A fast, reliable and cost-effective method to generate tumor organs for therapy screening in vivo

Innovative anticancer treatments continuously require tissue bioengineering models to test novel therapies. The increasing number of developments based on nanotechnology for cancer therapy or theragnostics demand simple, reliable, fast and cost-effective cancer in vivo models for preclinical testing. However, despite the many tumor models available, very few reproduce the complex intratumoral cell-to-cell interactions as well as the accompanying systemic whole body effects resulting of the tumor organ metabolic, hormonal or growth factor activities, all having critical implications in the success of cancer therapies. Here we describe a reliable tumor model that can be easily reproduced to generate visible solid malignant melanoma tumor organs within a defined period of 5–10 days recapitulating the tumor stroma that is essential for cancer development. These models can be easily evaluated in vivo or by anatomo-pathological procedures. This method provides a fast, reproducible, reliable and cost-effective way to generate solid tumors for in vivo therapy, drug, nanomaterial or imaging probe evaluation, diagnostic or theragnostic screening and validation

Neuronal accumulation of unrepaired DNA in a novel specific chromatin domain: structural, molecular and transcriptional characterization

There is growing evidence that defective DNA repair in neurons with accumulation of DNA lesions and loss of genome integrity underlies aging and many neurodegenerative disorders. An important challenge is to understand how neurons can tolerate the accumulation of persistent DNA lesions without triggering the apoptotic pathway. Here we study the impact of the accumulation of unrepaired DNA on the chromatin architecture, kinetics of the DNA damage response and transcriptional activity in rat sensory ganglion neurons exposed to 1-to-3 doses of ionizing radiation (IR). In particular, we have characterized the structural, molecular and transcriptional compartmentalization of unrepaired DNA in persistent DNA damaged foci (PDDF). IR induced the formation of numerous transient foci, which repaired DNA within the 24 h post-IR, and a 1-to-3 PDDF. The latter concentrate DNA damage signaling and repair factors, including ?H2AX, pATM, WRAP53 and 53BP1. The number and size of PDDF was dependent on the doses of IR administered. The proportion of neurons carrying PDDF decreased over time of post-IR, indicating that a slow DNA repair occurs in some foci. The fine structure of PDDF consisted of a loose network of unfolded 30 nm chromatin fiber intermediates, which may provide a structural scaffold accessible for DNA repair factors. Furthermore, the transcription assay demonstrated that PDDF are transcriptionally silent, although transcription occurred in flanking euchromatin. Therefore, the expression of ?H2AX can be used as a reliable marker of gene silencing in DNA damaged neurons. Moreover, PDDF were located in repressive nuclear environments, preferentially in the perinucleolar domain where they were frequently associated with Cajal bodies or heterochromatin clumps forming a structural triad. We propose that the sequestration of unrepaired DNA in discrete PDDF and the transcriptional silencing can be essential to preserve genome stability and prevent the synthesis of aberrant mRNA and protein products encoded by damaged genes

Nucleolar disruption and cajal body disassembly are nuclear hallmarks of DNA damage-induced neurodegeneration in purkinje cells

The Purkinje cell (PC) degeneration (pcd) phenotype results from mutation in nna1 gene and is associated with the degeneration and death of PCs during the postnatal life. Although the pcd mutation is a model of the ataxic mouse, it shares clinical and pathological characteristics of inherited human spinocerebellar ataxias. PC degeneration in pcd mice provides a useful neuronal system to study nuclear mechanisms involved in DNA damage-dependent neurodegeneration, particularly the contribution of nucleoli and Cajal bodies (CBs). Both nuclear structures are engaged in housekeeping functions for neuronal survival, the biogenesis of ribosomes and the maturation of snRNPs and snoRNPs required for pre-mRNA and pre-rRNA processing, respectively. In this study, we use ultrastructural analysis, in situ transcription assay and molecular markers for DNA damage, nucleoli and CB components to demonstrate that PC degeneration involves the progressive accumulation of nuclear DNA damage associated with disruption of nucleoli and CBs, disassembly of polyribosomes into monoribosomes, ribophagy and shut down of nucleolar and extranucleolar transcription. Microarray analysis reveals that four genes encoding repressors of nucleolar rRNA synthesis (p53, Rb, PTEN and SNF2) are upregulated in the cerebellum of pcd mice. Collectively, these data support that nucleolar and CB alterations are hallmarks of DNA damage-induced neurodegeneration.ACKNOWLEDGMENTS: The authors wish to thank Raquel García-Ceballos and Saray Pereda for technical assistance. This work was supported by the following grants: Dirección General de Investigación (BFU2008- 00175); Instituto de Salud Carlos III (CIBERNED, CB06/05/ 0037), Ministerio de Ciencia y Tecnología (BFU2010-18284), Ministerio de Sanidad, Política Social e Igualdad (Plan Nacional Sobre Drogas), Instituto de Formación e Investigación Marqués de Valdecilla (IFIMAV, FMV/UC09-02), Junta de Castilla y León, Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León and Fundación Memoria D. Samuel Solórzano-Barruso, all of them from Spain

Magnetic lipid nanovehicles synergize the controlled thermal release of chemotherapeutics with magnetic ablation while enabling non-invasive monitoring by MRI for melanoma theranostics

Nowadays, a number of promising strategies are being developed that aim at combining diagnostic and therapeutic capabilities into clinically effective formulations. Thus, the combination of a modified release provided by an organic encapsulation and the intrinsic physico-chemical properties from an inorganic counterpart opens new perspectives in biomedical applications. Herein, a biocompatible magnetic lipid nanocomposite vehicle was developed through an efficient, green and simple method to simultaneously incorporate magnetic nanoparticles and an anticancer drug (doxorubicin) into a natural nano-matrix. The theranostic performance of the final magnetic formulation was validated in vitro and in vivo, in melanoma tumors. The systemic administration of the proposed magnetic hybrid nanocomposite carrier enhanced anti-tumoral activity through a synergistic combination of magnetic hyperthermia effects and antimitotic therapy, together with MRI reporting capability. The application of an alternating magnetic field was found to play a dual role, (i) acting as an extra layer of control (remote, on-demand) over the chemotherapy release and (ii) inducing a local thermal ablation of tumor cells. This combination of chemotherapy with thermotherapy establishes a synergistic platform for the treatment of solid malignant tumors under lower drug dosing schemes, which may realize the dual goal of reduced systemic toxicity and enhanced anti-tumoral efficacy.Funding: This work was partially supported by NORTE 2020 (2014–2020 North Portugal Regional Operational Program), and the ERDF (European Regional Development Fund) under Grant NORTE-01-0145-FEDER-000019, by European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 686009, by “TAMs-targeted and externally controlled nanotheranostics of triple-negative-breast-cancer (Nanother)" project UTAPEXPL/NTec/0038/2017, by “Local specific treatment of triple-negativebreast-cancer through externally triggered target-less drug carriers (MagtargetON)" project NORTE-01-0145-FEDER-031142, co-funded by FCT and the ERDF through NORTE2020, and by 2014–2020 INTERREG Cooperation Programme Spain–Portugal (POCTEP) through the Project 0624_2IQBIONEURO_6_E. Co-authors also acknowledge support from Raman4clinics COST Action BM1401 and Radiomag COST action TD1402. ML-F also acknowledges the ERDF and the Spanish MINECO under project ref. PI19/00349 (AES 2019). LGH thanks the Instituto de Salud Carlos III for the Sara Borrell Grant (CD19/00035)

Persistent accumulation of unrepaired DNA damage in rat cortical neurons: nuclear organization and ChIP-seq analysis of damaged DNA

Neurons are highly vulnerable to DNA damage induced by genotoxic agents such as topoisomerase activity, oxidative stress, ionizing radiation (IR) and chemotherapeutic drugs. To avert the detrimental effects of DNA lesions in genome stability, transcription and apoptosis, neurons activate robust DNA repair mechanisms. However, defective DNA repair with accumulation of unrepaired DNA are at the basis of brain ageing and several neurodegenerative diseases. Understanding the mechanisms by which neurons tolerate DNA damage accumulation as well as defining the genomic regions that are more vulnerable to DNA damage or refractory to DNA repair and therefore constitute potential targets in neurodegenerative diseases are essential issues in the field. In this work we investigated the nuclear topography and organization together with the genome-wide distribution of unrepaired DNA in rat cortical neurons 15 days upon IR. About 5% of non-irradiated and 55% of irradiated cells accumulate unrepaired DNA within persistent DNA damage foci (PDDF) of chromatin. These PDDF are featured by persistent activation of DNA damage/repair signaling, lack of transcription and localization in repressive nuclear microenvironments. Interestingly, the chromatin insulator CTCF is concentrated at the PDDF boundaries, likely contributing to isolate unrepaired DNA from intact transcriptionally active chromatin. By confining damaged DNA, PDDF would help preserving genomic integrity and preventing the production of aberrant proteins encoded by damaged genes.ChIP-seq analysis of genome-wide ?H2AX distribution revealed a number of genomic regions enriched in ?H2AX signal in IR-treated cortical neurons. Some of these regions are in close proximity to genes encoding essential proteins for neuronal functions and human neurodegenerative disorders such as epm2a (Lafora disease), serpini1 (familial encephalopathy with neuroserpin inclusion bodies) and il1rpl1 (mental retardation, X-linked 21). Persistent ?H2AX signal close to those regions suggests that nearby genes could be either more vulnerable to DNA damage or more refractory to DNA repair.This work was supported by the following grants: “Dirección General de Investigación” (BFU2014–54754-P) and “Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas” (CIBERNED; CB06/05/0037) Spain

Gb3/cd77 Is a Predictive Marker and Promising Therapeutic Target for Head and Neck Cancer

Head and neck squamous cell carcinoma is the sixth leading cancer in the world. This cancer is difficult to treat and is characterized by recurrences that are often fatal. This cancer is generally removed surgically, but it often regrows from the edges of the lesion from where most recurrences reappear. In this study, we have investigated if the expression of GB3 in human cell lines, tissues from patient biopsies, and a murine animal model could be used as an early and determinant marker of HNC. We found that in all the investigated systems, this marker appears in neoplastic cells from the very early stages of their malignant transformation. Our conclusions support the hypothesis that GB3 is a reliable and independent target for HNC identification and selective delivery of treatments. Furthermore, we show that the level of expression of this marker correlates with the degree of malignancy of the tumor. These studies suggest that GB3 may provide the basis for the early identification and new targeted therapies for head and neck cancer.Funding: This research was funded by ISCIII Projects ref. PI19/00349, DTS19/00033, co-funded by ERDF/ESF, “Investing in your future”; NanoBioApp Research Network (MINECO-17-MAT2016-81955-REDT), COST action Nano2Clinic CA17140, and IDIVAL for the INNVAL 19/12 and INNVAL20/13 projects

Multiwalled Carbon Nanotubes inhibit tumor progression in a mouse model

Understanding the molecular mechanisms underlying the biosynthetic interactions between particular nanomaterials with specific cells or proteins opens new alternatives in nanomedicine and nanotoxicology. Multiwalled carbon nanotubes (MWCNTs) have long been explored as drug delivery systems and nanomedicines against cancer. There are high expectations for their use in therapy and diagnosis. These filaments can translocate inside cultured cells and intermingle with the protein nanofilaments of the cytoskeleton, interfering with the biomechanics of cell division mimicking the effect of traditional microtubule-binding anti-cancer drugs such as paclitaxel. Here, it is shown how MWCNTs can trigger significant anti-tumoral effects in vivo, in solid malignant melanomas produced by allograft transplantation. Interestingly, the MWCNT anti-tumoral effects are maintained even in solid melanomas generated from paclitaxel-resistant cells. These findings provide great expectation in the development of groundbreaking adjuvant synthetic microtubule-stabilizing chemotherapies to overcome drug resistance in cancer.Acknowledgements: We thank Dr. E. Flahaut for providing the MWCNTs. We are grateful to the Nikon A1R Laser Microscopy Unit of the IDIVAL Institute for the electron microscopy and confocal/time-lapse microscopy, and to M. Aramburu and J. Díaz-Gómez for their help. This work has been supported by the Spanish MINECO and European Union FEDER under Projects ref. PI13/01074 (AES 2013) and MAT2012-38664-C02-01. We especially thank the Fundación Eugenio Rodríguez Pascual (ref “Ayudas de investigación” 2014)

Reactive nucleolar and Cajal body responses to proteasome inhibition in sensory ganglion neurons

The dysfunction of the ubiquitin proteasome system has been related to a broad array of neurodegenerative disorders in which the accumulation of misfolded protein aggregates causes proteotoxicity. The ability of proteasome inhibitors to induce cell cycle arrest and apoptosis has emerged as a powerful strategy for cancer therapy. Bortezomib is a proteasome inhibitor used as an antineoplastic drug, although its neurotoxicity frequently causes a severe sensory peripheral neuropathy. In this study we used a rat model of bortezomib treatment to study the nucleolar and Cajal body responses to the proteasome inhibition in sensory ganglion neurons that are major targets of bortezomib-induced neurotoxicity. Treatment with bortezomib induced dose-dependent dissociation of protein synthesis machinery (chromatolysis) and nuclear retention of poly(A) RNA granules resulting in neuronal dysfunction. However, as a compensatory response to the proteotoxic stress, both nucleoli and Cajal bodies exhibited reactive changes. These include an increase in the number and size of nucleoli, strong nucleolar incorporation of the RNA precursor 5'-fluorouridine, and increased expression of both 45S rRNA and genes encoding nucleolar proteins UBF, fibrillarin and B23. Taken together, these findings appear to reflect the activation of the nucleolar transcription in response to proteotoxic stress Furthermore, the number of Cajal bodies, a parameter related to transcriptional activity, increases upon proteasome inhibition. We propose that nucleoli and Cajal bodies are important targets in the signaling pathways that are activated by the proteotoxic stress response to proteasome inhibition. The coordinating activity of these two organelles in the production of snRNA, snoRNA and rRNA may contribute to neuronal survival after proteasome inhibition. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease