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The current statistics on cancer show that 90% of all human cancers originate from epithelial cells. Breast and prostate cancer are examples of common tumors of epithelial origin that would benefit from improved drug treatment strategies. About 90% of preclinically approved drugs fail in clinical trials, partially due to the use of too simplified in vitro models and a lack of mimicking the tumor microenvironment in drug efficacy testing. This review focuses on the origin and mechanism of epithelial cancers, followed by experimental models designed to recapitulate the epithelial cancer structure and microenvironment, such as 2D and 3D cell culture models and animal models. A specific focus is put on novel technologies for cell culture of spheroids, organoids, and 3D-printed tissue-like models utilizing biomaterials of natural or synthetic origins. Further emphasis is laid on high-content imaging technologies that are used in the field to visualize in vitro models and their morphology. The associated technological advancements and challenges are also discussed. Finally, the review gives an insight into the potential of exploiting nanotechnological approaches in epithelial cancer research both as tools in tumor modeling and how they can be utilized for the development of nanotherapeutics.

Pharmacological inhibition of DOT1L blocks estrogen receptor signaling in breast cancer., Breast cancer (BC) resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor α (ERα) signaling, and ways to block ERα pathway in these tumors are sought after. We identified the H3K79 methyltransferase DOT1L as a novel cofactor of ERα in BC cell chromatin, where the two proteins colocalize to regulate estrogen target gene transcription. DOT1L blockade reduces proliferation of hormone-responsive BC cells in vivo and in vitro, consequent to cell cycle arrest and apoptotic cell death, with widespread effects on ER-dependent gene transcription, including ERα and FOXA1 gene silencing. Antiestrogen-resistant BC cells respond to DOT1L inhibition also in mouse xenografts, with reduction in ERα levels, H3K79 methylation, and tumor growth. These results indicate that DOT1L is an exploitable epigenetic target for treatment of endocrine therapy–resistant ERα-positive BCs.

Heat-shock factors (HSFs) are key transcriptional regulators in cell survival. Although HSF1 has been identified as a driver of carcinogenesis, HSF2 has not been explored in malignancies. Here, we report that HSF2 suppresses tumor invasion of prostate cancer (PrCa). In three-dimensional organotypic cultures and the in vivo xenograft chorioallantoic membrane model HSF2 knockdown perturbs organoid differentiation and promotes invasiveness. Gene expression profiling together with functional studies demonstrated that the molecular mechanism underlying the effect on tumor progression originates from HSF2 steering the switch between acinar morphogenesis and invasion. This is achieved by the regulation of genes connected to, for example, GTPase activity, cell adhesion, extracellular matrix and actin cytoskeleton dynamics. Importantly, low HSF2 expression correlates with high Gleason score, metastasis and poor survival of PrCa patients, highlighting the clinical relevance of our findings. Finally, the study was expanded beyond PrCa, revealing that the expression of HSF2 is decreased in a wide range of cancer types. This study provides the first evidence for HSF2 acting as a suppressor of invasion in human malignancies.

Wnt-11 promotes cancer cell migration and invasion independently of β-catenin but the receptors involved remain unknown. Here, we provide evidence that FZD8 is a major Wnt-11 receptor in prostate cancer that integrates Wnt-11 and TGF-β signals to promote EMT. FZD8 mRNA is upregulated in multiple prostate cancer datasets and in metastatic cancer cell lines in vitro and in vivo. Analysis of patient samples reveals increased levels of FZD8 in cancer, correlating with Wnt-11. FZD8 co-localizes and co-immunoprecipitates with Wnt-11 and potentiates Wnt-11 activation of ATF2-dependent transcription. FZD8 silencing reduces prostate cancer cell migration, invasion, three-dimensional (3D) organotypic cell growth, expression of EMT-related genes, and TGF-β/Smad-dependent signaling. Mechanistically, FZD8 forms a TGF-β-regulated complex with TGF-β receptors that is mediated by the extracellular domains of FZD8 and TGFBR1. Targeting FZD8 may therefore inhibit aberrant activation of both Wnt and TGF-β signals in prostate cancer., Wnt11 has been shown to play a role in invasion and metastasis of prostate cancer. Here the authors show that in prostate cancer cells Wnt11 signals through the Fzd8 receptor and report an interaction between Fzd8 and TGF-β receptors regulating the transcription of a subset of TGF-beta genes.

Intracellular drug delivery by mesoporous silica nanoparticles (MSNs) carrying hydrophilic and hydrophobic fluorophores as model drug cargo is demonstrated on 2D cellular and 3D tumor organoid level. Two different MSN designs, chosen on the basis of the characteristics of the loaded cargo, were used: MSNs with a surface-grown poly(ethylene imine), PEI, coating only for hydrophobic cargo and MSNs with lipid bilayers covalently coupled to the PEI layer as a diffusion barrier for hydrophilic cargo. First, the effect of hydrophobicity corresponding to loading degree (hydrophobic cargo) as well as surface charge (hydrophilic cargo) on intracellular drug release was studied on the cellular level. All incorporated agents were able to release to varying degrees from the endosomes into the cytoplasm in a loading degree (hydrophobic) or surface charge (hydrophilic) dependent manner as detected by live cell imaging. When administered to organotypic 3D tumor models, the hydrophilic versus hydrophobic cargo-carrying MSNs showed remarkable differences in labeling efficiency, which in this case also corresponds to drug delivery efficacy in 3D. The obtained results could thus indicate design aspects to be taken into account for the development of efficacious intracellular drug delivery systems, especially in the translation from standard 2D culture to more biologically relevant organotypic 3D cultures.

// Malin Akerfelt 1, 2 , Neslihan Bayramoglu 3 , Sean Robinson 4, 5, 6, 7 , Mervi Toriseva 1, 2, 8 , Hannu-Pekka Schukov 8 , Ville Harma 2 , Johannes Virtanen 2 , Raija Sormunen 9 , Mika Kaakinen 10 , Juho Kannala 3 , Lauri Eklund 10 , Janne Heikkila 3 , Matthias Nees 1, 2 1 Turku Centre for Biotechnology, University of Turku, Turku, FI-20520, Finland 2 VTT Technical Research Centre of Finland, Turku, FI-20521, Finland 3 Centre for Machine Vision Research, University of Oulu, Oulu, FI-90014, Finland 4 Department of Mathematics and Statistics, University of Turku, Turku, FI-20014, Finland 5 University Grenoble Alpes, iRTSV-BGE, Grenoble, F-38000, France 6 CEA, iRTSV-BGE, Grenoble, F-38000, France 7 INSERM, BGE, Grenoble, F-38000, France 8 Institute of Biomedicine, University of Turku, Turku, FI-20520, Finland 9 Biocenter Oulu and Department of Pathology, University of Oulu and Oulu University Hospital, Oulu, FI-90220, Finland 10 Oulu Center for Cell-Matrix Research, Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, FI-90014, Finland Correspondence to: Matthias Nees, e-mail: matthias.nees@btk.fi Keywords: 3D co-culture, cancer associated fibroblast (CAF), phenotypic screening, invasion, focal adhesion kinase (FAK) Received: March 21, 2015 Accepted: August 24, 2015 Published: September 03, 2015 ABSTRACT Cancer-associated fibroblasts (CAFs) constitute an important part of the tumor microenvironment and promote invasion via paracrine functions and physical impact on the tumor. Although the importance of including CAFs into three-dimensional (3D) cell cultures has been acknowledged, computational support for quantitative live-cell measurements of complex cell cultures has been lacking. Here, we have developed a novel automated pipeline to model tumor-stroma interplay, track motility and quantify morphological changes of 3D co-cultures, in real-time live-cell settings. The platform consists of microtissues from prostate cancer cells, combined with CAFs in extracellular matrix that allows biochemical perturbation. Tracking of fibroblast dynamics revealed that CAFs guided the way for tumor cells to invade and increased the growth and invasiveness of tumor organoids. We utilized the platform to determine the efficacy of inhibitors in prostate cancer and the associated tumor microenvironment as a functional unit. Interestingly, certain inhibitors selectively disrupted tumor-CAF interactions, e.g. focal adhesion kinase (FAK) inhibitors specifically blocked tumor growth and invasion concurrently with fibroblast spreading and motility. This complex phenotype was not detected in other standard in vitro models. These results highlight the advantage of our approach, which recapitulates tumor histology and can significantly improve cancer target validation in vitro .

Glandular epithelial cells differentiate into three-dimensional (3D) multicellular or acinar structures, particularly when embedded in laminin-rich extracellular matrix (ECM). The spectrum of different multicellular morphologies formed in 3D is a reliable indicator for the differentiation potential of normal, non-transformed cells compared to different stages of malignant progression. Motile cancer cells may actively invade the matrix, utilizing epithelial, mesenchymal, or mixed modes of motility. Dynamic phenotypic changes involved in 3D tumor cell invasion are also very sensitive to small-molecule inhibitors that, e.g., target the actin cytoskeleton. Our strategy is to recapitulate the formation and the histology of complex solid cancer tissues in vitro, based on cell culture technologies that promote the intrinsic differentiation potential of normal and transformed epithelial cells, and also including stromal fibroblasts and other key components of the tumor microenvironment. We have developed a streamlined stand-alone software solution that supports the detailed quantitative phenotypic analysis of organotypic 3D cultures. This approach utilizes the power of automated image analysis as a phenotypic readout in cell-based assays. AMIDA (Automated Morphometric Image Data Analysis) allows quantitative measurements of a large number of multicellular structures, which can form a multitude of different organoid shapes, sizes, and textures according to their capacity to engage in epithelial differentiation programs or not. At the far end of this spectrum of tumor-relevant differentiation properties, there are highly invasive tumor cells or multicellular structures that may rapidly invade the surrounding ECM, but fail to form higher-order epithelial tissue structures. Furthermore, this system allows us to monitor dynamic changes that can result from the extraordinary plasticity of tumor cells, e.g., epithelial-to-mesenchymal transition in live cell settings. Furthermore, AMIDA supports an automated workflow, and can be combined with quality control and statistical tools for data interpretation and visualization. Our approach supports the growing needs for user-friendly, straightforward solutions that facilitate cell-based organotypic 3D assays in basic research, drug discovery, and target validation.

Glandular epithelial cells differentiate into three-dimensional (3D) multicellular or acinar structures, particularly when embedded in laminin-rich extracellular matrix (ECM). The spectrum of different multicellular morphologies formed in 3D is a reliable indicator for the differentiation potential of normal, non-transformed cells compared to different stages of malignant progression. Motile cancer cells may actively invade the matrix, utilizing epithelial, mesenchymal, or mixed modes of motility. Dynamic phenotypic changes involved in 3D tumor cell invasion are also very sensitive to small-molecule inhibitors that, e.g., target the actin cytoskeleton. Our strategy is to recapitulate the formation and the histology of complex solid cancer tissues in vitro, based on cell culture technologies that promote the intrinsic differentiation potential of normal and transformed epithelial cells, and also including stromal fibroblasts and other key components of the tumor microenvironment. We have developed a streamlined stand-alone software solution that supports the detailed quantitative phenotypic analysis of organotypic 3D cultures. This approach utilizes the power of automated image analysis as a phenotypic readout in cell-based assays. AMIDA (Automated Morphometric Image Data Analysis) allows quantitative measurements of a large number of multicellular structures, which can form a multitude of different organoid shapes, sizes, and textures according to their capacity to engage in epithelial differentiation programs or not. At the far end of this spectrum of tumor-relevant differentiation properties, there are highly invasive tumor cells or multicellular structures that may rapidly invade the surrounding ECM, but fail to form higher-order epithelial tissue structures. Furthermore, this system allows us to monitor dynamic changes that can result from the extraordinary plasticity of tumor cells, e.g., epithelial-to-mesenchymal transition in live cell settings. Furthermore, AMIDA supports an automated workflow, and can be combined with quality control and statistical tools for data interpretation and visualization. Our approach supports the growing needs for user-friendly, straightforward solutions that facilitate cell-based organotypic 3D assays in basic research, drug discovery, and target validation.

Expression of follicle-stimulation hormone receptor (FSHR) is confined to gonads and at low levels to some extragonadal tissues like human umbilical vein endothelial cells (HUVEC). FSH-FSHR signaling was shown to promote HUVEC angiogenesis and thereafter suggested to have an influential role in pregnancy. We revisited hereby the expression and functionality of FSHR in HUVECs angiogenesis, and were unable to reproduce the FSHR expression in human umbilical cord, HUVECs or immortalized HUVECs (HUV-ST). Positive controls as granulosa cells and HEK293 cells stably transfected with human FSHR cDNA expressed FSHR signal. In contrast to positive control VEGF, FSH treatment showed no effects on tube formation, nitric oxide production, wound healing or cell proliferation in HUVEC/HUV-ST. Thus, it remains open whether the FSH-FSHR activation has a direct regulatory role in the angiogenesis of HUVECs.

The identification and validation of biomarkers for clinical applications remains an important issue for improving diagnostics and therapy in many diseases, including prostate cancer. Gene expression profiles are routinely applied to identify diagnostic and predictive biomarkers or novel targets for cancer. However, only few predictive markers identified in silico have also been validated for clinical, functional or mechanistic relevance in disease progression. In this study, we have used a broad, bioinformatics-based approach to identify such biomarkers across a spectrum of progression stages, including normal and tumor-adjacent, premalignant, primary and late stage lesions. Bioinformatics data mining combined with clinical validation of biomarkers by sensitive, quantitative reverse-transcription PCR (qRT-PCR), followed by functional evaluation of candidate genes in disease-relevant processes, such as cancer cell proliferation, motility and invasion. From 300 initial candidates, eight genes were selected for validation by several layers of data mining and filtering. For clinical validation, differential mRNA expression of selected genes was measured by qRT-PCR in 197 clinical prostate tissue samples including normal prostate, compared against histologically benign and cancerous tissues. Based on the qRT-PCR results, significantly different mRNA expression was confirmed in normal prostate versus malignant PCa samples (for all eight genes), but also in cancer-adjacent tissues, even in the absence of detectable cancer cells, thus pointing to the possibility of pronounced field effects in prostate lesions. For the validation of the functional properties of these genes, and to demonstrate their putative relevance for disease-relevant processes, siRNA knock-down studies were performed in both 2D and 3D organotypic cell culture models. Silencing of three genes (DLX1, PLA2G7 and RHOU) in the prostate cancer cell lines PC3 and VCaP by siRNA resulted in marked growth arrest and cytotoxicity, particularly in 3D organotypic cell culture conditions. In addition, silencing of PLA2G7, RHOU, ACSM1, LAMB1 and CACNA1D also resulted in reduced tumor cell invasion in PC3 organoid cultures. For PLA2G7 and RHOU, the effects of siRNA silencing on proliferation and cell-motility could also be confirmed in 2D monolayer cultures. In conclusion, DLX1 and RHOU showed the strongest potential as useful clinical biomarkers for PCa diagnosis, further validated by their functional roles in PCa progression. These candidates may be useful for more reliable identification of relapses or therapy failures prior to the recurrence local or distant metastases.

Heat shock factor 1 (HSF1) is an important transcription factor in cellular stress responses, cancer, aging, and developmental processes including gametogenesis. Disruption of Hsf1, together with another HSF family member, Hsf2, causes male sterility and complete lack of mature sperm in mice, but the specific role of HSF1 in spermatogenesis has remained unclear. Here, we show that HSF1 is transiently expressed in meiotic spermatocytes and haploid round spermatids in mouse testis. The Hsf1(-/-) male mice displayed regions of seminiferous tubules containing only spermatogonia and increased morphological abnormalities in sperm heads. In search for HSF1 target genes, we identified 742 putative promoters in mouse testis. Among them, the sex chromosomal multicopy genes that are expressed in postmeiotic cells were occupied by HSF1. Given that the sex chromatin mostly is repressed during and after meiosis, it is remarkable that HSF1 directly regulates the transcription of sex-linked multicopy genes during postmeiotic repression. In addition, our results show that HSF1 localizes to the sex body prior to the meiotic divisions and to the sex chromocenter after completed meiosis. To the best of our knowledge, HSF1 is the first known transcription factor found at the repressed sex chromatin during meiosis.

Organotypic 3D cell culture models combined with screening modalities and automated high-content image analyses provide tools to gain a spectrum of biologically relevant information simultaneously from drug responses in tumor cells. This includes information about cell growth, death, differentiation and tumor cell invasion. In this study, we examined the phenotypic drug responses of prostate cancer cell lines PC-3 and LNCaP, and breast cancer cell lines MDA-MB-231 (SA) and MCF-7 cultured in a miniaturized, imaging-optimized, Matrigel-based organotypic screening platform. We demonstrate the use of the 3D cell culture technology combined with automated morphometric image data analysis software AMIDA, for phenotypic, high-content screening. The emerging tumor organoids were treated with the cytostatic drugs doxorubicin, docetaxel and paclitaxel, a selective inhibitor of matrix metalloproteinase-13 (WAY170523), and with ROCK-inhibitors RKI-1447 and Y-27632. Treatments were conducted at seven different drug concentrations for 4-10 days. At the end point, confocal live cell images were captured and analyzed using AMIDA. Among others, the numerical data representing cell growth (Area) and cell invasion (Appendages) were visualized and used for statistics. EC50 values were calculated based on the Area-parameter derived from AMIDA analysis. All cell lines initially formed multicellular, round organoids. PC-3 cells formed round and well-differentiated structures but spontaneously converted around day 9 of culture into structures showing massive, string-like collective invasion into the surrounding matrix. A different pattern of cell invasion was observed in MDA-MB-231 organoids, which developed strong and multicellular extensions by day 7-8. Interestingly, both ROCK-inhibitors promoted the invasion of PC-3 cells, as detected by phenotypic analysis with AMIDA. In contrast, Y-27632 reduced the invasion of MDA-MB-231 cells, whereas both ROCK-inhibitors induced cell invasion in MCF-7 cells at high concentration. WAY170523 inhibited the invasion of PC-3 cells but not of MDA-MB-231 cells, pointing to a different mechanism of invasiveness. All cell lines were highly sensitive to doxorubicin, docetaxel and paclitaxel at nanomolar range, as detected by the decrease of cell invasion, reduced organoid size, and increased cell death. The breast cancer cells were more sensitive to taxanes than PC-3 cells. Organotypic 3D cultures combined with high-content phenotypic analysis with AMIDA software provide a quantitative view of drug effects and enable assessment of differential drug responses on various cell lines. Citation Format: Mervi Toriseva, Katja Fagerlund, Jesse Mattsson, Tiina E. Kähkönen, Ilmari Ahonen, Malin Åkerfelt, Jenni Bernoulli, Jussi M. Halleen, Matthias Nees, Jenni H. Mäki-Jouppila. Phenotypic screening using AMIDA identifies different drug responses in breast and prostate cancer cell lines in an organotypic cell culture model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1158.

Heat shock transcription factor 1 (HSF1) is the main regulator of the stress response that triggers the transcription of several genes encoding heat shock proteins (Hsps). Hsps act as molecular chaperones involved in protein folding, stability, and trafficking. HSF1 is highly expressed in oocytes and Hsf1 knock-out in mice revealed that in the absence of stress this factor plays an important role in female reproduction. We previously reported that Hsf1-/- females produce oocytes but no viable embryos. Consequently, we asked whether oocytes require HSF1 to regulate a particular set of Hsps necessary for them to develop. We find that Hsp90α (Hspaa1) is the major HSF1-dependent chaperone inasmuch as Hsf1 knock-out resulted in Hsp90-depleted oocytes. These oocytes exhibited delayed germinal vesicle breakdown (or G2/M transition), partial meiosis I block, and defective asymmetrical division. To probe the role of Hsp90α in this meiotic syndrome, we analyzed meiotic maturation in wild-type oocytes treated with a specific inhibitor of Hsp90, 17-allylamino-17-demethoxy-geldanamycin, and observed similar defects. At the molecular level we showed that, together with these developmental anomalies, CDK1 and MAPK, key meiotic kinases, were significantly disturbed. Thus, our data demonstrate that HSF1 is a maternal transcription factor essential for normal progression of meiosis.

The mammalian Y chromosome is essential for spermatogenesis, which is characterized by sperm cell differentiation and chromatin condensation for acquisition of correct shape of the sperm. Deletions of the male-specific region of the mouse Y chromosome long arm (MSYq), harboring multiple copies of a few genes, lead to sperm head defects and impaired fertility. Using chromatin immunoprecipitation on promoter microarray (ChIP-chip) on mouse testis, we found a striking in vivo MSYq occupancy by heat shock factor 2 (HSF2), a transcription factor involved in spermatogenesis. HSF2 was also found to regulate the transcription of MSYq resident genes, whose transcriptional regulation has been unknown. Importantly, disruption of Hsf2 caused a similar phenotype as the 2/3 deletion of MSYq, i.e., altered expression of the multicopy genes and increased mild sperm head abnormalities. Consequently, aberrant levels of chromatin packing proteins and more frequent DNA fragmentation were detected, implying that HSF2 is required for correct chromatin organization in the sperm. Our findings define a physiological role for HSF2 in the regulation of MSYq resident genes and the quality of sperm.

Targeting of the Hsp function in tumor cells is currently being assessed as potential anticancer therapy. An improved understanding of the molecular signals that trigger or attenuate the stress protein response is essential for advances to be made in this field. The present study provides evidence that the membrane fluidizer benzyl alcohol (BA), a documented nondenaturant, acts as a chaperone inducer in B16(F10) melanoma cells. It is demonstrated that this effect relies basically on heat shock transcription factor 1 (HSF1) activation. Under the conditions tested, the BA-induced Hsp response involves the up-regulation of a subset of hsp genes. It is shown that the same level of membrane fluidization (estimated in the core membrane region) attained with the closely analogous phenethyl alcohol (PhA) does not generate a stress protein signal. BA, at a concentration that activates heat shock genes, exerts a profound effect on the melting of raft-like cholesterol-sphingomyelin domains in vitro , whereas PhA, at a concentration equipotent with BA in membrane fluidization, has no such effect. Furthermore, through the in vivo labeling of melanoma cells with a fluorescein labeled probe that inserts into the cholesterol-rich membrane domains [fluorescein ester of polyethylene glycol-derivatized cholesterol (fPEG-Chol)], we found that, similarly to heat stress per se , BA, but not PhA, initiates profound alterations in the plasma membrane microdomain structure. We suggest that, apart from membrane hyperfluidization in the deep hydrophobic region, a distinct reorganization of cholesterol-rich microdomains may also be required for the generation and transmission of stress signals to activate hsp genes.

Organotypic, three dimensional (3D) cell culture models of epithelial tumour types such as prostate cancer recapitulate key aspects of the architecture and histology of solid cancers. Morphometric analysis of multicellular 3D organoids is particularly important when additional components such as the extracellular matrix and tumour microenvironment are included in the model. The complexity of such models has so far limited their successful implementation. There is a great need for automatic, accurate and robust image segmentation tools to facilitate the analysis of such biologically relevant 3D cell culture models. We present a segmentation method based on Markov random fields (MRFs) and illustrate our method using 3D stack image data from an organotypic 3D model of prostate cancer cells co-cultured with cancer-associated fibroblasts (CAFs). The 3D segmentation output suggests that these cell types are in physical contact with each other within the model, which has important implications for tumour biology. Segmentation performance is quantified using ground truth labels and we show how each step of our method increases segmentation accuracy. We provide the ground truth labels along with the image data and code. Using independent image data we show that our segmentation method is also more generally applicable to other types of cellular microscopy and not only limited to fluorescence microscopy. Public Library of Science open access

In this paper, we propose a novel feature description algorithm based on image statistics. The pipeline first performs independent component analysis on training image patches to obtain basis vectors (filters) for a lower dimensional representation. Then for a given image, a set of filter responses at each pixel is computed. Finally, a histogram representation, which considers the signs and magnitudes of the responses as well as the number of filters, is applied on local image patches. We propose to apply this idea to a microscopy image pixel identification system based on a learning framework. Experimental results show that the proposed algorithm performs better than the state-of-the-art descriptors in biomedical images of different microscopy modalities.

Heat-shock factors (HSFs) are associated with multiple developmental processes, but their mechanisms of action in these processes remain largely enigmatic. Hsf2-null mice display gametogenesis defects and brain abnormalities characterized by enlarged ventricles. Here, we show that Hsf2−/− cerebral cortex displays mispositioning of neurons of superficial layers. HSF2 deficiency resulted in a reduced number of radial glia fibers, the architectural guides for migrating neurons, and of Cajal-Retzius cells, which secrete the positioning signal Reelin. Therefore, we focused on the radial migration signaling pathways. The levels of Reelin and Dab1 tyrosine phosphorylation were reduced, suggesting that the Reelin cascade is affected in Hsf2−/− cortices. The expression of p35, an activator of cyclin-dependent kinase 5 (Cdk5), essential for radial migration, was dependent on the amount of HSF2 in gain- and loss-of-function systems. p39, another Cdk5 activator, displayed reduced mRNA levels in Hsf2−/− cortices, which, together with the lowered p35 levels, decreased Cdk5 activity. We demonstrate in vivo binding of HSF2 to the p35 promoter and thereby identify p35 as the first target gene for HSF2 in cortical development. In conclusion, HSF2 affects cellular populations that assist in radial migration and directly regulates the expression of p35, a crucial actor of radial neuronal migration.

Glandular epithelial cells differentiate into complex multicellular or acinar structures, when embedded in three-dimensional (3D) extracellular matrix. The spectrum of different multicellular morphologies formed in 3D is a sensitive indicator for the differentiation potential of normal, non-transformed cells compared to different stages of malignant progression. In addition, single cells or cell aggregates may actively invade the matrix, utilizing epithelial, mesenchymal or mixed modes of motility. Dynamic phenotypic changes involved in 3D tumor cell invasion are sensitive to specific small-molecule inhibitors that target the actin cytoskeleton. We have used a panel of inhibitors to demonstrate the power of automated image analysis as a phenotypic or morphometric readout in cell-based assays. We introduce a streamlined stand-alone software solution that supports large-scale high-content screens, based on complex and organotypic cultures. AMIDA (Automated Morphometric Image Data Analysis) allows quantitative measurements of large numbers of images and structures, with a multitude of different spheroid shapes, sizes, and textures. AMIDA supports an automated workflow, and can be combined with quality control and statistical tools for data interpretation and visualization. We have used a representative panel of 12 prostate and breast cancer lines that display a broad spectrum of different spheroid morphologies and modes of invasion, challenged by a library of 19 direct or indirect modulators of the actin cytoskeleton which induce systematic changes in spheroid morphology and differentiation versus invasion. These results were independently validated by 2D proliferation, apoptosis and cell motility assays. We identified three drugs that primarily attenuated the invasion and formation of invasive processes in 3D, without affecting proliferation or apoptosis. Two of these compounds block Rac signalling, one affects cellular cAMP/cGMP accumulation. Our approach supports the growing needs for user-friendly, straightforward solutions that facilitate large-scale, cell-based 3D assays in basic research, drug discovery, and target validation.

Automated image analysis is demanded in cell biology and drug development research. The type of microscopy is one of the considerations in the trade-offs between experimental setup, image acquisition speed, molecular labelling, resolution and quality of images. In many cases, phase contrast imaging gets higher weights in this optimization. And it comes at the price of reduced image quality in imaging 3D cell cultures. For such data, the existing state-of-the-art computer vision methods perform poorly in segmenting specific cell type. Low SNR, clutter and occlusions are basic challenges for blind segmentation approaches. In this study we propose an automated method, based on a learning framework, for detecting particular cell type in cluttered 2D phase contrast images of 3D cell cultures that overcomes those challenges. It depends on local features defined over super pixels. The method learns appearance based features, statistical features, textural features and their combinations. Also, the importance of each feature is measured by employing Random Forest classifier. Experiments show that our approach does not depend on training data and the parameters.

Prostate cancer (PrCa) is one of the most prevalent malignant diseases among men in Western countries. Despite good initial treatment response is observed in the vast majority of PrCa patients, tumor relapse is observed in about 7-10% of patients undergoing standard antihormonal therapies with anti-androgens and/or GnRH antagonists. There is currently no cure for castration-resistant prostate cancer (CRPC), and median survival for these patients is only about 18 months. The high mortality rate of advanced cases is closely associated with invasive carcinomas and systemic metastasis, most frequently to the bone; and CRPC is almost always coincident with overexpression/amplification of the androgen receptor (AR) gene. CRPCs fail to respond to all currently prescribed first line anti-hormones such as flutamide, nilutamide or casodex (bicalutamide). The second line anti-tumor treatment against CRPC, frequently taxanes like docetaxel, is administered to patients after the first or second relapse. Taxanes are often supported by zoledronic acid targeting bone metastases. These drugs and drug combinations are in most cases initially effective, but rarely curative. Although taxanes do show potent anti-tumor effects, advanced PrCa patients develop resistance and only gain several months of survival time. For decades, no major improvements have been made in the therapy of advanced PrCa. However, this situation has dramatically changed over the last few years with a number of novel, promising drug concepts in the pipeline, some of which have already entered the market. It is not expected that these drugs will be curative, and relapses and therapy failures are expected to develop in these cases which may remain fatal. Therefore, the demands will remain high to develop yet better drugs, and more faithful models that reliably mimic at least some of the key aspects of advanced prostate cancers. These models are required to recapitulate the mechanisms leading to therapeutic resistance & failure also for new compounds and drug combinations entering the clinics now. There will remain a need to mimic which mechanisms either in the tumor cells or in the tumor-associated microenvironment may have contributed to the resistance. This consistent high demand in improved models will go hand in hand with a better understanding of the pros and cons of the various models available. Continued development of improved model systems for PrCa and in particular CRPC could lead to an informed ranking according to the maximal throughput in drug screens that can be achieved, balanced against their true informative value and relevance for recapitulating clinical PrCa. A systematic side-by-side comparison of available models, e.g. cell lines, organotypic three-dimensional cultures and co-cultures, xenografts, tissue

Heat shock factors (HSFs) are essential for all organisms to survive exposures to acute stress. They are best known as inducible transcriptional regulators of genes encoding molecular chaperones and other stress proteins. Four members of the HSF family are also important for normal development and lifespan-enhancing pathways, and the repertoire of HSF targets has thus expanded well beyond the heat shock genes. These unexpected observations have uncovered complex layers of post-translational regulation of HSFs that integrate the metabolic state of the cell with stress biology, and in doing so control fundamental aspects of the health of the proteome and ageing.

International audience; Organisms respond to circumstances threatening the cellular protein homeostasis by activation of heat-shock transcription factors (HSFs), which play important roles in stress resistance, development, and longevity. Of the four HSFs in vertebrates (HSF1-4), HSF1 is activated by stress, whereas HSF2 lacks intrinsic stress responsiveness. The mechanism by which HSF2 is recruited to stress-inducible promoters and how HSF2 is activated is not known. However, changes in the HSF2 expression occur, coinciding with the functions of HSF2 in development. Here, we demonstrate that HSF1 and HSF2 form heterotrimers when bound to satellite III DNA in nuclear stress bodies, subnuclear structures in which HSF1 induces transcription. By depleting HSF2, we show that HSF1-HSF2 heterotrimerization is a mechanism regulating transcription. Upon stress, HSF2 DNA binding is HSF1 dependent. Intriguingly, when the elevated expression of HSF2 during development is mimicked, HSF2 binds to DNA and becomes transcriptionally competent. HSF2 activation leads to activation of also HSF1, revealing a functional interdependency that is mediated through the conserved trimerization domains of these factors. We propose that heterotrimerization of HSF1 and HSF2 integrates transcriptional activation in response to distinct stress and developmental stimuli.

International audience; Organisms must be able to sense and respond rapidly to changes in their environment in order to maintain homeostasis and survive. Induction of heat shock proteins (Hsps) is a common cellular defense mechanism for promoting survival in response to various stress stimuli. Heat shock factors (HSFs) are transcriptional regulators of Hsps, which function as molecular chaperones in protecting cells against proteotoxic damage. Mammals have three different HSFs that have been considered functionally distinct: HSF1 is essential for the heat shock response and is also required for developmental processes, whereas HSF2 and HSF4 are important for differentiation and development. Specifically, HSF2 is involved in corticogenesis and spermatogenesis, and HSF4 is needed for maintenance of sensory organs, such as the lens and the olfactory epithelium. Recent evidence, however, suggests a functional interplay between HSF1 and HSF2 in the regulation of Hsp expression under stress conditions. In lens formation, HSF1 and HSF4 have been shown to have opposite effects on gene expression. In this chapter, we present the different roles of the mammalian HSFs as regulators of cellular stress and developmental processes. We highlight the interaction between different HSFs and discuss the discoveries of novel target genes in addition to the classical Hsps.

The failure of many drugs in the clinic - drugs proven to be active in pre-clinical assays - has raised question marks on the predictive power of these models, often based on cell line data. The divergence of these cell lines from the original tumors, and their rapid growth as simple monocultures on 2D plastic were highlighted as potentially causing these issues. Implantation of the cell lines into mice adds complexity to these models, but the crosstalk with mouse stroma also leads to confounding results (eg, HGF, IL-6). PREDECT, a European consortium funded by the Innovative Medicines Initiative (IMI), has the goal to build and characterise more predictive Oncology model platforms for three pathologies (lung, breast and prostate). Signalling pathways and heterogeneity of these model platforms are compared to each other as well as to primary human tissue using a central TMA-based platform. For prostate cancer, a 3D tumor growth assay system was established recently using RFP/FLuc-labelled LNCaP, PC346C, or VCaP human prostate cancer cells grown in a matrix of Matrigel, collagen, or a mix of the two, in the presence or absence of GFP/RLuc labelled human stromal cells (WPMY, or an immortalised cancer-associated fibroblast cell line, CAF). Cell densities, ratios, and matrix concentrations/volumes were optimised for cells cultured for 3 weeks. In an approach to improve tumor-stroma crosstalk in vivo, LNCaP cells pre-grown in 3D cultures with or without human stromal cells were implanted orthotopically into SCID mice. Growth of the tumor and stromal cells was monitored by in vivo bioluminescence, and the effect of the co-implantation on the primary tumor and metastasis was analysed via ex-vivo luciferase assays and IHC. Whereas LNCaP and PC346C cells readily formed spheroids in 3D culture, VCaPs remained as single cells. Growth of VCaPs was facilitated by pre-forming spheroids in vitro, followed by matrix embedding. Addition of stromal cells stimulated growth of not only the tumor, but also the stromal cells in some conditions, as monitored using RFP and GFP live cell imaging. 3D, as well as stromal cell-mediated treatment resistance was observed. Using the fluorescent dyes Hoechst, EdU, and NucView, the proliferative status of individual cells within spheroids could be analysed in situ. Finally, paraffin-embedding of the 3D cultures allowed for TMA analysis and comparison to other models and patient samples.Results will also be shown on the orthotopic growth of LNCaP tumors and the effect of stromal cell co-implantation. Introducing complexity to cell culture may help to generate more realistic, and thus also more predictive models - in a first step using even standard cell lines. Citation Format: Suzana Vidic, Norbert Esser, Ronald de Hoogt, Ingrid Verberne, Ira Kogan-Sakin, Yan Stein, Varda Rotter, Michael Barbier, Yolanda Chong, Sabine De Breucker, Karine Smans, Malin Akerfelt, Matthias Nees, Peter King, Ian Hickson, Wytske van Weerden, Ralph Graeser. Complex in vitro and in vivo prostate cancer models for the PREDECT consortium. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2023. doi:10.1158/1538-7445.AM2014-2023

Prostate cancer (PrCa) is the most common malignancy in Western countries and account for the second highest mortality rate of all cancer forms in males. Specifically in late stage castration-resistant PrCa (CRPC), the signaling pathways that contribute to invasion and metastasis are poorly understood. The heat shock factors (HSFs) are a family of multifaceted transcriptional regulators that govern survival upon proteotoxic stress. This is achieved, mainly via HSF1, through up-regulation of genes critical for endurance under stressful conditions. Interestingly, the capacity to withstand stress is also readily utilized by cancer cells, which face a variety of stressful conditions. It thus seems that HSF1 allows cancer cells to rapidly modify their physiology, metabolism and protein homeostasis, thereby facilitating oncogenesis. Moreover, lack of HSF1 reduces the susceptibility of mice to mutation- or carcinogen-driven tumors and reduces the growth of human cancer cell lines. Recently, examination of a cohort of breast cancer patients showed that high expression of HSF1 correlated with decreased survival rate, highlighting the clinical significance of HSF1. The aim of this study was to elucidate novel molecular pathways that contribute to the detrimental development of CRPC. Our initial in silico analyses showed that in PrCa, HSF1 expression levels correlate with poor survival and high Gleason grades. Most intriguingly, HSF2, thus far unassociated with cancer, showed decreased expression in the same set of tumors. We used real time live cell imaging in combination with endpoint immunofluorescent stainings of 3-dimensional (3D) organotypic cultures of PC-3 cells that spontaneously undergo an invasive switch, leading to the disintegration of epithelial structures and the basal lamina, and formation of invadopodia and invasive structures. This model demonstrates the highly dynamic nature of epithelial plasticity, and the functional studies of 3D cultures implied roles for both of these factors in the invasive behavior of tumors. In fact, we found that HSF1 and HSF2 show opposing effect on tumor cell invasion. Tumor spheroids lacking HSF1 grew more slowly, were more differentiated and polarized, and were devoid of any invasive behavior. In contrast, downregulation of HSF2 seemed to increase the invasive properties, and HSF2 could therefore act as a tumor suppressor. In combination with bioinformatics analyses, these results were further validated in human pathology using tissue microarray and the molecular pathways behind these observations were elucidated by transcriptome analyses. Our results reveal involvement of two novel players in PrCa, HSF1 and HSF2 that intriguingly seem to have opposite roles in tumor invasion. We conclude that HSF1 and HSF2 have significant implications for the molecular understanding of cancer progression and invasion. Citation Format: Malin Åkerfelt, Johanna Björk, Tuomas Mirtti, Vidal Fey, Lea Sistonen, Matthias Nees. Opposing effects of heat shock factors 1 and 2 on prostate cancer invasion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A46.