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Bladder Cancer (BLCa) inter-patient heterogeneity is considered the primary cause of tumor reoccurrence and treatment failure, suggesting that BLCa patients could benefit from a more personalized treatment approach. Patient-derived organoids (PDOs) have been successfully used as a functional model for predicting drug response in different cancer types. In our study, we established BLCa PDO cultures from different BLCa stages. BLCa PDOs preserve the histological and molecular heterogeneity of the parental tumors, including their multiclonal genetic landscapes. BLCa PDOs consistently share key genetic alterations detected in parental tumors, mirroring tumor evolution in longitudinal sampling. Our drug screening pipeline was implemented using BLCa PDOs, testing both standard-of-care and additional FDA-approved compounds for other solid tumors. Integrative analysis of drug response profiles with matched PDO genomic analysis was used to determine enrichment thresholds for candidate markers of therapy resistance and sensitivity. By assessing the clinical history of longitudinally sampled cases, the clonal evolution of the disease could be determined and matched with drug response profiles. In conclusion, we have developed a clinically relevant pipeline for drug response profile assessment and discovery of candidate markers of therapy resistance.

Introduction: Interpatient heterogeneity is one of the causes of treatment failure in bladder cancer (BLCa) patients, who would therefore profit from tailor-made therapies. Toward the direction of precision medicine, patient-derived organoids (PDOs), which retain parental tumor (PT) features, have been successfully tested in different cancer types and shown to predict patients’ responses. Therefore, our study aimed to explore the ability of BLCa PDOs to retain PT features and to determine patient drug sensitivity. We correlated PDO drug response profiles to their genomic profiles and the patient's clinical history. Methods: PDOs were derived from the suspension culture of single cells. Whole exome sequencing was performed on genomic DNA and scRNA sequencing on 3 PT/PDO pairs. Histological evaluation of PDO and PT marker expression and morphology was performed. PDOs were treated for 48h and drug response was evaluated by viability assay. Results: We derived PDOs from non-muscle-invasive and muscle-invasive BLCa. Genomic analysis of 15 representative PT/PDOs pairs showed high similarity in terms of copy number variations and single nucleotide variants (SNVs) with preservation of peculiar BLCa alterations. PT genomic heterogeneity was preserved in PDOs as demonstrated by the correlation between SNVs clonality profiles of PT/PDOs pairs. Moreover, marker analyses at the scRNA and protein levels supported that PDOs retained the main tumor phenotype. Interestingly, PDO grew accordingly to three morphologies linked to marker expression and PT stage.PDOs were used in drug screen assays for testing standard-of-care (SOC) and FDA-approved drugs. PDO responses were highly heterogeneous highlighting interpatient heterogeneity. In 2 case, PDO sensitivity mimicked the patient’s response to SOC and in a few cases correlated with the sample-specific genomic background. Due to the high relapse rate of BLCa, we performed 2 longitudinal studies. In the first one, PDOs from a relapse collected after epirubicin treatment showed less sensitivity to the drug compared to the baseline sample. Moreover, the SNVs' mean clonality of relapse PDOs was significantly higher than the baseline PDOs which were characterized by mutations in DNA damage repair genes (e.g., ATM, FANC1, ERCC2, and BRCA2). We hypothesize a drug-induced selection of a pre-existing epirubicin-resistant population. In the second study, PDOs from the cystectomy and the following relapse showed similar genomic and drug sensitivity profiles suggesting that the first sample could be informative for adjuvant therapy. Conclusion: We established PDOs from different BLCa stages and grades which preserve PT features and can be implemented in drug screening assays for personalized approaches. The power of PDOs in precision medicine was highlighted by the longitudinal studies that showed that PDOs mirrored patient response and tumor evolution in vitro. Citation Format: Martina Minoli, Thomas Cantore, Daniel Hanhart, Mirjam Kiener, Tarcisio Fedrizzi, Federico La Manna, Sofia Karkampouna, Panagiotis Chouvardas, Vera Genitsch, José Antonio Rodriguez-Calero, Eva Compérat, Irena Klima, Paola Gasperini, Bernhard Kiss, Roland Seiler-Blarer, Francesca Demichelis, George N. Thalmann, Marianna Kruithof-de Julio. Bladder cancer organoids as a functional system to model different disease stages and therapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 182.

During disease progression from primary towards metastatic prostate cancer (PCa), and in particular bone metastases, the tumor microenvironment (TME) evolves in parallel with the cancer clones, altering extracellular matrix composition (ECM), vasculature architecture, and recruiting specialized tumor-supporting cells that favor tumor spread and colonization at distant sites. We introduce the clinical profile of advanced metastatic PCa in terms of common genetic alterations. Findings from recently developed models of PCa metastatic spread are discussed, focusing mainly on the role of the TME (mainly matrix and fibroblast cell types), at distinct stages: premetastatic niche orchestrated by the primary tumor towards the metastatic site and bone metastasis. We report evidence of premetastatic niche formation, such as the mechanisms of distant site conditioning by extracellular vesicles, chemokines and other tumor-derived mechanisms, including altered cancer cell-ECM interactions. Furthermore, evidence supporting the similarities of stroma alterations among the primary PCa and bone metastasis, and contribution of TME to androgen deprivation therapy resistance are also discussed. We summarize the available bone metastasis transgenic mouse models of PCa from a perspective of pro-metastatic TME alterations during disease progression and give an update on the current diagnostic and therapeutic radiological strategies for bone metastasis clinical management.

Oncofetal protein, CRIPTO, is silenced during homeostatic postnatal life and often re-expressed in different neoplastic processes, such as hepatocellular carcinoma. Given the reactivation of CRIPTO in pathological conditions reported in various adult tissues, the aim of this study was to explore whether CRIPTO is expressed during liver fibrogenesis and whether this is related to the disease severity and pathogenesis of fibrogenesis. Furthermore, we aimed to identify the impact of CRIPTO expression on fibrogenesis in organs with high versus low regenerative capacity, represented by murine liver fibrogenesis and adult murine heart fibrogenesis. Circulating CRIPTO levels were measured in plasma samples of patients with cirrhosis registered at the waitlist for liver transplantation (LT) and 1 year after LT. The expression of CRIPTO and fibrotic markers (αSMA, collagen type I) was determined in human liver tissues of patients with cirrhosis (on a basis of viral hepatitis or alcoholic disease), in cardiac tissue samples of patients with end-stage heart failure, and in mice with experimental liver and heart fibrosis using immuno-histochemical stainings and qPCR. Mouse models with experimental chronic liver fibrosis, induced with multiple shots of carbon tetrachloride (CCl4) and acute liver fibrosis (one shot of CCl4), were evaluated for CRIPTO expression and fibrotic markers. CRIPTO was overexpressed in vivo (Adenoviral delivery) or functionally sequestered by ALK4Fc ligand trap in the acute liver fibrosis mouse model. Murine heart tissues were evaluated for CRIPTO and fibrotic markers in three models of heart injury following myocardial infarction, pressure overload, and ex vivo induced fibrosis. Patients with end-stage liver cirrhosis showed elevated CRIPTO levels in plasma, which decreased 1 year after LT. Cripto expression was observed in fibrotic tissues of patients with end-stage liver cirrhosis and in patients with heart failure. The expression of CRIPTO in the liver was found specifically in the hepatocytes and was positively correlated with the Model for End-stage Liver Disease (MELD) score for end-stage liver disease. CRIPTO expression in the samples of cardiac fibrosis was limited and mostly observed in the interstitial cells. In the chronic and acute mouse models of liver fibrosis, CRIPTO-positive cells were observed in damaged liver areas around the central vein, which preceded the expression of αSMA-positive stellate cells, i.e., mediators of fibrosis. In the chronic mouse models, the fibrosis and CRIPTO expression were still present after 11 weeks, whereas in the acute model the liver regenerated and the fibrosis and CRIPTO expression resolved. In vivo overexpression of CRIPTO in this model led to an increase in fibrotic markers, while blockage of CRIPTO secreted function inhibited the extent of fibrotic areas and marker expression (αSMA, Collagen type I and III) and induced higher proliferation of residual healthy hepatocytes. CRIPTO expression was also upregulated in several mouse models of cardiac fibrosis. During myocardial infarction CRIPTO is upregulated initially in cardiac interstitial cells, followed by expression in αSMA-positive myofibroblasts throughout the infarct area. After the scar formation, CRIPTO expression decreased concomitantly with the αSMA expression. Temporal expression of CRIPTO in αSMA-positive myofibroblasts was also observed surrounding the coronary arteries in the pressure overload model of cardiac fibrosis. Furthermore, CRIPTO expression was upregulated in interstitial myofibroblasts in hearts cultured in an ex vivo model for cardiac fibrosis. Our results are indicative for a functional role of CRIPTO in the induction of fibrogenesis as well as a potential target in the antifibrotic treatments and stimulation of tissue regeneration. ispartof: CELLS vol:10 issue:12 ispartof: location:Switzerland status: published

INTRODUCTION AND OBJECTIVE:Prostate cancer (PCa) is the most frequent malignancy detected in men and the second leading cause of cancer-related male death in Europe. Current markers frequently fail...

There exists a set of factors termed oncofetal proteins that play key roles in ontogeny before they decline or disappear as the organism’s tissues achieve homeostasis, only to then re-emerge in cancer. Although the unique therapeutic potential presented by such factors has been recognized for more than a century, their clinical utility has yet to be fully realized1. This review highlights the small signaling protein CRIPTO encoded by the tumor derived growth factor 1 (TDGF1/Tdgf1) gene, an oft cited oncofetal protein whose presence in the cancer literature as a tumor promoter, diagnostic marker and viable therapeutic target continues to grow. We touch lightly on features well established and well-reviewed since its discovery more than 30 years ago, including CRIPTO’s early developmental roles and modulation of SMAD2/3 activation by a selected set of transforming growth factor β (TGF-β) family ligands. We predominantly focus instead on more recent and less well understood additions to the CRIPTO signaling repertoire, on its potential upstream regulators and on new conceptual ground for understanding its mode of action in the multicellular and often stressful contexts of neoplastic transformation and progression. We ask whence it re-emerges in cancer and where it ‘hides’ between the time of its fetal activity and its oncogenic reemergence. In this regard, we examine CRIPTO’s restriction to rare cells in the adult, its potential for paracrine crosstalk, and its emerging role in inflammation and tissue regeneration—roles it may reprise in tumorigenesis, acting on subsets of tumor cells to foster cancer initiation and progression. We also consider critical gaps in knowledge and resources that stand between the recent, exciting momentum in the CRIPTO field and highly actionable CRIPTO manipulation for cancer therapy and beyond.

BackgroundOncofetal protein, Cripto, is silenced during postnatal life and often re-expressed in different neoplastic processes. In the present study we investigated the potential role of Cripto in hepatic and cardiac fibrosis. In this study, the aim was to explore whether Cripto is expressed during liver fibrogenesis and whether this is related to the disease severity and pathogenesis of fibrogenesis. Furthermore, we aimed to identify the impact of Cripto expression on fibrogenesis in organs with high versus low regenerative capacity, represented by murine liver fibrogenesis and adult murine heart fibrogenesisMethodsCirculating CRIPTO levels were measured in plasma samples of patients with cirrhosis registered at the waitlist for liver transplantation (LT) and one year after LT. The expression of Cripto and fibrotic markers (aSMA, collagen I) were determined in human liver tissues of patients with cirrhosis (on a basis of viral hepatitis or alcoholic disease), in cardiac tissue samples of patients with end-stage heart failure and of mice with experimental liver and heart fibrosis using immuno-histochemical stainings and qPCR. Mouse models with experimental chronic liver fibrosis, induced with multiple shots of carbon tetrachloride (CCl4) and acute liver fibrosis (one shot of CCl4) were evaluated for Cripto expression and fibrotic markers. Cripto was overexpressed in vivo (Adenoviral delivery) or functionally sequestered by ALK4Fc ligand trap in the acute liver fibrosis mouse model. Murine heart tissues were evaluated for Cripto and fibrotic markers, in three models of heart injury; following myocardial infarction, pressure overload and ex vivo induced fibrosis.ResultsPatients with end-stage liver cirrhosis showed elevated Cripto levels in plasma, which had decreased one year after LT. Cripto expression was observed in fibrotic tissues of patients with end-stage liver cirrhosis and in patients with heart failure. The expression of Cripto in the liver was found specifically in the hepatocytes and was positively correlated with the Model for End-stage Liver Disease (MELD) score for end-stage liver disease. Cripto expression in the samples of cardiac fibrosis was limited and mostly observed in the interstitial cells. In the chronic and acute mouse models of liver fibrosis, Cripto-positve cells were observed in damaged liver areas around the central vein, which preceded the expression of aSMA-positive stellate cells, i.e. mediators of fibrosis. Whereas in the chronic mouse models the fibrosis and Cripto expression was still present after 11 weeks, in the acute model the liver regenerated and the fibrosis and Cripto expression resolved. In vivo overexpression of Cripto in this model, led to an increase in fibrotic markers while blockage of Cripto secreted function inhibited the extend of fibrotic areas and marker expression (αSMA, Collagen type I and III) and induced higher proliferation of residual healthy hepatocytes. Cripto expression was also upregulated in several mouse models of cardiac fibrosis. During myocardial infarction Cripto is upregulated initially in cardiac interstitial cells, followed by expression in αSMA-positive myofibroblasts throughout the infarct area. After the scar formation, Cripto expression decreased concomitantly with the aSMA expression. Temporal expression of Cripto in αSMA-positive myofibroblasts was also observed surrounding the coronary arteries in the pressure overload model of cardiac fibrosis. Furthermore, Cripto expression was upregulated in interstitial myofibroblasts in hearts cultured in an ex vivo model for cardiac fibrosis.ConclusionOur results are indicative for a functional role of Cripto in induction of fibrogenesis and potential applications in antifibrotic treatments and stimulation of tissue regeneration.

Tumors strongly depend on their surrounding tumor microenvironment (TME) for growth and progression, since stromal elements are required to generate the optimal conditions for cancer cell proliferation, invasion, and possibly metastasis. Prostate cancer (PCa), though easily curable during primary stages, represents a clinical challenge in advanced stages because of the acquisition of resistance to anti-cancer treatments, especially androgen-deprivation therapies (ADT), which possibly lead to uncurable metastases such as those affecting the bone. An increasing number of studies is giving evidence that prostate TME components, especially cancer-associated fibroblasts (CAFs), which are the most abundant cell type, play a causal role in PCa since the very early disease stages, influencing therapy resistance and metastatic progression. This is highlighted by the prognostic value of the analysis of stromal markers, which may predict disease recurrence and metastasis. However, further investigations on the molecular mechanisms of tumor–stroma interactions are still needed to develop novel therapeutic approaches targeting stromal components. In this review, we report the current knowledge of the characteristics and functions of the stroma in prostate tumorigenesis, including relevant discussion of normal prostate homeostasis, chronic inflammatory conditions, pre-neoplastic lesions, and primary and metastatic tumors. Specifically, we focus on the role of CAFs, to point out their prognostic and therapeutic potential in PCa.

Cripto is a small glycosylphosphatidylinisitol (GPI)-anchored and secreted oncofetal protein that plays important roles in regulating normal physiological processes, including stem cell differentiation, embryonal development, and tissue growth and remodeling, as well as pathological processes such as tumor initiation and progression. Cripto functions as a co-receptor for TGF-β ligands such as Nodal, GDF1, and GDF3. Soluble and secreted forms of Cripto also exhibit growth factor-like activity and activate SRC/MAPK/PI3K/AKT pathways. Glucose-Regulated Protein 78 kDa (GRP78) binds Cripto at the cell surface and has been shown to be required for Cripto signaling via both TGF-β and SRC/MAPK/PI3K/AKT pathways. To provide a comprehensive overview of the scientific literature related to Cripto, we performed, for the first time, a bibliometric analysis of the biological roles of Cripto as reported in the scientific literature covering the last 10 years. We present different fields of knowledge in comprehensive areas of research on Cripto, ranging from basic to translational research, using a keyword-driven approach. Our ultimate aim is to aid the scientific community in conducting targeted research by identifying areas where research has been conducted so far and, perhaps more importantly, where critical knowledge is still missing.

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, due to lack of experimental models that mimic different disease stages. We describe a novel androgen-dependent PCa patient-derived xenograft (PDX) model from treatment-naïve, soft tissue metastasis (PNPCa). RNA and whole-exome sequencing of the PDX tissue and organoids confirmed transcriptomic and genomic similarity to primary tumor. PNPCa harboursBRCA2 and CHD1somatic mutations, shows anSPOP/FOXA1-like transcriptomic signature and microsatellite instability, which occurs in 3% of advanced PCa and has never been modelledin vivo. Comparison of the treatment-naïve PNPCa with additional metastatic PDXs (BM18, LAPC9), in a medium-throughput organoid screen of FDA-approved compounds, revealed differential drug sensitivities. Multikinase inhibitors (ponatinib, sunitinib, sorafenib) were broadly effective on all PDX- and patient-derived organoids from advanced cases with acquired resistance to standard-of-care compounds. This proof-of-principle study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified, repurposed compounds.

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, due to lack of experimental models that mimic different disease stages. We describe an androgen-dependent PCa patient-derived xenograft (PDX) model from treatment-naïve, soft tissue metastasis (PNPCa). RNA and whole-exome sequencing of the PDX tissue and organoids confirmed transcriptomic and genomic similarity to primary tumor. PNPCa harbors BRCA2 and CHD1 somatic mutations, shows an SPOP/FOXA1-like transcriptomic signature and microsatellite instability, which occurs in 3% of advanced PCa and has never been modeled in vivo. Comparison of the treatment-naïve PNPCa with additional metastatic PDXs (BM18, LAPC9), in a medium-throughput organoid screen of FDA-approved compounds, revealed differential drug sensitivities. Multikinase inhibitors (ponatinib, sunitinib, sorafenib) were broadly effective on all PDX- and patient-derived organoids from advanced cases with acquired resistance to standard-of-care compounds. This proof-of-principle study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified, repurposed compounds., To date, patients still succumb to cancer, due to tumors not responding to therapy or ultimately acquiring resistance. Here the authors show that by exploiting patient derived organoids and a treatment-naïve patient derived xenograft, patient therapy can be personalized.

Simple Summary Currently, there is a need for prognostic tools that can stratify patients, who present with primary disease, based on whether they are at low or high risk for drug resistant and hormone-independent lethal metastatic prostate cancer. The aim of our study was to assess the potentially added value of tumor microenvironment (stroma) components for the characterisation of prostate cancer. By utilising patient derived-xenograft models we show that the molecular properties of the stroma cells are highly responsive to androgen hormone levels, and considerable ECM remodelling processes take place not only in androgen-dependent but also in androgen-independent tumor models. Transcriptomic mechanisms linked to osteotropism are conserved in bone metastatic xenografts, even when implanted in a different microenvironment. A stroma-specific gene list signature was identified, which highly correlates with Gleason score, metastasis progression and progression-free survival, and thus could potentially complement current patient stratification methods. Abstract Resistance acquisition to androgen deprivation treatment and metastasis progression are a major clinical issue associated with prostate cancer (PCa). The role of stroma during disease progression is insufficiently defined. Using transcriptomic and proteomic analyses on differentially aggressive patient-derived xenografts (PDXs), we investigated whether PCa tumors predispose their microenvironment (stroma) to a metastatic gene expression pattern. RNA sequencing was performed on the PCa PDXs BM18 (castration-sensitive) and LAPC9 (castration-resistant), representing different disease stages. Using organism-specific reference databases, the human-specific transcriptome (tumor) was identified and separated from the mouse-specific transcriptome (stroma). To identify proteomic changes in the tumor (human) versus the stroma (mouse), we performed human/mouse cell separation and subjected protein lysates to quantitative Tandem Mass Tag labeling and mass spectrometry. Tenascin C (TNC) was among the most abundant stromal genes, modulated by androgen levels in vivo and highly expressed in castration-resistant LAPC9 PDX. The tissue microarray of primary PCa samples (n = 210) showed that TNC is a negative prognostic marker of the clinical progression to recurrence or metastasis. Stroma markers of osteoblastic PCa bone metastases seven-up signature were induced in the stroma by the host organism in metastatic xenografts, indicating conserved mechanisms of tumor cells to induce a stromal premetastatic signature. A 50-gene list stroma signature was identified based on androgen-dependent responses, which shows a linear association with the Gleason score, metastasis progression and progression-free survival. Our data show that metastatic PCa PDXs, which differ in androgen sensitivity, trigger differential stroma responses, which show the metastasis risk stratification and prognostic biomarker potential.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre-loxP-controlled lentiviral vectors expressing CRIPTO in cell line-derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft-derived ex vivo tumour slices. CRIPTO-overexpressing patient-derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial-to-mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2-CRIPTO cells formed tumours when injected into immune-compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High-level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO-expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Lysosomal sequestration of anti-cancer compounds reduces drug availability at intracellular target sites, thereby limiting drug-sensitivity and inducing chemoresistance. For hepatocellular carcinoma (HCC), sorafenib (SF) is the first line systemic treatment, as well as a simultaneous activator of autophagy-induced drug resistance. The purpose of this study is to elucidate how combination therapy with the FDA-approved photosensitizer verteporfin (VP) can potentiate the antitumor effect of SF, overcoming its acquired resistance mechanisms. HCC cell lines and patient-derived in vitro and in vivo preclinical models were used to identify the molecular mechanism of action of VP alone and in combination with SF. We demonstrate that SF is lysosomotropic and increases the total number of lysosomes in HCC cells and patient-derived xenograft model. Contrary to the effect on lysosomal stability by SF, VP is not only sequestered in lysosomes, but induces lysosomal pH alkalinization, lysosomal membrane permeabilization (LMP) and tumor-selective proteotoxicity. In combination, VP-induced LMP potentiates the antitumor effect of SF, further decreasing tumor proliferation and progression in HCC cell lines and patient-derived samples in vitro and in vivo. Our data suggest that combination of lysosome-targeting compounds, such as VP, in combination with already approved chemotherapeutic agents could open a new avenue to overcome chemo-insensitivity caused by passive lysosomal sequestration of anti-cancer drugs in the context of HCC.

Dupuytren’s disease is a connective tissue disorder of the hand causing excessive palmar fascial fibrosis with associated finger contracture and disability. The aetiology of the disease is heterogeneous, with both genetic and environmental components. The connective tissue is abnormally infiltrated by myofibroblasts that deposit collagen and other extracellular matrix proteins. We describe the clinical profile of Dupuytren’s disease along with current therapeutic schemes. Recent findings on molecular and cellular parameters that are dysregulated in Dupuytren’s disease, which may contribute to the onset of the disease, and the role of resident inflammation promoting fibrosis, are highlighted. We review recent literature focusing on non-myofibroblast cell types (stem cell-like cells), their pro-inflammatory and pro-fibrotic role that may account for abnormal wound healing response.

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, mainly due to the lack of experimental models representing different disease stages. We aim to provide functional experimental and preclinical drug tools applicable for direct use with patient-derived material. Biopsies from metastatic PCa were used for the establishment of patient-derived xenografts (PDXs) by subcutaneous implantation. In vivo tumor growth kinetic in response to androgens was assessed by surgical castration and testosterone supplementation. RNA and whole-exome sequencing (WES) and organoid drug screens were used to characterize the genomic, transcriptomic, and functional properties. Organoid culture methodology was adapted in order to set up an automated medium-throughput organoid drug screen (Nexus Theragnostics automated platform) for screening standard-of-care compounds and candidates for drug repurposing. A novel case of PCa xenograft model derived from soft tissue metastasis (PNPCa) was established. RNAseq and WES confirmed transcriptomic and genomic similarity to the primary tumor. PNPCa harbors BRAC2 and CHD1 mutations and shows SPOP-like and FOXA1-like transcriptomic signature with microsatellite instability. PNPCa tumor growth is inhibited after androgen deprivation by castration, while androgen replacement leads to tumor reformation. No spontaneous tumor growth occurred after prolonged period of castration; however, scarce micrometastases were found in the bone marrow and lymph nodes. The treatment-naive and androgen-dependent properties of the PNPCa made it a candidate model for identification of potent drug compounds. PNPCa-derived organoid screens on standard-of-care and 74 FDA-approved compounds showed that mTOR pathway and multi-tyrosine kinase inhibitors, used for clinical treatment of other cancer types, have high impact on PCa organoid viability. Application of the organoid drug screen panel on two additional metastatic PDXs of advanced disease has allowed shortlisting of compounds for repurposing use in patient-derived organoid screens that can be routinely performed in a timeframe of few weeks, and thus, provide information on treatment decision. Treatment response of a treatment-naive, early metastatic PCa case (PNPCa PDX) highlights the potential of organoid screens to assess therapy response with extended applications for personalized screens. Citation Format: Sofia Karkampouna, Federico la Manna, Maria R. De Filippo, Mirjam Kiener, Marta De Menna, Eugenio Zoni, Joel Grosjean, Irena Klima, Andrea Garofoli, Marco Bolis, Jean-Philippe Theurillat, Vera Genitsch, David Keller, Booij Tijmen, Christian U. Stirnimann, Andrea Sboner, Charlotte K.Y. Ng, Salvatore Piscuoglio, Martin Spahn, Mark A. Rubin, George N. Thalmann, Marianna Kruithof-de Julio. Patient-derived xenograft and organoids models of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B18.

Prostate cancer (PCa) is the most common malignancy detected in males and the second most common cause of cancer death in western countries. The development of the prostate gland, is finely regulated by androgens which modulate also its growth and function. Importantly, androgens exert a major role in PCa formation and progression and one of the hypothesized mechanism proposed has been linked to the chromosomal rearrangement of the androgen regulated gene TMPRSS2 with ERG. Androgens have been therefore used as main target for therapies in the past. However, despite the development of endocrine therapies (e.g. androgen ablation), when PCa progress, tumors become resistant to this therapeutic castration and patients develop incurable metastases. A strategy to better understand how patients respond to therapy, in order to achieve a better patient stratification, consists in monitoring the levels of small noncoding RNAs (microRNAs). microRNAs are a class of small molecules that regulate protein abundance and their application as biomarkers to monitor disease progression has been intensely studied in the last years. In this review, we highlight the interactions between microRNAs and endocrine-related aspects of PCa in tissues. We focus on the modulation of TMPRSS2-ERG and Glucocorticoid Receptor (GR) by microRNAs and detail the influence of steroidal hormonal therapies on microRNAs expression.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Despite increasing treatment options for this disease, prognosis remains poor. CRIPTO (TDGF1) protein is expressed at high levels in several human tumours and promotes oncogenic phenotype. Its expression has been correlated to poor prognosis in HCC. In this study, we aimed to elucidate the basis for the effects of CRIPTO in HCC. We investigated CRIPTO expression levels in three cohorts of clinical cirrhotic and HCC specimens. We addressed the role of CRIPTO in hepatic tumourigenesis using Cre-loxP-controlled lentiviral vectors expressing CRIPTO in cell line-derived xenografts. Responses to standard treatments (sorafenib, doxorubicin) were assessed directly on xenograft-derived ex vivo tumour slices. CRIPTO-overexpressing patient-derived xenografts were established and used for ex vivo drug response assays. The effects of sorafenib and doxorubicin treatment in combination with a CRIPTO pathway inhibitor were tested in ex vivo cultures of xenograft models and 3D cultures. CRIPTO protein was found highly expressed in human cirrhosis and hepatocellular carcinoma specimens but not in those of healthy participants. Stable overexpression of CRIPTO in human HepG2 cells caused epithelial-to-mesenchymal transition, increased expression of cancer stem cell markers, and enhanced cell proliferation and migration. HepG2-CRIPTO cells formed tumours when injected into immune-compromised mice, whereas HepG2 cells lacking stable CRIPTO overexpression did not. High-level CRIPTO expression in xenograft models was associated with resistance to sorafenib, which could be modulated using a CRIPTO pathway inhibitor in ex vivo tumour slices. Our data suggest that a subgroup of CRIPTO-expressing HCC patients may benefit from a combinatorial treatment scheme and that sorafenib resistance may be circumvented by inhibition of the CRIPTO pathway. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.

CRIPTO (CR-1, TDGF1) is a cell surface/secreted oncoprotein actively involved in development and cancer. Here, we report that high expression of CRIPTO correlates with poor survival in stratified risk groups of prostate cancer (PCa) patients. CRIPTO and its signaling partner glucose-regulated protein 78 (GRP78) are highly expressed in PCa metastases and display higher levels in the metastatic ALDH(high) sub-population of PC-3M-Pro4Luc2 PCa cells compared with non-metastatic ALDH(low). Coculture of the osteotropic PC-3M-Pro4Luc2 PCa cells with differentiated primary human osteoblasts induced CRIPTO and GRP78 expression in cancer cells and increases the size of the ALDH(high) sub-population. Additionally, CRIPTO or GRP78 knockdown decreases proliferation, migration, clonogenicity and the size of the metastasis-initiating ALDH(high) sub-population. CRIPTO knockdown reduces the invasion of PC-3M-Pro4Luc2 cells in zebrafish and inhibits bone metastasis in a preclinical mouse model. These results highlight a functional role for CRIPTO and GRP78 in PCa metastasis and suggest that targeting CRIPTO/GRP78 signaling may have significant therapeutic potential.

Liver exhibits a remarkable maintenance of functional homeostasis in the presence of a variety of damaging toxic factors. Tissue regeneration involves cell replenishment and extracellular matrix remodeling. Key regulator of homeostasis is the transforming growth factor-β (TGFβ) cytokine. To understand the role of TGFβ during liver regeneration, we used the single-dose carbon tetrachloride (CCl4) treatment in mice as a model of acute liver damage. We combined this with in vivo inhibition of the TGFβ pathway by a small molecule inhibitor, LY364947, which targets the TGFβ type I receptor kinase [activin receptor-like kinase 5 (ALK5)] in hepatocytes but not in activated stellate cells. Co-administration of LY364947 inhibitor and CCl4 toxic agent resulted in enhanced liver regeneration; cell proliferation (measured by PCNA, phosphorylated histone 3, p21) levels were increased in CCl4 + LY364947 versus CCl4-treated mice. Recovery of CCl4-metabolizing enzyme CYP2E1 expression in hepatocytes is enhanced 7 days after CCl4 intoxication in the mice that received also the TGFβ inhibitor. In summary, a small molecule inhibitor that blocks ALK5 downstream signaling and halts the cytostatic role of TGFβ pathway results in increased cell regeneration and improved liver function during acute liver damage. Thus, in vivo ALK5 modulation offers insight into the role of TGFβ, not only in matrix remodeling and fibrosis, but also in cell regeneration.

Introduction: Androgen-deprivation therapy is the standard treatment for prostate cancer. Despite the initial response, a fraction of cases manifest progression to castration-resistant prostate cancer. The paradigm of PCa recurrence is the existence of androgen-independent cancer stem cells, which differentiate into androgen-dependent cancer cells that reinitiate tumor growth. The phenotypic properties of cancer stem cells versus highly proliferative progenitor cells/facultative stem cells remain to be further characterized in terms of androgen receptor signaling, quiescence/proliferation, tumorigenicity, and therapy resistance. Methods: To address the molecular basis of CSC switch from androgen-dependency to independency we have employed two patient-derived xenograft models (LAPC-9 and BM-18) that have different androgen sensitivity properties. We have performed microarray and proteomic analysis of BM-18 tumor tissues prior to and following castration as well as androgen replacement. To characterize the cancer stem cells in these models we have isolated different subpopulations based on combination of selected markers and assessed the transcriptomic changes, proteomic profile, and in vivo self-renewal. Results: Castration induces a rapid tumor volume decrease in the BM-18 and a stabilization of tumor burden in the LAPC-9, reflecting different androgen-dependent cell states. Proteomic analysis of bulk BM-18 tumors (intact, 14 days post castration, 50 days post androgen replacement) indicates an enrichment of stem cell markers upon castration; CD44, NKX3.1, and ALDH1 isoforms. Microarray analysis has confirmed upregulation of CD44 and ALDH1A1 at castrated state, and downregulation of expression upon androgen replacement at early time points (24 hours). We have isolated CD44+/-ALDHhigh/low subpopulation by flow cytometry and analyzed their transcriptome and proteomic changes. Castration in the BM-18 model induces an increase in the subpopulations of CD44+/ALDHlow (from 0.44% to 2.3%) and CD44+/ALDHhigh (from 0.06% to 0.38%). However, the same subpopulations are decreased in the LAPC-9 model upon castration, while only the CD44-/ALDHhigh subset is enriched (from 3.5% to 7.1%). Conclusions: Different androgen-independent cancer stem cell subpopulations may be distinguished by the ALDH activity status in combination with CD44. The androgen-independent cells in the BM-18 androgen-dependent model are reflected by enrichment of CD44 expression, as well as a rare double positive population. In the androgen-independent LAPC-9 model, CD44 expression is contrastingly decreased, potentially reflecting androgen-dependent CD44+ cells, and ALDH activity increased in CD44- cells. Ongoing analysis may elucidate the molecular mechanisms controlling the different cancer stem cell fates and their androgen sensitivity. Cancer stem cell potential of the different cell populations will have to be elucidated by transplantation experiments. Citation Format: Sofia Karkampouna, Marta de Menna, Markus Germann, Jöel Grosjean, Peter C. Gray, George N. Thalmann, Marianna Kruithof-de Julio. Molecular characterization of cancer stem cells from patient-derived xenografts of advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B060.

Androgen-deprivation therapy is the standard treatment for prostate cancer. Despite the initial response, a fraction of cases manifest progression to castration-resistant prostate cancer. PCa recurrence is possibly due to androgen-independent cancer stem cells that reinitiate tumor growth. The properties of cancer stem cells versus highly proliferative progenitor cells remain to be further characterized at the cellular and gene expression level. To address the molecular basis of CSC switch from androgen dependency to independence, we have employed patient-derived xenograft models (LAPC-9 and BM-18) that have different androgen sensitivity properties. We have performed microarray and proteomic analysis of BM-18 tumor tissues prior to and following castration as well as androgen replacement. To characterize the cancer stem cells in these models we have isolated different subpopulations by FACS cytometry, based on combination of selected markers (CD44 and ALDH activity measured by ALDELFUOR assay) aiming to assess the transcriptomic profile of these different populations. To assess response of these CD44+/-/ALDH+/- cells to androgen targeting compounds we have established an androgen-sensitive xenograft model (PNPCa) and tested it by ex vivo tissue culture and organoid viability assays. Proteomic and microarray analysis of bulk BM-18 tumors indicates enrichment of stem cell markers upon castration: CD44, NKX3.1 and ALDH1 isoforms. Readministration of testosterone induces decrease of these markers. Castration induces a rapid tumor volume decrease in the BM-18 as opposed to LAPC-9, reflecting different androgen-dependent cell states. CD44+/-/ALDHhigh/low subpopulations were isolated by flow cytometry and for transcriptomic analysis. Castration in the BM18 model induces an increase in the subpopulations of CD44+/ALDHlow and CD44+/ALDHhigh, while ALDHsingle and ALDHhigh total populations are decreased. Both CD44 expression and ALDH activity is increased in the LAPC-9 model upon castration. Treatment of PNPCa tumor parts ex vivo with enzalutamide inhibited expression of androgen receptor and keratin 8 expression. CD44+ organoids derived from the same tissue did not show altered viability after enzalutamide treatment. Different androgen-independent cancer stem cell populations may be distinguished by ALDH activity status and CD44. Androgen-independent cells in the BM-18 androgen-dependent model are reflected by enrichment of CD44 expression. The least abundant subpopulation CD44+/ALDHhigh is present in the LAPC-9 and expanded upon castration, while in the BM-18 model it is exclusively present after castration, reflecting different androgen sensitivity. Ongoing analysis may elucidate the molecular mechanisms controlling cancer stem cell fates, androgen sensitivity and drug resistance. Citation Format: Sofia Karkampouna, Marta De Menna, Markus Germann, Joel Grosjean, Peter Clark Gray, George Niklaus Thalmann, Marianna Kruithof-de Julio. Characterization of cancer stem cell subpopulations from patient-derived xenografts of advanced prostate cancer [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 3053.