the control of prostate cancer cell proliferation, migration, DNA repair, and radioresistance. ALDH1A1 gain in prostate cancer bone metastases is associated with high PLK3 expression. Conclusion: This report provides the first evidence that ALDH1A1 and PLK3 could serve as biomarkers to predict metastatic dissemination and radiotherapy resistance in patients with prostate cancer and could be potential therapeutic targets to eliminate metastasis-initiating and radioresistant tumor cell populations.

Spontaneous metastasis xenograft models link CD44 isoform 4 to angiogenesis, hypoxia, EMT and mitochondria-related pathways in colorectal cancer.

Hematogenous metastasis limits the survival of colorectal cancer (CRC) patients. Here, we illuminated the roles of CD44 isoforms in this process. Isoforms 3 and 4 were predominantly expressed in CRC patients. CD44 isoform 4 indicated poor outcome and correlated with epithelial-mesenchymal transition (EMT) and decreased oxidative phosphorylation (OxPhos) in patients; opposite associations were found for isoform 3. Pan-CD44 knockdown (kd) independently impaired primary tumor formation and abrogated distant metastasis in CRC xenografts. The xenograft tumors mainly expressed the clinically relevant CD44 isoforms 3 and 4. Both isoforms were enhanced in the paranecrotic, hypoxic tumor regions but were generally absent in lung metastases. Upon CD44 kd, tumor angiogenesis was increased in the paranecrotic areas, accompanied by reduced hypoxia-inducible factor-1α and CEACAM5 but increased E-cadherin expression. Mitochondrial genes and proteins were induced upon pan-CD44 kd, as were OxPhos genes. Hypoxia increased VEGF release from tumor spheres, particularly upon CD44 kd. Genes affected upon CD44 kd in xenografts specifically overlapped concordantly with genes correlating with CD44 isoform 4 (but not isoform 3) in patients, validating the clinical relevance of the used model and highlighting the metastasis-promoting role of CD44 isoform 4.

[Targeted therapy for pulmonary arterial hypertension in patients without comorbidities]. / Gezielte medikamentöse Therapie der pulmonalarteriellen Hypertonie bei Patient*innen ohne Komorbiditäten.

The 2022 guidelines on pulmonary hypertension from the European Society of Cardiology (ESC) and the European Respiratory Society (ERS) provide therapeutic strategies that account for the variability in the clinical presentation of newly diagnosed patients. We summarize treatment recommendations for pulmonary arterial hypertension (PAH) in patients without significant comorbidities, particularly for idiopathic, hereditary, drug/toxin-induced, or connective tissue disease-associated PAH. In this group of patients, multidimensional assessments for short-term mortality risk guide initial treatment decisions and treatment decisions during follow-up. Upfront dual combination therapy (phosphodiesterase type-5 inhibitor and endothelin receptor antagonist) is recommended for low- and intermediate-risk patients, and triple therapy including a parenteral prostacyclin should be considered in high- or intermediate-high-risk patients. If a low or intermediate-low-risk profile cannot be achieved during therapy, sequential add-on therapy escalation with parenteral prostacyclin or a prostacyclin receptor agonist should be considered, and switching from a phosphodiesterase type-5 inhibitor to a guanylate cyclase stimulator may also be considered.

[Medication treatment in pulmonary arterial hypertension with comorbidities - Which phenotypes and points to consider]. / Medikamentöse Therapie der pulmonalarteriellen Hypertonie mit Komorbiditäten ­ Welche Phänotypen gibt es, was gibt es zu beachten?

Within the last decade, the age at diagnosis of patients with pulmonary arterial hypertension has increased, which led to a change of the clinical phenoype being associated with more comorbidities. Cluster analyses of registry data have identified cardiac, cardio-pulmonary and classical phenotypes of pulmonary arterial hypertension.Subgroup analyses of randomised controlled trials and registry data indicate, that in patients with pulmonary arterial hypertension and cardiac comorbidities, especially the left-heart phenotype, a closely supervised combination treatment may be considered. The 4-strata model may be used for monitoring and risk stratification in these patients. Individual treatment decisions should be made in the pulmonary hypertension centre. Factors such as hemodynamics, age, phenotype, number and severity of comorbidities, therapy response, adverse reactions and the wish of the patient should be considered.Prospective, randomized studies to assess the efficacy and safety profile of pulmonary arterial hypertension treatments are desirable. Patients with a mainly pulmonary phenotype (smoking, diffusion capacity of the lung < 45 % and/or lung parenchymal changes) may have less benefit of oral medication.

Treatment strategies and survival of patients with connective tissue disease and pulmonary arterial hypertension: A COMPERA analysis.

OBJECTIVES: Pulmonary arterial hypertension (PAH) occurs in various connective tissue diseases (CTDs). We sought to assess contemporary treatment patterns and survival of patients with various forms of CTD-PAH. METHODS: We analysed data from COMPERA, a European pulmonary hypertension registry, to describe treatment strategies and survival in patients with newly diagnosed PAH associated with systemic sclerosis (SSc), systemic lupus erythematosus (SLE), mixed CTD (MCTD), undifferentiated CTD (UCTD), and other types of CTD. All-cause mortality was analysed according to the underlying CTD. For patients with SSc-PAH, we also assessed survival according to initial therapy with endothelin receptor antagonists (ERA), phosphodiesterase type 5 inhibitors (PDE5i), or a combination of these two drug classes. RESULTS: This analysis included 607 patients with CTD-PAH. Survival estimates at 1-, 3- and 5-years for SSc-PAH (n = 390) were 85%, 59% and 42%; for SLE-PAH (n = 34), 97%, 77% and 61%; for MCTD-PAH (n = 33), 97%, 70% and 59%, for UCTD (n = 60), 88%, 67%, and 52%; and for other CTD (n = 90), 92%, 69%, and 55%, respectively. After multivariable adjustment, the survival of patients with SSc-PAH was significantly worse compared with the other conditions (p= 0.001). In these patients, the survival estimates were significantly better with initial ERA/PDE5i combination therapy than with initial ERA or PDE5i monotherapy (p= 0.016 and p= 0.012, respectively). CONCLUSIONS: Mortality remains high in patients with CTD-PAH, especially for patients with SSc-PAH. However, for patients with SSc-PAH, our results suggest that long-term survival may be improved with initial ERA/PDE5i combination therapy compared with initial monotherapy.

Identification of potential classes of glycoligands mediating dynamic endothelial adhesion of human tumor cells.

One critical step of metastasis formation is the extravasation of circulating tumor cells from the bloodstream. This process requires the dynamic interaction of cell adhesion molecules like E-selectin on endothelial cells with carbohydrate ligands on tumor cells. To characterize these glycans in a comprehensible approach, the rolling, tethering, and firm adhesion of nine human tumor cell lines on human umbilical vein endothelial cells was analyzed using laminar flow adhesion assays. The tumor cell lines were grouped into three subsets by their canonical E-selectin ligand status (sialyl-Lewis A and X +/+, -/+, -/-) and their adhesiveness was compared after enzymatic, pharmacologic, chemical treatment or antibody blockade of the tumor cells or endothelial cells, respectively. Tumor cells were also screened regarding their glycosyltransferase expression profile. We found that although E-selectin and terminal α2,3-sialic acid largely determined firm adhesion, adhesive events did not exclusively depend on the presence of sialyl-Lewis A and/or sialyl-Lewis X. Nevertheless, two of the three sialyl-Lewis A/X-/- tumor cells additionally or fully depended on vascular cell adhesion molecule-1 for firm adhesion. The significance of O-GalNAc- and N-glycans for adhesion varied remarkably among the tumor cells. The sialyl-Lewis A/X+/+ subset showed glycoprotein-independent adhesion, suggesting a role of glycolipids as well. All sialyl-Lewis A/X-/- tumor cells lacked FUT3 and FUT7 expression as opposed to sialyl-Lewis A/X+/+ or -/+ cell lines. In summary, the glycans on tumor cells mediating endothelial adhesion are not as much restricted to sialyl-Lewis A /X as previously assumed. The present study specifically suggests α2,3-linked sialic acid, O-GalNAc glycans, glycosphingolipids, and FUT3/FUT7 products as promising targets for future studies.

Lipidomics for diagnosis and prognosis of pulmonary hypertension.

Background: Pulmonary hypertension (PH) poses a significant health threat with high morbidity and mortality, necessitating improved diagnostic tools for enhanced management. Current biomarkers for PH lack functionality and comprehensive diagnostic and prognostic capabilities. Therefore, there is a critical need to develop biomarkers that address these gaps in PH diagnostics and prognosis. Methods: To address this need, we employed a comprehensive metabolomics analysis in 233 blood based samples coupled with machine learning analysis. For functional insights, human pulmonary arteries (PA) of idiopathic pulmonary arterial hypertension (PAH) lungs were investigated and the effect of extrinsic FFAs on human PA endothelial and smooth muscle cells was tested in vitro. Results: PA of idiopathic PAH lungs showed lipid accumulation and altered expression of lipid homeostasis-related genes. In PA smooth muscle cells, extrinsic FFAs caused excessive proliferation and endothelial barrier dysfunction in PA endothelial cells, both hallmarks of PAH.In the training cohort of 74 PH patients, 30 disease controls without PH, and 65 healthy controls, diagnostic and prognostic markers were identified and subsequently validated in an independent cohort. Exploratory analysis showed a highly impacted metabolome in PH patients and machine learning confirmed a high diagnostic potential. Fully explainable specific free fatty acid (FFA)/lipid-ratios were derived, providing exceptional diagnostic accuracy with an area under the curve (AUC) of 0.89 in the training and 0.90 in the validation cohort, outperforming machine learning results. These ratios were also prognostic and complemented established clinical prognostic PAH scores (FPHR4p and COMPERA2.0), significantly increasing their hazard ratios (HR) from 2.5 and 3.4 to 4.2 and 6.1, respectively. Conclusion: In conclusion, our research confirms the significance of lipidomic alterations in PH, introducing innovative diagnostic and prognostic biomarkers. These findings may have the potential to reshape PH management strategies.

The chemicals between us-First results of the cluster analyses on anatomy embalming procedures in the German-speaking countries.

Hands-on courses utilizing preserved human tissues for educational training offer an important pathway to acquire basic anatomical knowledge. Owing to the reevaluation of formaldehyde limits by the European Commission, a joint approach was chosen by the German-speaking anatomies in Europe (Germany, Austria, Switzerland) to find commonalities among embalming protocols and infrastructure. A survey comprising 537 items was circulated to all anatomies in German-speaking Europe. Clusters were established for "ethanol"-, formaldehyde-based ("FA"), and "other" embalming procedures, depending on the chemicals considered the most relevant for each protocol. The logistical framework, volumes of chemicals, and infrastructure were found to be highly diverse between the groups and protocols. Formaldehyde quantities deployed per annum were three-fold higher in the "FA" (223 L/a) compared to the "ethanol" (71.0 L/a) group, but not for "other" (97.8 L/a), though the volumes injected per body were similar. "FA" was strongly related to table-borne air ventilation and total fixative volumes ≤1000 L. "Ethanol" was strongly related to total fixative volumes >1000 L, ceiling- and floor-borne air ventilation, and explosion-proof facilities. Air ventilation was found to be installed symmetrically in the mortuary and dissection facilities. Certain predictors exist for the interplay between the embalming used in a given infrastructure and technical measures. The here-established cluster analysis may serve as decision supportive tool when considering altering embalming protocols or establishing joint protocols between institutions, following a best practice approach to cater toward best-suited tissue characteristics for educational purposes, while simultaneously addressing future demands on exposure limits.

Refined risk stratification, current treatment, and new therapeutic approaches in pulmonary arterial hypertension.

The 2022 European Society of Cardiology/European Respiratory Society (ESC/ERS) guidelines for pulmonary hypertension have introduced a refined risk stratification to guide both initial and subsequent treatment of pulmonary arterial hypertension (PAH). The risk stratification at PAH diagnosis still comprises three risk categories (low, intermediate, high) and lists some new parameters. As the estimated 1­year mortality is more than 20% in high-risk patients after diagnosis, an initial triple-combination therapy including parenteral prostacyclin analogues is recommended for this group. All other patients should receive a dual-combination therapy with an endothelin receptor antagonist and a phosphodiesterase­5 inhibitor. However, this approach of initial combination therapy is only recommended for classic PAH, while monotherapy followed by regular follow-up and individualized therapy should be used for patients with cardiopulmonary comorbidities. For PAH patients without cardiopulmonary comorbidities, it is recommended to assess their risk at follow-up with a new 4­strata classification, where the intermediate-risk group is split on the basis of three noninvasive parameters. Importantly, changes from intermediate-high to intermediate-low risk have been shown to be associated with a better prognosis. In addition, the recommendations on treatment escalation became more precise with the addition of a prostacyclin receptor agonist or switching a phosphodiesterase­5 inhibitor to a soluble guanylate cyclase stimulator for intermediate-low risk and proceeding to triple-combination therapy with parenteral prostacyclin analogues already for intermediate-high risk. With sotatercept, the first non-vasodilator PAH treatment will become available in the near future to further enrich our treatment options for this chronic and still severe disease.

Efficacy of zoledronic acid for the elimination of disseminated tumor cells in a clinically relevant, spontaneously metastatic prostate cancer xenograft model.

Bone metastases develop in >90 % of patients with castration-resistant prostate cancer (PCa) through complex interactions between the bone microenvironment and tumor cells. Previous androgen-deprivation therapy (ADT), which is known to cause bone loss, as well as anti-resorptive agents such as zoledronic acid (ZA), used to prevent skeletal complications, may influence these interactions and thereby the growth of disseminated tumor cells (DTC) in the bone marrow (BM). Here, a spontaneously metastatic xenograft tumor model of human PCa was further optimized to mimic the common clinical situation of ADT (castration) combined with primary tumor resection in vivo. The effects of these interventions, alone or in combination with ZA treatment, on tumor cell dissemination to the BM and other distant sites were analyzed. Metastatic burden was quantified by human-specific Alu-qPCR, bioluminescence imaging (BLI), and immunohistochemistry. Further, bone remodeling was assessed by static histomorphometry and serum parameters. Initial comparative analysis between NSG and SCID mice showed that spontaneous systemic dissemination of subcutaneous PC-3 xenograft tumors was considerably enhanced in NSG mice. Primary tumor resection and thereby prolonged observational periods resulted in a higher overall metastatic cell load at necropsy and tumor growth alone caused significant bone loss, which was further augmented by surgical castration. In addition, castrated mice showed a strong trend towards higher bone metastasis loads. Weekly treatment of mice with ZA completely prevented castration- and tumor-induced bone loss but had no effect on bone metastasis burden. Conversely, the total lung metastasis load as determined by BLI was significantly decreased upon ZA treatment. These findings provide a basis for future research on the role of ZA not only in preventing skeletal complications but also in reducing metastasis to other organs.

Tumor cell integrin ß4 and tumor stroma E-/P-selectin cooperatively regulate tumor growth in vivo.

BACKGROUND: The immunological composition of the tumor microenvironment has a decisive influence on the biological course of cancer and is therefore of profound clinical relevance. In this study, we analyzed the cooperative effects of integrin ß4 (ITGB4) on tumor cells and E-/P-selectin on endothelial cells within the tumor stroma for regulating tumor growth by shaping the local and systemic immune environment. METHODS: We used several preclinical mouse models for different solid human cancer types (xenograft and syngeneic) to explore the role of ITGB4 (shRNA-mediated knockdown in tumor cells) and E-/P-selectins (knockout in mice) for tumor growth; effects on apoptosis, proliferation and intratumoral signaling pathways were determined by histological and biochemical methods and 3D in vitro experiments; changes in the intratumoral and systemic immune cell composition were determined by flow cytometry and immunohistochemistry; chemokine levels and their attracting potential were measured by ELISA and 3D invasion assays. RESULTS: We observed a very robust synergism between ITGB4 and E-/P-selectin for the regulation of tumor growth, accompanied by an increased recruitment of CD11b+ Gr-1Hi cells with low granularity (i.e., myeloid-derived suppressor cells, MDSCs) specifically into ITGB4-depleted tumors. ITGB4-depleted tumors undergo apoptosis and actively attract MDSCs, well-known to promote tumor growth in several cancers, via increased secretion of different chemokines. MDSC trafficking into tumors crucially depends on E-/P-selectin expression. Analyses of clinical samples confirmed an inverse relationship between ITGB4 expression in tumors and number of tumor-infiltrating leukocytes. CONCLUSIONS: These findings suggest a distinct vulnerability of ITGB4Lo tumors for MDSC-directed immunotherapies.

Dynamics of CXCR4 positive circulating tumor cells in prostate cancer patients during radiotherapy.

Ablative radiotherapy is a highly efficient treatment modality for patients with metastatic prostate cancer (PCa). However, a subset of patients does not respond. Currently, this subgroup with bad prognosis cannot be identified before disease progression. We hypothesize that markers indicative of radioresistance, stemness and/or bone tropism may have a prognostic potential to identify patients profiting from metastases-directed radiotherapy. Therefore, circulating tumor cells (CTCs) were analyzed in patients with metastatic PCa (n = 24) during radiotherapy with CellSearch, multicolor flow cytometry and imaging cytometry. Analysis of copy-number alteration indicates a polyclonal CTC population that changes after radiotherapy. CTCs were found in 8 out of 24 patients (33.3%) and were associated with a shorter time to biochemical progression after radiotherapy. Whereas the total CTC count dropped after radiotherapy, a chemokine receptor CXCR4-expressing subpopulation representing 28.6% of the total CTC population remained stable up to 3 months. At once, we observed higher chemokine CCL2 plasma concentrations and proinflammatory monocytes. Additional functional analyses demonstrated key roles of CXCR4 and CCL2 for cellular radiosensitivity, tumorigenicity and stem-like potential in vitro and in vivo. Moreover, a high CXCR4 and CCL2 expression was found in bone metastasis biopsies of PCa patients. In summary, panCK+ CXCR4+ CTCs may have a prognostic potential in patients with metastatic PCa treated with metastasis-directed radiotherapy.

Ex Vivo Model of Neuroblastoma Plasticity.

Tumor plasticity is essential for adaptation to changing environmental conditions, in particular during the process of metastasis. In this study, we compared morphological and biochemical differences between LAN-1 neuroblastoma (NB) cells recovered from a subcutaneous xenograft primary tumor (PT) and the corresponding three generations of bone metastasis (BM I-III). Moreover, growth behavior, as well as the response to chemotherapy and immune cells were assessed. For this purpose, F-actin was stained, mRNA and protein expression assessed, and lactate secretion analyzed. Further, we measured adhesion to collagen I, the growth rate of spheroids in the presence and absence of vincristine, and the production of IL-6 by peripheral blood mononuclear cells (PBMCs) co-incubated with PT or BM I-III. Analysis of PT and the three BM generations revealed that their growth rate decreased from PT to BM III, and accordingly, PT cells reacted most sensitively to vincristine. In addition, morphology, adaption to hypoxic conditions, as well as transcriptomes showed strong differences between the cell lines. Moreover, BM I and BM II cells exhibited a significantly different ability to stimulate human immune cells compared to PT and BM III cells. Interestingly, the differences in immune cell stimulation corresponded to the expression level of the cancer-testis antigen MAGE-A3. In conclusion, our ex vivo model allows to analyze the adaption of tumor populations to different microenvironments and clearly demonstrates the strong alteration of tumor cell populations during this process.