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The Bacillus Calmette-Guérin (BCG) vaccine is the oldest cancer immunotherapeutic agent in use. Despite its effectiveness, its initial mechanisms of action remain largely unknown. Here, we elucidate the earliest cellular mechanisms involved in BCG-induced tumor clearance. We developed a fast preclinical in vivo assay to visualize in real time and at single-cell resolution the initial interactions among bladder cancer cells, BCG and innate immunity using the zebrafish xenograft model. We show that BCG induced the recruitment and polarization of macrophages towards a pro-inflammatory phenotype, accompanied by induction of the inflammatory cytokines tnfa, il1b and il6 in the tumor microenvironment. Macrophages directly induced apoptosis of human cancer cells through zebrafish TNF signaling. Macrophages were crucial for this response as their depletion completely abrogated the BCG-induced phenotype. Contrary to the general concept that macrophage anti-tumoral activities mostly rely on stimulating an effective adaptive response, we demonstrate that macrophages alone can induce tumor apoptosis and clearance. Thus, our results revealed an additional step to the BCG-induced tumor immunity model, while providing proof-of-concept experiments demonstrating the potential of this unique model to test innate immunomodulators.
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Apoptosis , Vacuna BCG , Macrófagos , Transducción de Señal , Neoplasias de la Vejiga Urinaria , Pez Cebra , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/inmunología , Animales , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Vacuna BCG/farmacología , Vacuna BCG/uso terapéutico , Transducción de Señal/efectos de los fármacos , Humanos , Línea Celular Tumoral , Apoptosis/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Microambiente TumoralRESUMEN
Ovarian cancer (OC) is an umbrella term for cancerous malignancies affecting the ovaries, yet treatment options for all subtypes are predominantly derived from high-grade serous ovarian cancer, the largest subgroup. The concept of "functional precision medicine" involves gaining personalized insights on therapy choice, based on direct exposure of patient tissues to drugs. This especially holds promise for rare subtypes like low-grade serous ovarian cancer (LGSOC). This study aims to establish an in vivo model for LGSOC using zebrafish embryos, comparing treatment responses previously observed in mouse PDX models, cell lines and 3D tumor models. To address this goal, a well-characterized patient-derived LGSOC cell line with the KRAS mutation c.35 G>T (p.(Gly12Val)) was used. Fluorescently labeled tumor cells were injected into the perivitelline space of 2 days' post-fertilization zebrafish embryos. At 1 day post-injection, xenografts were assessed for tumor size, followed by random allocation into treatment groups with trametinib, luminespib and trametinib + luminespib. Subsequently, xenografts were euthanized and analyzed for apoptosis and proliferation by confocal microscopy. Tumor cells formed compact tumor masses (n = 84) in vivo, with clear Ki67 staining, indicating proliferation. Zebrafish xenografts exhibited sensitivity to trametinib and luminespib, individually or combined, within a two-week period, establishing them as a rapid and complementary tool to existing in vitro and in vivo models for evaluating targeted therapies in LGSOC.
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Metastatic colonization by circulating cancer cells is a highly inefficient process. To colonize distant organs, disseminating cancer cells must overcome many obstacles in foreign microenvironments, and only a small fraction of them survives this process. How these disseminating cancer cells cope with stress and initiate metastatic process is not fully understood. In this study, we report that the metastatic onset of prostate cancer cells is associated with the dynamic conversion of metabolism signaling pathways governed by the energy sensors AMPK and mTOR. While in circulation in blood flow, the disseminating cancer cells display decreased mTOR and increased AMPK activities that protect them from stress-induced death. However, after metastatic onset, the mTOR-AMPK activities are reversed, enabling mTOR-dependent tumor growth. Suppression of this dynamic conversion by co-targeting of AMPK and mTOR signaling significantly suppresses prostate cancer cell and tumor organoid growth in vitro and experimental metastasis in vivo, suggesting that this can be a therapeutic approach against metastasizing prostate cancer.
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Proteínas Quinasas Activadas por AMP , Neoplasias de la Próstata , Masculino , Humanos , Proteínas Quinasas Activadas por AMP/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Transducción de Señal , Neoplasias de la Próstata/patología , Microambiente TumoralRESUMEN
Starting with pregnenolone, a 20-carbonyl group was converted into an amino group through a series of chemical reactions. This amino group was further converted into selenocyanoalkylamide, leading to the synthesis of six pregnenolone selenocyanoalkylamide derivatives. These compounds were then screened for antitumor activity in vitro, yielding promising results. Compounds 4b-4f show higher inhibitory activity than the positive control abiraterone and 2-methoxyestradiol, with IC50 values lower than 10 µmol/L against breast, ovarian, and cervical cancer cell lines that closely related to human hormone expression levels. The Annexin V assay of compound 4f revealed that compounds inhibited tumor cell proliferation primarily through the induction of programmed apoptosis. The zebrafish test results indicated that compound 4d had significant inhibitory activity against MCF-7 cell xenografts in vivo. Moreover, the antibacterial test indicated that compounds 4a and 4d-4e had better inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) than the positive controls vancomycin and ampicillin. These results suggest that these compounds may hold promise as novel antitumor agents or antimicrobial agents for further study.
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Antineoplásicos , Staphylococcus aureus Resistente a Meticilina , Animales , Humanos , Vancomicina , Pregnenolona/farmacología , Pez Cebra , Antibacterianos/farmacologíaRESUMEN
Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.
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Sarcoma de Ewing , Humanos , Animales , Ratones , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Pez Cebra/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Evaluación Preclínica de Medicamentos , Xenoinjertos , Apoptosis , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Línea Celular TumoralRESUMEN
Patient-derived xenografts (PDXs), also called "avatars," are generated by the implantation of human primary tumor cells or tissues into a host animal. Given the complexity and unique characteristics of each tumor, PDXs are models of choice in cancer research and precision medicine. In this context, the zebrafish PDX model (zPDX or zAvatar) has been recognized as a promising in vivo model to directly challenge patient cells with anti-cancer therapies in a personalized manner. The assay relies on the injection of tumor cells from patients into zebrafish embryos to then test and identify the best available drug combination for a particular patient. Compared to mouse PDXs, zAvatar assays take less time and do not require in vitro or in vivo cell expansion. The present article describes how to generate zAvatars from resected digestive cancer from surgeries and how to then use them for anti-cancer therapy screening. We describe the steps for tumor sample collection and cryopreservation, sample preparation and fluorescent labeling for microinjection into zebrafish embryos, drug administration, and analysis of tumor behavior by single-cell confocal imaging. We provide detailed protocols and helpful tips for performing this assay, and we address the technical challenges associated with the workflow. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Patient tumor sample collection and cryopreservation Basic Protocol 2: Generation of zAvatars and anti-cancer treatment Basic Protocol 3: Whole-mount immunofluorescence Basic Protocol 4: Confocal imaging and analysis.
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Neoplasias Gastrointestinales , Pez Cebra , Animales , Modelos Animales de Enfermedad , Detección Precoz del Cáncer , Humanos , Ratones , Medicina de Precisión/métodos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Metastatic uveal melanoma (MUM) is characterized by poor patient survival. Unfortunately, current treatment options demonstrate limited benefits. In this study, we evaluate the efficacy of ACY-1215, a histone deacetylase inhibitor (HDACi), to attenuate growth of primary ocular UM cell lines and, in particular, a liver MUM cell line in vitro and in vivo, and elucidate the underlying molecular mechanisms. A significant (p = 0.0001) dose-dependent reduction in surviving clones of the primary ocular UM cells, Mel270, was observed upon treatment with increasing doses of ACY-1215. Treatment of OMM2.5 MUM cells with ACY-1215 resulted in a significant (p = 0.0001), dose-dependent reduction in cell survival and proliferation in vitro, and in vivo attenuation of primary OMM2.5 xenografts in zebrafish larvae. Furthermore, flow cytometry revealed that ACY-1215 significantly arrested the OMM2.5 cell cycle in S phase (p = 0.0001) following 24 h of treatment, and significant apoptosis was triggered in a time- and dose-dependent manner (p < 0.0001). Additionally, ACY-1215 treatment resulted in a significant reduction in OMM2.5 p-ERK expression levels. Through proteome profiling, the attenuation of the microphthalmia-associated transcription factor (MITF) signaling pathway was linked to the observed anti-cancer effects of ACY-1215. In agreement, pharmacological inhibition of MITF signaling with ML329 significantly reduced OMM2.5 cell survival and viability in vitro (p = 0.0001) and reduced OMM2.5 cells in vivo (p = 0.0006). Our findings provide evidence that ACY-1215 and ML329 are efficacious against growth and survival of OMM2.5 MUM cells.
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BACKGROUND: Cancers of the pancreas and biliary tree remain one of the most aggressive oncological malignancies, with most patients relying on systemic chemotherapy. However, effective biomarkers to predict the best therapy option for each patient are still lacking. In this context, an assay able to evaluate individual responses prior to treatment would be of great value for clinical decisions. Here we aimed to develop such a model using zebrafish xenografts to directly challenge pancreatic cancer cells to the available chemotherapies. METHODS: Zebrafish xenografts were generated from a Panc-1 cell line to optimize the pancreatic setting. Pancreatic surgical resected samples, without in vitro expansion, were used to establish zebrafish patient-derived xenografts (zAvatars). Upon chemotherapy exposure, zAvatars were analyzed by single-cell confocal microscopy. RESULTS: We show that Panc-1 zebrafish xenografts are able to reveal tumor responses to both FOLFIRINOX and gemcitabine plus nanoparticle albumin-bound (nab)-paclitaxel in just 4 days. Moreover, we established pancreatic and ampullary zAvatars with patient-derived tumors representative of different histological types. CONCLUSION: Altogether, we provide a short report showing the feasibility of generating and analyzing with single-cell resolution zAvatars from pancreatic and ampullary cancers, with potential use for future preclinical studies and personalized treatment.
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Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Albúminas/uso terapéutico , Animales , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Quimioterapia Combinada , Fluorouracilo/uso terapéutico , Irinotecán/uso terapéutico , Leucovorina/uso terapéutico , Oxaliplatino/uso terapéutico , Paclitaxel/uso terapéutico , Neoplasias Pancreáticas/patología , Células Tumorales Cultivadas , Pez Cebra , GemcitabinaRESUMEN
Tremendous data have been accumulated in the effort to understand chemoresistance of triple negative breast cancer (TNBC). However, modifications in cancer cells surviving combined and sequential treatment still remain poorly described. In order to mimic clinical neoadjuvant treatment, we first treated MDA-MB-231 and SUM159-PT TNBC cell lines with epirubicin and cyclophosphamide for 2 days, and then with paclitaxel for another 2 days. After 4 days of recovery, persistent cells surviving the treatment were characterized at both cellular and molecular level. Persistent cells exhibited increased growth and were more invasive in vitro and in zebrafish model. Persistent cells were enriched for vimentinhigh sub-population, vimentin knockdown using siRNA approach decreased the invasive and sphere forming capacities as well as Akt phosphorylation in persistent cells, indicating that vimentin is involved in chemotherapeutic treatment-induced enhancement of TNBC aggressiveness. Interestingly, ectopic vimentin overexpression in native cells increased cell invasion and sphere formation as well as Akt phosphorylation. Furthermore, vimentin overexpression alone rendered the native cells resistant to the drugs, while vimentin knockdown rendered them more sensitive to the drugs. Together, our data suggest that vimentin could be considered as a new targetable player in the ever-elusive status of drug resistance and recurrence of TNBC.
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Resistencia a Antineoplásicos/fisiología , Neoplasias de la Mama Triple Negativas/metabolismo , Vimentina/fisiología , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Ciclofosfamida/farmacología , Modelos Animales de Enfermedad , Quimioterapia/métodos , Epirrubicina/farmacología , Transición Epitelial-Mesenquimal , Femenino , Humanos , Terapia Neoadyuvante/métodos , Invasividad Neoplásica/patología , Recurrencia Local de Neoplasia , Paclitaxel/uso terapéutico , Neoplasias de la Mama Triple Negativas/patología , Vimentina/metabolismo , Pez CebraRESUMEN
Telomerase reverse transcriptase (TERT) maintains telomere homeostasis, thus ensuring chromosome stability and cell proliferation. In addition, several telomere-independent functions of human TERT have been described. In this study, we report that TERT binds directly to the TCF binding elements located upstream of the oncomiR miR500A, and induces its transcription. This function was independent of the telomerase activity, as shown with experiments using catalytically inactive TERT and inhibitors of TERT and the TERT RNA component. miR500A was in turn found to target three key components of the Hedgehog signalling pathway: Patched 1; Gli family zinc finger 3; and Cullin 3, thereby promoting cell invasion. Our results point to the crucial role of the TERT-miR500A-Hedgehog axis in tumour aggressiveness and highlight the therapeutic potential of targeting noncanonical TERT functions in cancer.
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Neoplasias , Telomerasa , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Neoplasias/genética , Transducción de Señal/genética , Telomerasa/genética , Telomerasa/metabolismo , Telómero/metabolismoRESUMEN
Cancer is the second leading cause of death in the world. Given that cancer is a highly individualized disease, predicting the best chemotherapeutic treatment for individual patients can be difficult. Ex vivo models such as mouse patient-derived xenografts (PDX) and organoids are being developed to predict patient-specific chemosensitivity profiles before treatment in the clinic. Although promising, these models have significant disadvantages including long growth times that introduce genetic and epigenetic changes to the tumor. The zebrafish xenograft assay is ideal for personalized medicine. Imaging of the small, transparent fry is unparalleled among vertebrate organisms. In addition, the speed (5-7 days) and small patient tissue requirements (100-200 cells per animal) are unique features of the zebrafish xenograft model that enable patient-specific chemosensitivity analyses.
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Antineoplásicos/farmacología , Descubrimiento de Drogas/métodos , Neoplasias/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Pez Cebra , Animales , Animales Modificados Genéticamente , Antineoplásicos/uso terapéutico , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Embrión no Mamífero , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Ratones , Neoplasias/genética , Neoplasias/patología , Medicina de Precisión/métodos , Factores de Tiempo , Quimera por TrasplanteRESUMEN
Chimeric antigen receptor (CAR) T cells have proven to be a powerful cellular therapy for B cell malignancies. Massive efforts are now being undertaken to reproduce the high efficacy of CAR T cells in the treatment of other malignancies. Here, predictive preclinical model systems are important, and the current gold standard for preclinical evaluation of CAR T cells are mouse xenografts. However, mouse xenograft assays are expensive and slow. Therefore, an additional vertebrate in vivo assay would be beneficial to bridge the gap from in vitro to mouse xenografts. Here, we present a novel assay based on embryonic zebrafish xenografts to investigate CAR T cell-mediated killing of human cancer cells. Using a CD19-specific CAR and Nalm-6 leukemia cells, we show that live observation of killing of Nalm-6 cells by CAR T cells is possible in zebrafish embryos. Furthermore, we applied Fiji macros enabling automated quantification of Nalm-6 cells and CAR T cells over time. In conclusion, we provide a proof-of-principle study that embryonic zebrafish xenografts can be used to investigate CAR T cell-mediated killing of tumor cells. This assay is cost-effective, fast, and offers live imaging possibilities to directly investigate CAR T cell migration, engagement, and killing of effector cells.
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Gomisin M2 isolated from Schisandra viridis A. C. Smith has potential anti-tumor effects on certain cancers, including breast cancer. However, only a few investigations have been conducted on the effects of Gomisin M2 on breast cancer stem cells (CSCs), which have the ability to self-renew and differentiate, as a possible strategy to resolve cancer cell resistance to apoptosis and to improve treatments. It is essential to investigate the effects of Gomisin M2 on breast cancer stem cells (BCSCs). In this study, we enriched breast cancer stem cells with CD44+/CD24- from MDA-MB-231 and HCC1806 cells through magnetic-activated cell sorting and cultured these in serum-free medium. The ability of Gomisin M2 to kill breast cancer stem cells was evaluated in vitro and in vivo. Gomisin M2 significantly inhibited the proliferation of the triple-negative breast cancer cell lines and mammosphere formation in breast CSCs and downregulated the Wnt/ß-catenin self-renewal pathway. Moreover, Gomisin M2 induced apoptosis and blocked the mitochondrial membrane potential of BCSCs. Gomisin M2 suppressed the proliferation of MDA-MB-231 and HCC1806 xenografts in zebrafish. Together, these findings suggest that the anti-BCSC activity of Gomisin M2 could become a promising starting point for the discovery of novel BCSC-targeting drugs.
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Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama , Proliferación Celular/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Schisandra/química , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Medicamentos Herbarios Chinos/farmacología , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Pez CebraRESUMEN
Cancer stem cells are involved in cancer establishment, progression, and resistance to current treatments. We demonstrated the in vitro and in vivo anti-breast cancer effect of bakuchiol in a previous study. However, the ability of bakuchiol to target breast cancer stem cells (BCSCs) and inhibit breast cancer metastasis remains unknown. In the current study, we used the cell surface markers CD44 and CD24 to distinguish BCSCs from MCF-7 cells. Bakuchiol inhibited mammosphere formation and aldehyde dehydrogenase activity in BCSCs. Moreover, bakuchiol induced apoptosis and suppressed the mitochondrial membrane potential of BCSCs. Bakuchiol upregulated the expression levels of pro-apoptotic genes, BNIP3 and DAPK2. Bakuchiol induced oxidative stress and altered lipogenesis in BCSCs. In zebrafish xenografts, bakuchiol inhibited breast cancer cell metastasis in vivo. In addition, bakuchiol altered the expression levels of metastasis-related genes through upregulating CK18 and downregulating Notch3, FASN, TGFBR1, and ACVR1B. Our study provides evidence for the anti-breast cancer potential of bakuchiol.
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Polysulfanes show chemopreventive effects against gastrointestinal tumors. We identified diallyl tetrasulfide and its derivative, dibenzyl tetrasulfide (DBTTS), to be mitotic inhibitors and apoptosis inducers. Here, we translate their application in colorectal cancer (CRC). MALDI-TOF-MS analysis identified both compounds as reversible tubulin binders, validated by in cellulo α-tubulin degradation. BRAF(V600E)-mutated HT-29 cells were resistant to DBTTS, as evidenced by mitotic arrest for 48 h prior to apoptosis induction compared to KRAS(G12V)-mutated SW480/620 cells, which committed to death earlier. The prolonged mitotic block correlated with autophagy impairment and p62 protein accumulation in HT-29 but not in SW480/620 cells, whereas siRNA-mediated p62 inhibition sensitized HT-29 cells to death. In silico analysis with 484 colorectal cancer patients associated higher p62 expression and reduced autophagic flux with greater overall survival. Accordingly, we hypothesized that DBTTS targets CRC survival/death through autophagy interference in cell types with differential autophagic capacities. We confirmed the therapeutic potential of DBTTS by the inhibition of spheroid and colony formation capacities in CRC cells, as well as in HT-29 zebrafish xenografts in vivo.