An Extracellular Matrix Overlay Model for Bioluminescence Microscopy to Measure Single-Cell Heterogeneous Responses to Antiandrogens in Prostate Cancer Cells.

Prostate cancer (PCa) displays diverse intra-tumoral traits, impacting its progression and treatment outcomes. This study aimed to refine PCa cell culture conditions for dynamic monitoring of androgen receptor (AR) activity at the single-cell level. We introduced an extracellular matrix-Matrigel (ECM-M) culture model, enhancing cellular tracking during bioluminescence single-cell imaging while improving cell viability. ECM-M notably tripled the traceability of poorly adherent PCa cells, facilitating robust single-cell tracking, without impeding substrate permeability or AR response. This model effectively monitored AR modulation by antiandrogens across various PCa cell lines. Single-cell imaging unveiled heterogeneous antiandrogen responses within populations, correlating non-responsive cell proportions with drug IC50 values. Integrating ECM-M culture with the PSEBC-TSTA biosensor enabled precise characterization of ARi responsiveness within diverse cell populations. Our ECM-M model stands as a promising tool to assess heterogeneous single-cell treatment responses in cancer, offering insights to link drug responses to intracellular signaling dynamics. This approach enhances our comprehension of the nuanced and dynamic nature of PCa treatment responses.

Cancer-Associated Fibroblasts in a 3D Engineered Tissue Model Induce Tumor-like Matrix Stiffening and EMT Transition.

A tumor microenvironment is characterized by its altered mechanical properties. However, most models remain unable to faithfully recreate the mechanical properties of a tumor. Engineered models based on the self-assembly method have the potential to better recapitulate the stroma architecture and composition. Here, we used the self-assembly method based on a bladder tissue model to engineer a tumor-like environment. The tissue-engineered tumor models were reconstituted from stroma-derived healthy primary fibroblasts (HFs) induced into cancer-associated fibroblast cells (iCAFs) along with an urothelium overlay. The iCAFs-derived extracellular matrix (ECM) composition was found to be stiffer, with increased ECM deposition and remodeling. The urothelial cells overlaid on the iCAFs-derived ECM were more contractile, as measured by quantitative polarization microscopy, and displayed increased YAP nuclear translocation. We further showed that the proliferation and expression of epithelial-to-mesenchymal transition (EMT) marker in the urothelial cells correlate with the increased stiffness of the iCAFs-derived ECM. Our data showed an increased expression of EMT markers within the urothelium on the iCAFs-derived ECM. Together, our results demonstrate that our tissue-engineered tumor model can achieve stiffness levels comparable to that of a bladder tumor, while triggering a tumor-like response from the urothelium.

Bladder cancer cell lines adapt their aggressiveness profile to oxygen tension.

During the process of tumor growth, cancer cells will be subjected to intermittent hypoxia. This results from the delay in the development of the vascular network in relation to the proliferation of cancer cells. The hypoxic nature of a tumor has been demonstrated as a negative factor for patient survival. To evaluate the impact of hypoxia on the survival and migration properties of low and high-grade bladder cancer cell lines, two low-grade (MGHU-3 and SW-780) and two high-grade (SW-1710 and T24) bladder cancer cell lines were cultured in normoxic (20% O2) or hypoxic atmospheric conditions (2% O2). The response of bladder cancer cell lines to hypoxic atmospheric cell culture conditions was examined under several parameters, including epithelial-mesenchymal transition, doubling time and metabolic activities, thrombospondin-1 expression, whole Matrix Metallo-Proteinase activity, migration and resistance to oxidative stress. The low-grade cell line response to hypoxia was heterogeneous even if it tended to adopt a more aggressive profile. Hypoxia enhanced migration and pro-survival properties of MGHU-3 cells, whereas these features were reduced for the SW-780 cell line cultured under low oxygen tension. The responses of tested high-grade cell lines were more homogeneous and tended to adopt a less aggressive profile. Hypoxia drastically changed some of the bladder cancer cell line properties, for example matrix metalloproteinases expression for all cancer cells but also switch in glycolytic metabolism of low grade cancer cells. Overall, studying bladder cancer cells in hypoxic environments are relevant for the translation from in vitro findings to in vivo context.

GLUT1 expression in high-risk prostate cancer: correlation with 18F-FDG-PET/CT and clinical outcome.

BACKGROUND: Tumour 18F-FDG-uptake is of prognostic value in high-risk and metastatic prostate cancer (PCa). The aim of this study is to investigate the underlying glucose metabolism mechanisms of 18F-FDG-uptake on PET/CT imaging in PCa. METHODS: Retrospective analysis was conducted for 94 patients diagnosed with a Gleason sum ≥8 adenocarcinoma of the prostate at biopsy between July 2011 and July 2014 who underwent 18F-FDG-PET/CT imaging before radical prostatectomy (RP). 18F-FDG-uptake in primary lesion was measured by a blinded reader using maximum standardised uptake value (SUVmax). GLUT1, GLUT12 and HK2 expression were blindly scored after immunohistochemistry on specimens RP by three pathologists. Correlations between GLUT1, GLUT12 and HK2, and SUVmax were assessed using Spearman's rank correlation test. Survival probabilities were based on the Kaplan-Meier method. RESULTS: With a median follow-up of 4.5 years, 56% (n = 53) of patients had biochemical recurrence (BCR), 7% (n = 7) progressed to castration-resistant prostate cancer (CRPC) disease, 13% (n = 12) developed metastasis and 6% (n = 6) died. Correlation was found between GLUT1 expression and SUVmax level (r = 0.25, p = 0.02). In addition, SUVmax was significantly higher in tumours with high GLUT1 expression (n = 17, 5.74 ± 1.67) than tumours with low GLUT1 expression (n = 71, 2.68 ± 0.31, p = 0.004). Moreover, a significant association was found between GLUT1 expression levels and SUVmax level (p = 0.005), lymph node status (p = 0.05), volume of cancer (p = 0.01), CRPC disease progression (p = 0.02) and metastasis development (p = 0.04). No significant difference between GLUT12 and HEX2 expression and SUVmax have been found. CONCLUSIONS: GLUT1 expression in PCa tumours correlates with 18F-FDG-uptake and poor prognostic factors. These results suggest that this transporter is involved in the molecular mechanism of 18F-FDG-uptake in high-risk PCa and raise interest in targeting metabolic dependencies of PCa cells as a selective anticancer strategy.

Modeling human bladder cancer.

INTRODUCTION: Bladder cancer is a major public health concern and the treatment options available are unable to significantly prevent disease recurrence and progression. The need for experimental tumor models to efficiently reproduce the pathology of human cancers has prompted researchers to attempt various approaches. METHODS: A PubMed search combining the MeSH bladder cancer and models was executed in March 2017. RESULTS: We review the advantages and limitations of currently available in vitro 2D and 3D bladder cancer models as well as in vivo rodent models. To date, despite the description of a variety of animal models (including transplantable, carcinogen-induced and genetically engineered models), the establishment of reliable, simple, practicable and reproducible animal models remains an ongoing challenge. Recently, sophisticated 3D culture systems have been designed to better recapitulate the phenotypic and cellular heterogeneity as well as microenvironmental aspects of in vivo tumor growth, while being more flexible to conduct repeated experiments. CONCLUSION: Selecting the most appropriate model for a specific application will maximize the conversion of potential therapies from the laboratory to clinical practice and requires an understanding of the various models available.

Exosomes Induce Fibroblast Differentiation into Cancer-Associated Fibroblasts through TGFß Signaling.

A particularly important tumor microenvironment relationship exists between cancer cells and surrounding stromal cells. Fibroblasts, in response to cancer cells, become activated and exhibit myofibroblastic characteristics that favor invasive growth and metastasis. However, the mechanism by which cancer cells promote activation of healthy fibroblasts into cancer-associated fibroblasts (CAF) is still not well understood. Exosomes are nanometer-sized vesicles that shuttle proteins and nucleic acids between cells to establish intercellular communication. Here, bladder cancer-derived exosomes were investigated to determine their role in the activation of healthy primary vesical fibroblasts. Exosomes released by bladder cancer cells are internalized by fibroblasts and promoted the proliferation and expression of CAF markers. In addition, cancer cell-derived exosomes contain TGFß and in exosome-induced CAFs SMAD-dependent signaling is activated. Furthermore, TGFß inhibitors attenuated CAF marker expression in healthy fibroblasts. Therefore, these data demonstrate that bladder cancer cells trigger the differentiation of fibroblasts to CAFs by exosomes-mediated TGFß transfer and SMAD pathway activation. Finally, exosomal TGFß localized inside the vesicle and contributes 53.4% to 86.3% of the total TGFß present in the cancer cell supernatant. This study highlights a new function for bladder cancer exosomes as novel modulators of stromal cell differentiation.Implication: This study identifies exosomal TGFß as new molecular mechanism involved in cancer-associated fibroblast activation. Mol Cancer Res; 16(7); 1196-204. ©2018 AACR.

Tissue-engineered human 3D model of bladder cancer for invasion study and drug discovery.

The tumour microenvironment is critical to both the initiation and maintenance of tumorigenesis. Reconstitution of the microenvironment is a major challenge for in vitro cancer models. Indeed, conventional 2D culture systems cannot replicate the complexity, diversity and dynamic nature of the tumour microenvironment. In this study, we have developed a 3D endotheliazed vesical equivalent by using tissue engineering from primary human cells in which non-invasive or invasive bladder cancer (BCa) cell lines, cultured as compact spheroids, were incorporated. Invasive BCa cells cross the basement membrane and invade the stromal compartment whereas non-invasive BCa cells are confined to the urothelium. Our 3D BCa model could be used as a reliable model for assessing drug responses, potentially reducing or partially replacing animal experiments, and thus should have applications in the identification of novel targets as well as toxicological evaluation of anti-cancer therapies.

Brain bioavailability of human intravenous immunoglobulin and its transport through the murine blood-brain barrier.

Intravenous immunoglobulin (IVIg) is currently evaluated in clinical trials for the treatment of various disorders of the central nervous system. To assess its capacity to reach central therapeutic targets, the brain bioavailability of IVIg must be determined. We thus quantified the passage of IVIg through the blood-brain barrier (BBB) of C57Bl/6 mice using complementary quantitative and qualitative methodologies. As determined by enzyme-linked immunosorbent assay, a small proportion of systemically injected IVIg was detected in the brain of mice (0.009±0.001% of injected dose in the cortex) whereas immunostaining revealed localization mainly within microvessels and less frequently in neurons. Pharmacokinetic analyses evidenced a low elimination rate constant (0.0053 per hour) in the cortex, consistent with accumulation within cerebral tissue. In situ cerebral perfusion experiments revealed that a fraction of IVIg crossed the BBB without causing leakage. A dose-dependent decrease of brain uptake was consistent with a saturable blood-to-brain transport mechanism. Finally, brain uptake of IVIg after a subchronic treatment was similar in the 3xTg-AD mouse model of Alzheimer disease compared with nontransgenic controls. In summary, our results provide evidence of BBB passage and bioavailability of IVIg into the brain in the absence of BBB leakage and in sufficient concentration to interact with the therapeutic targets.