Human stromal cells are required for an anti-breast cancer effect of zoledronic acid

Previous studies suggested that bisphosphonate zoledronic acid exerts an antitumor effect by interacting with the microenvironment. In this study, we aimed to elucidate the mechanism behind the anti-breast cancer effect of zoledronic acid.Here we showed that zoledronic acid did not influence in vitro human breast cancer cell survival, but did affect human stromal cell survival. Breast cancer cell death in co-culture with stromal cells was analyzed in vitro by fluorescent microscopy and flowcytometry analysis. In co-culture, the addition of stromal cells to breast cancer cells induced tumor cell death by zoledronic acid, which was abolished by transforming growth factor (TGF)-beta. In the in vivo chicken chorioallantoic membrane model, zoledronic acid reduced the breast cancer cells fraction per tumor only in the presence of human stromal cells. Zoledronic acid decreased TGF-beta excretion by stromal cells and co-cultures. Moreover, supernatant of zoledronic acid treated stromal cells reduced phospho-Smad2 protein levels in breast cancer cells. Thus, zoledronic acid exerts an anti-breast cancer effect via stromal cells, accompanied by decreased stromal TGF-beta excretion and reduced TGF-beta signaling in cancer cells.</p

Effect of COPD treatments on MRP1-mediated transport in bronchial epithelial cells

Margaretha van der Deen1, Sandra Homan1, Hetty Timmer-Bosscha1, Rik J Scheper2, Wim Timens3, Dirkje S Postma4, Elisabeth G de Vries1Departments of 1Medical Oncology, 3Pathology, 4Pulmonary Diseases, University Medical Center Groningen and University of Groningen, The Netherlands; 2Department of Pathology, VU University Medical Center, Amsterdam, The NetherlandsBackground: Smoking is the principle risk factor for development of chronic obstructive pulmonary disease (COPD). Multidrug resistance-associated protein 1 (MRP1) is known to protect against toxic compounds and oxidative stress, and might play a role in protection against smoke-induced disease progression. We questioned whether MRP1-mediated transport is influenced by pulmonary drugs that are commonly prescribed in COPD.Methods: The immortalized human bronchial epithelial cell line 16HBE14o- was used to analyze direct in vitro effects of budesonide, formoterol, ipratropium bromide and N-acetylcysteine (NAC) on MRP1-mediated transport. Carboxyfluorescein (CF) was used as a model MRP1 substrate and was measured with functional flow cytometry.Results: Formoterol had a minor effect, whereas budesonide concentration-dependently decreased CF transport by MRP1. Remarkably, addition of formoterol to the highest concentration of budesonide increased CF transport. Ipratropium bromide inhibited CF transport at low concentrations and tended to increase CF transport at higher levels. NAC increased CF transport by MRP1 in a concentration-dependent manner.Conclusions: Our data suggest that, besides their positive effects on respiratory symptoms, budesonide, formoterol, ipratropium bromide, and NAC modulate MRP1 activity in bronchial epithelial cells. Further studies are required to assess whether stimulation of MRP1 activity is beneficial for long-term treatment of COPD.Keywords: bronchus epithelium, COPD, drugs, MRP1, multidrug resistance, oxidative stres

Assessing the role of tumour-associated macrophage subsets in breast cancer subtypes using digital image analysis

Purpose: The number of M1-like and M2-like tumour-associated macrophages (TAMs) and their ratio can play a role in breast cancer development and progression. Early clinical trials using macrophage targeting compounds are currently ongoing. However, the most optimal detection method of M1-like and M2-like macrophage subsets and their clinical relevance in breast cancer is still unclear. We aimed to optimize the assessment of TAM subsets in different breast cancer subtypes, and therefore related TAM subset numbers and ratio to clinicopathological characteristics and clinical outcome. Methods: Tissue microarrays of 347 consecutive primary Luminal-A, Luminal-B, HER2-positive and triple-negative tumours of patients with early-stage breast cancer were serially sectioned and immunohistochemically stained for the pan-macrophage marker CD68 and the M2-like macrophage markers CD163, CSF-1R and CD206. TAM numbers were quantified using a digital image analysis algorithm. M1-like macrophage numbers were calculated by subtracting M2-like TAM numbers from the total TAM number. Results: M2-like markers CD163 and CSF-1R showed a moderate positive association with each other and with CD68 (r ≥ 0.47), but only weakly with CD206 (r ≤ 0.06). CD68 + , CD163 + and CSF-1R + macrophages correlated with tumour grade in Luminal-B tumours (P < 0.001). Total or subset TAM numbers did not correlate with disease outcome in any breast cancer subtype. Conclusion: In conclusion, macrophages and their subsets can be detected by means of a panel of TAM markers and are related to unfavourable clinicopathological characteristics in Luminal-B breast cancer. However, their impact on outcome remains unclear. Preferably, this should be determined in prospective series

Reduced inflammatory response in cigarette smoke exposed Mrp1/Mdr1a/1b deficient mice

<p>Abstract</p> <p>Background</p> <p>Tobacco smoke is the principal risk factor for chronic obstructive pulmonary disease (COPD), though the mechanisms of its toxicity are still unclear. The ABC transporters multidrug resistance-associated protein 1 (MRP1) and P-glycoprotein (P-gp/MDR1) extrude a wide variety of toxic substances across cellular membranes and are highly expressed in bronchial epithelium. Their impaired function may contribute to COPD development by diminished detoxification of noxious compounds in cigarette smoke.</p> <p>Methods</p> <p>We examined whether triple knock-out (TKO) mice lacking the genes for <it>Mrp1 </it>and <it>Mdr1a/1b </it>are more susceptible to develop COPD features than their wild-type (WT) littermates. TKO and WT mice (six per group) were exposed to 2 cigarettes twice daily by nose-only exposure or room air for 6 months. Inflammatory infiltrates were analyzed in lung sections, cytokines and chemokines in whole lung homogenates, emphysema by mean linear intercept. Multiple linear regression analysis with an interaction term was used to establish the statistical significances of differences.</p> <p>Results</p> <p>TKO mice had lower levels of interleukin (IL)-7, KC (mouse IL-8), IL-12p70, IL-17, TNF-alpha, G-CSF, GM-CSF and MIP-1-alpha than WT mice independent of smoke exposure (<it>P </it>< 0.05). IL-1-alpha, IL-6, IL-8, IL-13, IL-17, TNF-alpha, G-CSF, GM-CSF and MCP-1 increased after smoke exposure in both groups, but the increase in IL-8 was lower in TKO than WT mice (<it>P </it>< 0.05) with a same trend for G-CSF (<it>P </it>< 0.10). Smoke-induced increase in pulmonary inflammatory cells in WT mice was almost absent in TKO mice. The mean linear intercept was not different between groups.</p> <p>Conclusion</p> <p><it>Mrp1/Mdr1a/1b </it>knock-out mice have a reduced inflammatory response to cigarette smoke. In addition, the expression levels of several cytokines and chemokines were also lower in lungs of <it>Mrp1/Mdr1a/1b </it>knock-out mice independent of smoke exposure. Further studies are required to determine whether dysfunction of MRP1 and/or P-gp contribute to the pathogenesis of COPD.</p

Fluorescent image-guided surgery in breast cancer by intravenous application of a quenched fluorescence activity-based probe for cysteine cathepsins in a syngeneic mouse model

PURPOSE: The reoperation rate for breast-conserving surgery is as high as 15-30% due to residual tumor in the surgical cavity after surgery. In vivo tumor-targeted optical molecular imaging may serve as a red-flag technique to improve intraoperative surgical margin assessment and to reduce reoperation rates. Cysteine cathepsins are overexpressed in most solid tumor types, including breast cancer. We developed a cathepsin-targeted, quenched fluorescent activity-based probe, VGT-309, and evaluated whether it could be used for tumor detection and image-guided surgery in syngeneic tumor-bearing mice. METHODS: Binding specificity of the developed probe was evaluated in vitro. Next, fluorescent imaging in BALB/c mice bearing a murine breast tumor was performed at different time points after VGT-309 administration. Biodistribution of VGT-309 after 24 h in tumor-bearing mice was compared to control mice. Image-guided surgery was performed at multiple time points tumors with different clinical fluorescent camera systems and followed by ex vivo analysis. RESULTS: The probe was specifically activated by cathepsins X, B/L, and S. Fluorescent imaging revealed an increased tumor-to-background contrast over time up to 15.1 24 h post probe injection. In addition, VGT-309 delineated tumor tissue during image-guided surgery with different optical fluorescent imaging camera systems. CONCLUSION: These results indicate that optical fluorescent molecular imaging using the cathepsin-targeted probe, VGT-309, may improve intraoperative tumor detection, which could translate to more complete tumor resection when coupled with commercially available surgical tools and techniques

High hepatocyte growth factor expression in primary tumor predicts better overall survival in male breast cancer

Background Breast cancer is rare in men, but management is focused on tumor characteristics commonly found in female breast cancer. The tumor microenvironment of male breast cancer is less well understood, and insight may improve male breast cancer management. The hepatocyte growth factor (HGF)/c-MET axis and the stromal cell-derived factor-1 (CXCL12)/C-X-C chemokine receptor type 4 (CXCR4) axis are prognostic in women with breast cancer. We aimed to investigate these factors in male breast cancer and correlate them with patient survival. Methods From 841 Dutch males with breast cancer who were enrolled in the EORTC 10085/TBCRC/BIG/NABCG International Male Breast Cancer Program (NCT01101425) and diagnosed between 1990 and 2010, archival primary tumor samples were collected. Tissue microarrays were constructed with 3 cores per sample and used for immunohistochemical analysis of HGF, c-MET, CXCL12, and CXCR4. Overall survival (OS) of the patients without metastases (M0) was analyzed using the Kaplan-Meier method. The value of the markers regarding OS was determined using univariable and multivariable Cox regression analyses, providing hazard ratios (HRs) and 95% confidence intervals (95% CIs). Results Of 720 out of 841 patients, sufficient tissue was available for analysis; 487 out of 720 patients had M0 disease. Patients with high HGF expression and high CXCL12 expression had a superior OS (low vs high expression of both markers, 7.5 vs 13.0 years, hazard ratio [HR] 0.64, 95% CI 0.49-0.84, P = 0.001 [HGF]; 9.1 vs 15.3 years, HR 0.63, 95% CI 0.45-0.87, P = 0.005 [CXCL12]). Multivariate analysis identified HGF as an independent predictor for OS (HR 0.64, 95% CI 0.47-0.88, P = 0.001). Conclusions HGF and CXCL12 tumor expression appear to identify male breast cancer patients with a relatively good prognosis. Possibly, this could support male breast cancer-specific management strategies in the future

Comprehensive characterization of the prostate tumor microenvironment identifies CXCR4/CXCL12 crosstalk as a novel antiangiogenic therapeutic target in prostate cancer

Background: Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. Methods: TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell-cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. Results: Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. Conclusions: Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa

Studying cancer metastasis:Existing models, challenges and future perspectives

Cancer metastasis causes most cancer-related deaths. Several model systems to study the complex and multi step process of metastasis exist, including in vitro systems, ex-vivo organ slices, Drosophila Melanogaster and zebrafish models and the use of the chorio allantoic membrane (CAM) of fertilized chicken eggs. These models are relatively easy and cheap but often lack the opportunity to study the complete metastasis cascade. More complex but also more expensive is the use of animal models including the more recently developed patient derived tumor xenografts (PDTX). In this review, we give an overview of the existing metastatic models, discuss the challenges of improving current models to enhance translation from the preclinical to the clinical setting and consider future perspectives. (C) 2015 Elsevier Ireland Ltd. All rights reserved