Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer.

Inflammation is a common condition of prostate tissue, whose impact on carcinogenesis is highly debated. Microbial colonization is a well-documented cause of a small percentage of prostatitis cases, but it remains unclear what underlies the majority of sterile inflammation reported. Here, androgen- independent fluctuations of PSA expression in prostate cells have lead us to identify a prominent function of the Transient Receptor Potential Cation Channel Subfamily M Member 8 (TRPM8) gene in sterile inflammation. Prostate cells secret TRPM8 RNA into extracellular vesicles (EVs), which primes TLR3/NF-kB-mediated inflammatory signaling after EV endocytosis by epithelial cancer cells. Furthermore, prostate cancer xenografts expressing a translation-defective form of TRPM8 RNA contain less collagen type I in the extracellular matrix, significantly more infiltrating NK cells, and larger necrotic areas as compared to control xenografts. These findings imply sustained, androgen-independent expression of TRPM8 constitutes as a promoter of anticancer innate immunity, which may constitute a clinically relevant condition affecting prostate cancer prognosis.

Molecular profiling of osteoprogenitor cells reveals FOS as a master regulator of bone non-union.

Despite the advances in bone fracture treatment, a significant fraction of fracture patients will develop non-union. Most non-unions are treated with surgery since identifying the molecular causes of these defects is exceptionally challenging. In this study, compared with marrow bone, we generated a transcriptional atlas of human osteoprogenitor cells derived from healing callus and non-union fractures. Detailed comparison among the three conditions revealed a substantial similarity of callus and nonunion at the gene expression level. Nevertheless, when assayed functionally, they showed different osteogenic potential. Utilizing longitudinal transcriptional profiling of the osteoprogenitor cells, we identified FOS as a putative master regulator of non-union fractures. We validated FOS activity by profiling a validation cohort of 31 tissue samples. Our work identified new molecular targets for non-union classification and treatment while providing a valuable resource to better understand human bone healing biology.

Bladder cancer organoids as a functional system to model different disease stages and therapy response.

Bladder Cancer (BLCa) inter-patient heterogeneity is the primary cause of treatment failure, suggesting that 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 cancers. In our study, we establish PDO cultures from different BLCa stages and grades. PDOs preserve the histological and molecular heterogeneity of the parental tumors, including their multiclonal genetic landscapes, and consistently share key genetic alterations, mirroring tumor evolution in longitudinal sampling. Our drug screening pipeline is implemented using PDOs, testing standard-of-care and FDA-approved compounds for other tumors. Integrative analysis of drug response profiles with matched PDO genomic analysis is used to determine enrichment thresholds for candidate markers of therapy response and resistance. Finally, by assessing the clinical history of longitudinally sampled cases, we can determine whether the disease clonal evolution matched with drug response.

A model for network-based identification and pharmacological targeting of aberrant, replication-permissive transcriptional programs induced by viral infection.

SARS-CoV-2 hijacks the host cell transcriptional machinery to induce a phenotypic state amenable to its replication. Here we show that analysis of Master Regulator proteins representing mechanistic determinants of the gene expression signature induced by SARS-CoV-2 in infected cells revealed coordinated inactivation of Master Regulators enriched in physical interactions with SARS-CoV-2 proteins, suggesting their mechanistic role in maintaining a host cell state refractory to virus replication. To test their functional relevance, we measured SARS-CoV-2 replication in epithelial cells treated with drugs predicted to activate the entire repertoire of repressed Master Regulators, based on their experimentally elucidated, context-specific mechanism of action. Overall, 15 of the 18 drugs predicted to be effective by this methodology induced significant reduction of SARS-CoV-2 replication, without affecting cell viability. This model for host-directed pharmacological therapy is fully generalizable and can be deployed to identify drugs targeting host cell-based Master Regulator signatures induced by virtually any pathogen.

A Model for Network-Based Identification and Pharmacological Targeting of Aberrant, Replication-Permissive Transcriptional Programs Induced by Viral Infection.

Precise characterization and targeting of host cell transcriptional machinery hijacked by viral infection remains challenging. Here, we show that SARS-CoV-2 hijacks the host cell transcriptional machinery to induce a phenotypic state amenable to its replication. Specifically, analysis of Master Regulator (MR) proteins representing mechanistic determinants of the gene expression signature induced by SARS-CoV-2 in infected cells revealed coordinated inactivation of MRs enriched in physical interactions with SARS-CoV-2 proteins, suggesting their mechanistic role in maintaining a host cell state refractory to virus replication. To test their functional relevance, we measured SARS-CoV-2 replication in epithelial cells treated with drugs predicted to activate the entire repertoire of repressed MRs, based on their experimentally elucidated, context-specific mechanism of action. Overall, >80% of drugs predicted to be effective by this methodology induced significant reduction of SARS-CoV-2 replication, without affecting cell viability. This model for host-directed pharmacological therapy is fully generalizable and can be deployed to identify drugs targeting host cell-based MR signatures induced by virtually any pathogen.

Tumor microenvironment mechanisms and bone metastatic disease progression of prostate cancer.

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.

Lineage-specific mechanisms and drivers of breast cancer chemoresistance revealed by 3D biomimetic culture.

To improve the success rate of current preclinical drug trials, there is a growing need for more complex and relevant models that can help predict clinical resistance to anticancer agents. Here, we present a three-dimensional (3D) technology, based on biomimetic collagen scaffolds, that enables the modeling of the tumor hypoxic state and the prediction of in vivo chemotherapy responses in terms of efficacy, molecular alterations, and emergence of resistance mechanisms. The human breast cancer cell lines MDA-MB-231 (triple negative) and MCF-7 (luminal A) were treated with scaling doses of doxorubicin in monolayer cultures, 3D collagen scaffolds, or orthotopically transplanted murine models. Lineage-specific resistance mechanisms were revealed by the 3D tumor model. Reduced drug uptake, increased drug efflux, and drug lysosomal confinement were observed in triple-negative MDA-MB-231 cells. In luminal A MCF-7 cells, the selection of a drug-resistant subline from parental cells with deregulation of p53 pathways occurred. These cells were demonstrated to be insensitive to DNA damage. Transcriptome analysis was carried out to identify differentially expressed genes (DEGs) in treated cells. DEG evaluation in breast cancer patients demonstrated their potential role as predictive biomarkers. High expression of the transporter associated with antigen processing 1 (TAP1) and the tumor protein p53-inducible protein 3 (TP53I3) was associated with shorter relapse in patients affected by ER+ breast tumor. Likewise, the same clinical outcome was associated with high expression of the lysosomal-associated membrane protein 1 LAMP1 in triple-negative breast cancer. Hypoxia inhibition by resveratrol treatment was found to partially re-sensitize cells to doxorubicin treatment. Our model might improve preclinical in vitro analysis for the translation of anticancer compounds as it provides: (a) more accurate data on drug efficacy and (b) enhanced understanding of resistance mechanisms and molecular drivers.

Corrigendum: Dual-mTOR Inhibitor Rapalink-1 Reduces Prostate Cancer Patient-Derived Xenograft Growth and Alters Tumor Heterogeneity.

[This corrects the article .].

Patient-derived xenografts and organoids model therapy response in prostate cancer.

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.

Dual-mTOR Inhibitor Rapalink-1 Reduces Prostate Cancer Patient-Derived Xenograft Growth and Alters Tumor Heterogeneity.

Bone metastasis is the leading cause of prostate cancer (PCa) mortality, frequently marking the progression to castration-resistant PCa. Dysregulation of the androgen receptor pathway is a common feature of castration-resistant PCa, frequently appearing in association with mTOR pathway deregulations. Advanced PCa is also characterized by increased tumor heterogeneity and cancer stem cell (CSC) frequency. CSC-targeted therapy is currently being explored in advanced PCa, with the aim of reducing cancer clonal divergence and preventing disease progression. In this study, we compared the molecular pathways enriched in a set of bone metastasis from breast and prostate cancer from snap-frozen tissue. To further model PCa drug resistance mechanisms, we used two patient-derived xenografts (PDX) models of bone-metastatic PCa, BM18, and LAPC9. We developed in vitro organoids assay and ex vivo tumor slice drug assays to investigate the effects of mTOR- and CSC-targeting compounds. We found that both PDXs could be effectively targeted by treatment with the bivalent mTORC1/2 inhibitor Rapalink-1. Exposure of LAPC9 to Rapalink-1 but not to the CSC-targeting drug disulfiram blocked mTORC1/2 signaling, diminished expression of metabolic enzymes involved in glutamine and lipid metabolism and reduced the fraction of CD44+ and ALDEFluorhigh cells, in vitro. Mice treated with Rapalink-1 showed a significantly delayed tumor growth compared to control and cells recovered from the tumors of treated animals showed a marked decrease of CD44 expression. Taken together these results highlight the increased dependence of advanced PCa on the mTOR pathway, supporting the development of a targeted approach for advanced, bone metastatic PCa.

A Multidisciplinary Review of the Roles of Cripto in the Scientific Literature Through a Bibliometric Analysis of its Biological Roles.

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-ß 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-ß 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.

A biomimetic 3D model of hypoxia-driven cancer progression.

The fate of tumors depends both on the cancer cells' intrinsic characteristics and on the environmental conditions where the tumors reside and grow. Engineered in vitro models have led to significant advances in cancer research, allowing the investigation of cells in physiological environments and the study of disease mechanisms and processes with enhanced relevance. Here we present a biomimetic cancer model based on a collagen matrix synthesized through a biologically inspired process. We compared in this environment the responses of two breast tumor lineages characterized by different molecular patterns and opposite clinical behaviors: MCF-7 that belong to the luminal A subtype connected to an indolent course, and basal-like MDA-MB-231 connected to high-grade and aggressive disease. Cancer cells in the biomimetic matrix recreate a hypoxic environment that affects their growth dynamics and phenotypic features. Hypoxia induces apoptosis and the selection of aggressive cells that acquire expression signatures associated with glycolysis, angiogenesis, cell-matrix interaction, epithelial to mesenchymal transition and metastatic ability. In response to hypoxia MDA-MB-231 migrate on the collagen fibrils and undergo cellular senescence, while MCF-7 do not exhibit these behaviors. Our biomimetic model mimics the evolution of tumors with different grade of aggressiveness fostered by a hypoxic niche and provides a relevant technology to dissect the events involved in cancer progression.

Tumor-Stroma Crosstalk in Bone Tissue: The Osteoclastogenic Potential of a Breast Cancer Cell Line in a Co-Culture System and the Role of EGFR Inhibition.

Although bone metastases represent a major challenge in the natural history of breast cancer (BC), the complex interactions involved have hindered the development of robust in vitro models. The aim of this work is the development of a preclinical model of cancer and bone stromal cells to mimic the bone microenvironment. We studied the effects on osteoclastogenesis of BC cells and Mesenchymal stem cells (MSC) cultured alone or in combination. We also analyzed: (a) whether the blockade of the Epithelial Growth Factor Receptor (EGFR) pathway modified their influence on monocytes towards differentiation, and (b) the efficacy of bone-targeted therapy on osteoclasts. We evaluated the osteoclastogenesis modulation of human peripheral blood monocytes (PBMC) indirectly induced by the conditioned medium (CM) of the human BC cell line SCP2, cultured singly or with MSC. Osteoclastogenesis was evaluated by TRAP analysis. The effect of the EGFR blockade was assessed by treating the cells with gefitinib, and analyzed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Western Blot (WB). We observed that SCP2 co-cultured with MSC increased the differentiation of PBMC. This effect was underpinned upon pre-treatment of the co-culture with gefitinib. Co-culture of SCP2 with MSC increased the expression of both the bone-related marker Receptor Activator of Nuclear Factor κB (RANK) and EGFR in BC cells. These upregulations were not affected by the EGFR blockade. The effects of the CM obtained by the cells treated with gefitinib in combination with the treatment of the preosteoclasts with the bone-targeted agents and everolimus enhanced the inhibition of the osteoclastogenesis. Finally, we developed a fully human co-culture system of BC cells and bone progenitor cells. We observed that the interaction of MSC with cancer cells induced in the latter molecular changes and a higher power of inducing osteoclastogenesis. We found that blocking EGFR signaling could be an efficacious strategy for breaking the interactions between cancer and bone cells in order to inhibit bone metastasis.

Innovative approaches to establish and characterize primary cultures: an ex vivo 3D system and the zebrafish model.

Patient-derived specimens are an invaluable resource to investigate tumor biology. However, in vivo studies on primary cultures are often limited by the small amount of material available, while conventional in vitro systems might alter the features and behavior that characterize cancer cells. We present our data obtained on primary dedifferentiated liposarcoma cells cultured in a 3D scaffold-based system and injected into a zebrafish model. Primary cells were characterized in vitro for their morphological features, sensitivity to drugs and biomarker expression, and in vivo for their engraftment and invasiveness abilities. The 3D culture showed a higher enrichment in cancer cells than the standard monolayer culture and a better preservation of liposarcoma-associated markers. We also successfully grafted primary cells into zebrafish, showing their local migratory and invasive abilities. Our work provides proof of concept of the ability of 3D cultures to maintain the original phenotype of ex vivo cells, and highlights the potential of the zebrafish model to provide a versatile in vivo system for studies with limited biological material. Such models could be used in translational research studies for biomolecular analyses, drug screenings and tumor aggressiveness assays.

RANKL: A promising circulating marker for bone metastasis response.

Bone metastases are a frequent event in patients with solid tumors. Although great advances have been made in the treatment of these patients, the identification of novel, accurate indicators of bone response would greatly facilitate the clinical management of the disease. The receptor activator of nuclear factor-κB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) signaling pathway is significantly involved in bone metastasis formation. The main aim of the present study was to evaluate the role of circulating RANK, RANKL and OPG levels in predicting bone response. Marker accuracy was also compared with that of the conventional tumor marker N-terminal telopeptide of type I collagen (NTX). A prospective study was performed on 49 patients with bone metastases from breast, lung and prostate cancer, who were undergoing treatment with zoledronic acid. Patients were monitored for 1 year with blood tests, clinical evaluation and instrumental exams according to the response evaluation criteria of the University of Texas M. D. Anderson Cancer Center (Houston, TX, USA) and the Positron Emission Tomography Response Criteria in Solid Tumors. Circulating RANK/RANKL/OPG transcripts and NTX levels were evaluated by reverse transcription-quantitative polymerase chain reaction and immune enzymatic assay, respectively. The baseline RANKL levels differed significantly between responders and non-responders, whereas no differences in NTX levels were observed between the two groups. Receiver operating characteristic curve evaluation for all markers revealed that RANKL was the most accurate marker, with an area under the curve of 0.74 (95% confidence interval, 0.54-0.93). In addition, RANKL, which is the target of the novel monoclonal antibody denosumab, was the most accurate predictor of bone response in the present series of patients with bone metastases. Thus, the use of RANKL as a marker could potentially improve clinical practice, as current bone response evaluation is still somewhat problematic.

Development of a Patient-Derived Xenograft (PDX) of Breast Cancer Bone Metastasis in a Zebrafish Model.

Bone metastasis is a complex process that needs to be better understood in order to help clinicians prevent and treat it. Xenografts using patient-derived material (PDX) rather than cancer cell lines are a novel approach that guarantees more clinically realistic results. A primary culture of bone metastasis derived from a 67-year-old patient with breast cancer was cultured and then injected into zebrafish (ZF) embryos to study its metastatic potential. In vivo behavior and results of gene expression analyses of the primary culture were compared with those of cancer cell lines with different metastatic potential (MCF7 and MDA-MB-231). The MCF7 cell line, which has the same hormonal receptor status as the bone metastasis primary culture, did not survive in the in vivo model. Conversely, MDA-MB-231 disseminated and colonized different parts of the ZF, including caudal hematopoietic tissues (CHT), revealing a migratory phenotype. Primary culture cells disseminated and in later stages extravasated from the vessels, engrafting into ZF tissues and reaching the CHT. Primary cell behavior reflected the clinical course of the patient's medical history. Our results underline the potential for using PDX models in bone metastasis research and outline new methods for the clinical application of this in vivo model.

First-line chemotherapy in patients with metastatic gastroenteropancreatic neuroendocrine carcinoma.

BACKGROUND AND AIM: To investigate the efficacy of platinum-based chemotherapy in patients with metastatic gastroenteropancreatic neuroendocrine carcinoma (mGEP-NEC) and define predictive and prognostic factors. METHODS: Twenty mGEP-NEC patients were treated with cisplatin or carboplatin/etoposide between April 2010 and October 2014. Both large-cell and small-cell histologies were included. Cisplatin 25 mg/m(2) was administered on days 1-3 followed by etoposide 100 mg/m(2) on days 1-3 every 21 days. Carboplatin 300 mg/m(2) was administered on day 1 followed by etoposide 100 mg/m(2) on days 1-3. RESULTS: Of the 19 evaluable patients, 13 obtained a partial response, four showed stable disease, and two progressed. Median overall survival (mOS) was 13.5 months and median progression-free survival (mPFS) was 10.9 months. Gallium-68 positron emission tomography/computerizsed tomography-positive patients had a higher, albeit not significantly, OS than those with negative results (75% vs 34.3% at 18 months; P=0.06). mPFS was 19.3 and 6.3 months (P<0.01) in mGEP-NEC patients with Ki67 ≤55% or >55%, respectively. mOS was 8.1 months in the latter group but was not reached in the Ki67 ≤55% group (P-value =0.039). Patients with a lower body mass index (BMI) had a better prognosis in terms of both OS and PFS. Patients with BMI ≥25 had a mOS of 11.7 months (P=0.0293) and a mPFS of 6.2 months (P=0.0057). CONCLUSION: Platinum-based chemotherapy showed good efficacy in mGEP-NEC patients. Those with Ki67 ≤55%, positive Gallium-68 positron emission tomography/computerized tomography and BMI <25 had a better prognosis.

Long-lasting activity of trabectedin in refractory uterine leiomyosarcoma: a case report.

BACKGROUND: Leiomyosarcoma (LMS) is an aggressive soft tissue sarcoma derived from smooth muscle cells typically of uterine, gastrointestinal or soft tissue origin. The prognosis for this tumor is poor, with survival rates among the lowest of all soft tissue sarcomas. Surgery is the best approach for localized disease. The principal role of chemotherapy is prevalently in the treatment of metastatic disease. Trabectedin, a promising new DNA-damaging agent with a mechanism of action that differs from that of traditional alkylating agents, has been approved in Europe for the treatment of patients with advanced soft tissue sarcoma after failure of anthracyclines and ifosfamide, CASE PRESENTATION: We report the case of a 53-year-old woman with metastatic well differentiated uterine leiomyosarcoma refractory to multiple treatments who underwent 22 cycles of trabectedin over 30 months, obtaining a partial response according to RECIST (Response Evaluation Criteria in Solid Tumors) criteria, with good tolerability, and maintaining the response for 10 months after trebectedin withdrawal. CONCLUSION: This very prolonged response, which persisted after drug discontinuation, suggests that trabectedin exerts an oncostatic effect rather than the cytotoxic one produced by other chemotherapeutic agents. Our experience also raises the question of the best way to evaluate trabectedin efficacy.