establishment-zebrafish-patient-derived-xenografts-from-pancreatic

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...

University of Pisa

Bcl-2 protein family is constituted by multidomain members originally identified as modulators of programmed cell death and whose expression is frequently misbalanced in cancer cells. The lead member Bcl-2 and its homologue Bcl-xL proteins are characterized by the presence of all four conserved BH domain and exert their antiapoptotic role mainly through the involvement of BH1, BH2 and BH3 homology domains, that mediate the interaction with the proapoptotic members of the same Bcl-2 family. The N-terminal BH4 domain of Bcl-2 and Bcl-xL is responsible for the interaction with other proteins that do not belong to Bcl-2 protein family. Beyond a classical role in inhibiting apoptosis, BH4 domain has been characterized as a crucial regulator of other important cellular functions attributed to Bcl-2 and Bcl-xL, including proliferation, autophagy, differentiation, DNA repair, cell migration, tumor progression and angiogenesis. During the last two decades a strong effort has been made to dissect the molecular pathways involved the capability of BH4 domain to regulate the canonical antiapoptotic and the non-canonical activities of Bcl-2 and Bcl-xL, creating the basis for the development of novel anticancer agents targeting this domain. Indeed, recent evidences obtained on in vitro and in vivo model of different cancer histotypes are confirming the promising therapeutic potential of BH4 domain inhibitors supporting their future employment as a novel anticancer strategy.

Non-canonical roles of Bcl-2 and Bcl-xL proteins: relevance of BH4 domain.

By using human melanoma and glioblastoma cell lines and their derivative BCL-X overexpressing clones, we investigated the role of BCL-X in aggressive features of these two tumor histotypes. We found that in both models, BCL-X overexpression increased in vitro cell migration and invasion and facilitated tumor cells to form de novo vasculogenic structures. Furthermore, BCL-X overexpressing cells exhibited higher tumors sphere formation capacity and expressed higher levels of some stem cell markers, supporting the concept that BCL-X plays essential roles in the maintenance of cancer stem cell phenotype. BCL-X expression reduction by siRNA, the exposure to a BCL-X-specific inhibitor and the use of a panel of human melanoma cell lines corroborated the evidence that BCL-X regulates tumor progression-associated properties. Finally, the vascular markers and the vasculogenic mimicry were up-regulated in the BCL-X overexpressing xenografts derived from both tumor histotypes. In conclusion, our work brings further support to the understanding of the malignant actions of BCL-X and, in particular, to the concept that BCL-X promotes stemness and contributes to the aggressiveness of both melanoma and glioblastoma.

BCL-X overexpression promotes tumor progression-associated properties.

Skin cutaneous melanoma (SKCM) is the most lethal form of skin cancer and while incidence rates are declining for most cancers, they have been steadily rising for SKCM. Serine protease inhibitor, kunitz-type, 1 (SPINT1) is a type II transmembrane serine protease inhibitor that has been shown to be involved in the development of several types of cancer, such as squamous cell carcinoma and colorectal cancer.

Zebrafish modeling reveals that SPINT1 regulates the aggressiveness of skin cutaneous melanoma and its crosstalk with tumor immune microenvironment.

Understanding the signalling pathways involved in angiogenesis, and developing anti-angiogenic drugs are one of the major focuses on cancer research. Herein, we assessed the effect of CPTH6, a lysine acetyltransferase inhibitor and anti-tumoral compound, on angiogenesis-related properties of both endothelial and cancer cells.

Inhibition of lysine acetyltransferases impairs tumor angiogenesis acting on both endothelial and tumor cells.

The plant-derived natural alkaloid berberine displays therapeutic potential to treat several pathological conditions, including dyslipidemias, diabetes and cardiovascular disorders. However, data on berberine effects during embryonic development are scarce and in part controversial. In this study, using zebrafish embryos as vertebrate experimental model, we address the effects of berberine treatment on cardiovascular system development and functionality. Starting from the observation that berberine induces developmental toxicity and pericardial edema in a time- and concentration-dependent manner, we found that treated embryos display cardiac looping defects and, at later stages, present an abnormal heart characterized by a stretched morphology and atrial endocardial/myocardial detachment. Furthermore, berberine affected cardiac functionality of the embryos, promoting bradycardia and reducing the cardiac output, the atrial shortening fraction percentage and the atrial stroke volume. We also found that, during development, berberine interferes with the angiogenic process, without altering vascular permeability. These alterations are associated with increased levels of vascular endothelial growth factor aa (vegfaa) mRNA, suggesting an important role for Vegfaa as mediator of berberine-induced cardiovascular defects. Altogether, these data indicate that berberine treatment during vertebrate development leads to an impairment of cardiovascular system morphogenesis and functionality, suggesting a note of caution in its use during pregnancy and lactation.

Exposure to the natural alkaloid Berberine affects cardiovascular system morphogenesis and functionality during zebrafish development.

Haploinsufficiency of the  gene, encoding a SET domain-containing histone methyltransferase, has been identified as a cause of intellectual disability and Autism Spectrum Disorder (ASD). Recently, the zebrafish has emerged as a valuable model to study neurodevelopmental disorders because of its genetic tractability, robust behavioral traits and amenability to high-throughput drug screening. To model human  haploinsufficiency, we generated zebrafish  mutants using the CRISPR/Cas9 technology and characterized their morphological, behavioral and molecular phenotypes. According to our observation that  is expressed in adult zebrafish brain, including those areas controlling social behavior, we found that  heterozygous mutants exhibit defective aggregation and coordination abilities required for shoaling interactions, as well as indifference to social stimuli. Interestingly, impairment in social interest is rescued by risperidone, an antipsychotic drug used to treat behavioral traits in ASD individuals. The molecular analysis underscored the downregulation of genes encoding proteins involved in the synaptic structure and function in the adult brain, thus suggesting that brain hypo-connectivity could be responsible for the social impairments of  mutant fishes. The zebrafish  mutants display ASD-like features and are a promising  haploinsufficiency model for drug screening aimed at reversing the behavioral phenotypes.

CRISPR/Cas9-Induced Inactivation of the Autism-Risk Gene  Leads to Social Impairments in Zebrafish.

Neuroprotective drug delivery to the posterior segment of the eye represents a major challenge to counteract vision loss. This work focuses on the development of a polymer-based nanocarrier, specifically designed for targeting the posterior eye. Polyacrylamide nanoparticles (ANPs) were synthesised and characterised, and their high binding efficiency was exploited to gain both ocular targeting and neuroprotective capabilities, through conjugation with peanut agglutinin (ANP:PNA) and neurotrophin nerve growth factor (ANP:PNA:NGF). The neuroprotective activity of ANP:PNA:NGF was assessed in an oxidative stress-induced retinal degeneration model using the teleost zebrafish. Upon nanoformulation, NGF improved the visual function of zebrafish larvae after the intravitreal injection of hydrogen peroxide, accompanied by a reduction in the number of apoptotic cells in the retina. Additionally, ANP:PNA:NGF counteracted the impairment of visual behaviour in zebrafish larvae exposed to cigarette smoke extract (CSE). Collectively, these data suggest that our polymeric drug delivery system represents a promising strategy for implementing targeted treatment against retinal degeneration.

Article	Year
Non-canonical roles of Bcl-2 and Bcl-xL proteins: relevance of BH4 domain. Carcinogenesis\| PubMed ID: 28203756	2017
BCL-X overexpression promotes tumor progression-associated properties. Cell death & disease\| PubMed ID: 29238043	2017
Zebrafish modeling reveals that SPINT1 regulates the aggressiveness of skin cutaneous melanoma and its crosstalk with tumor immune microenvironment. Journal of experimental & clinical cancer research : CR\| PubMed ID: 31519199	2019
Inhibition of lysine acetyltransferases impairs tumor angiogenesis acting on both endothelial and tumor cells. Journal of experimental & clinical cancer research : CR\| PubMed ID: 32498717	2020
Exposure to the natural alkaloid Berberine affects cardiovascular system morphogenesis and functionality during zebrafish development. Scientific reports\| PubMed ID: 33060638	2020
CRISPR/Cas9-Induced Inactivation of the Autism-Risk Gene Leads to Social Impairments in Zebrafish. International journal of molecular sciences\| PubMed ID: 36613611	2022
Neuroprotective Nanoparticles Targeting the Retina: A Polymeric Platform for Ocular Drug Delivery Applications. Pharmaceutics\| PubMed ID: 37111581	2023

Chiara Gabellini

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