Single-Cell Recovery from Tumor Cell Xenotransplanted Zebrafish Embryos for the Study of Metastasis-Initiating Cells.

The study of metastasis-competent cells at the single-cell level represents an opportunity to decipher the molecular mechanisms associated with the metastatic cascade as well as to understand the functional and molecular heterogeneity of these cells. In this context, preclinical in vivo models of cancer metastasis are valuable tools to understand the behavior of cancer cells throughout the process. Here we describe a detailed protocol for the isolation and recovery of individual viable human metastatic cells from zebrafish embryos xenotransplanted with cancer cells for downstream molecular analysis. We cover the critical steps for the dissociation of the xenografted zebrafish embryos to generate a single-cell suspension, and the micromanipulation for their recovery as single cells.

Isolation of Single Circulating Tumor Cells Using VyCAP Puncher System.

Tumor heterogeneity has a major role in the development of tumor evasion and resistance to treatments. To study and understand the intrinsic heterogeneity of cancer cells, the use of single-cell isolation technology has had a major boost in recent years, gaining ground to bulk analysis in the study of solid tumors. In the liquid biopsy field, the use of technologies for single-cell analysis has represented a major advance in the study of the heterogeneity of circulating tumor cells (CTCs), providing relevant information about therapy-resistant CTCs. However, single-cell analysis of CTCs is still challenging due to the weakness and scarcity of these cells. In this chapter, we describe a protocol for CTCs isolation at a single-cell level using the VyCAP Puncher system.

Models to study CTCs and CTC culture methods.

The vast majority of cancer-related deaths are due to the presence of disseminated disease. Understanding the metastatic process is key to achieving a reduction in cancer mortality. Particularly, there is a need to understand the molecular mechanisms that drive cancer metastasis, which will allow the identification of curative treatments for metastatic cancers. Liquid biopsies have arisen as a minimally invasive approach to gain insights into the biology of metastasis. Circulating tumour cells (CTCs), shed to the circulation from the primary tumour or metastatic lesions, are a key component of liquid biopsy. As metastatic precursors, CTCs hold the potential to unravel the mechanisms involved in metastasis formation as well as new therapeutic strategies for treating metastatic disease. However, the complex biology of CTCs together with their low frequency in circulation are factors hampering an in-depth mechanistic investigation of the metastatic process. To overcome these problems, CTC-derived models, including CTC-derived xenograft (CDX) and CTC-derived ex vivo cultures, in combination with more traditional in vivo models of metastasis, have emerged as powerful tools to investigate the biological features of CTCs facilitating cancer metastasis and uncover new therapeutic opportunities. In this chapter, we provide an up to date view of the diverse models used in different cancers to study the biology of CTCs, and of the methods developed for CTC culture and expansion, in vivo and ex vivo. We also report some of the main challenges and limitations that these models are facing.

Consecuencias judiciales de lesiones en instalaciones deportivas murales y opinión docente sobre seguridad en escalada escolar / Legal consequences of injuries in wall sports facilities and teachers views on school climbing safety

La prevención en el ámbito escolar y deportivo abarca una pluralidad de fuentes y factores presentes en los centros educativos e instalaciones deportivas. Pese a que el espacio de mayor siniestralidad es el patio y la propia cancha deportiva, son también destacados los accidentes en centros escolares o deportivos que implican estructuras murales, tanto en interior como exterior. Cualquier recinto o edificio, máxime cuando esté dedicado a una utilización formativa, deportiva o lúdica, debería estar sometido a una evaluación periódica, tanto por sus responsables directos, como de los propietarios. Se empleó una metodología combinada, de revisión no sistemática y observacional. Para ello, se realizó una búsqueda jurídica desde 1998-2023 en el Centro de Documentación Judicial del Consejo General del Poder Judicial de las sentencias sobre accidentes en instalaciones murales escolares y deportivas; a la par también se envió un cuestionario sobre seguridad y gestión de riesgo para conocer la opinión del profesorado de educación primaria y secundaria en actividades de trepa-escalada. Se localizaron 10 condenas que cumplían los criterios necesarios, relativas sobre todo de accidentes, habitualmente con indemnización por daños. Se concluye que la mayoría de lesiones no intencionadas en el ámbito escolar y deportivo en instalaciones murales deportivas y escolares, no son graves. La jurisprudencia existente, centrada en rocódromos y pistas de pádel, advierte que se debe mejorar la gestión y mantenimiento de las paredes para evitar reclamaciones. En esta línea, el profesorado de educación primaria y secundaria participante en el estudio se queja de que la falta y escaso mantenimiento de sus instalaciones de trepa-escalada, así como de adecuada formación y recursos para un mejor control de uso. En general, son partidarios de compartir parcialmente la gestión de riesgos con el alumnado, evitando situaciones de riesgo alto o fuera de control. (AU)

Prevention in the school and sports environment encompasses a plurality of sources and factors present in schools and sports facilities. Although the area with the highest accident rate is the playground and the sports field itself, there are also accidents in schools and sports centres involving wall structures, both indoors and outdoors. Any site or building, especially when it is used for educational, sporting or recreational purposes, should be subject to periodic evaluation, both by those directly responsible for it and by the owners. A combined methodology of non-systematic and observational review was used. To this end, a search was carried out from 1998-2023 in the Judicial Documentation Centre of the General Council of the Judiciary for judgements on accidents in school and sports facilities; at the same time, a questionnaire on safety and risk management was also sent out to find out the opinion of primary and secondary school teachers in climbing activities. Ten convictions were found that met the necessary criteria, mainly relating to accidents, usually with compensation for damages. It is concluded that the majority of unintentional injuries in the school and sports environment in school and sports wall facilities are not serious. Existing case law, focusing on climbing walls and paddle tennis courts, warns that the management and maintenance of walls must be improved to avoid claims. In this line, the primary and secondary school teachers participating in the study complain about the lack and poor maintenance of their climbing facilities, as well as the lack of adequate training and resources for better control of use. In general, they are in favour of partially sharing risk management with pupils, avoiding high-risk or out-of-control situations. (AU)

Correction: Effectiveness of a novel gene nanotherapy based on putrescine for cancer treatment.

Correction for 'Effectiveness of a novel gene nanotherapy based on putrescine for cancer treatment' by Saínza Lores et al., Biomater. Sci., 2023, https://doi.org/10.1039/d2bm01456d.

Modelling metastasis in zebrafish unveils regulatory interactions of cancer-associated fibroblasts with circulating tumour cells.

The dynamic intercommunication between tumour cells and cells from the microenvironment, such as cancer-associated fibroblast (CAFs), is a key factor driving breast cancer (BC) metastasis. Clusters of circulating tumour cells (CTCs), known to bare a higher efficiency at establishing metastases, are found in the blood of BC patients, often accompanied by CAFs in heterotypic CTC-clusters. Previously we have shown the utility of CTC-clusters models and the zebrafish embryo as a model of metastasis to understand the biology of breast cancer CTC-clusters. In this work, we use the zebrafish embryo to study the interactions between CTCs in homotypic clusters and CTC-CAFs in heterotypic CTC-clusters to identify potential pro-metastatic traits derived from CTC-CAF communication. We found that upon dissemination CAFs seem to exert a pro-survival and pro-proliferative effect on the CTCs, but only when CTCs and CAFs remain joined as cell clusters. Our data indicate that the clustering of CTC and CAF allows the establishment of physical interactions that when maintained over time favour the selection of CTCs with a higher capacity to survive and proliferate upon dissemination. Importantly, this effect seems to be dependent on the survival of disseminated CAFs and was not observed in the presence of normal fibroblasts. Moreover, we show that CAFs can exert regulatory effects on the CTCs without being involved in promoting tumour cell invasion. Lastly, we show that the physical communication between BC cells and CAFs leads to the production of soluble factors involved in BC cell survival and proliferation. These findings suggest the existence of a CAF-regulatory effect on CTC survival and proliferation sustained by cell-to-cell contacts and highlight the need to understand the molecular mechanisms that mediate the interaction between the CTCs and CAFs in clusters enhancing the metastatic capacity of CTCs.

Effectiveness of a novel gene nanotherapy based on putrescine for cancer treatment.

Gene therapy has long been proposed for cancer treatment. However, the use of therapeutic nucleic acids presents several limitations such as enzymatic degradation, rapid clearance, and poor cellular uptake and efficiency. In this work we propose the use of putrescine, a precursor for higher polyamine biosynthesis for the preparation of cationic nanosystems for cancer gene therapy. We have formulated and characterized putrescine-sphingomyelin nanosystems (PSN) and studied their endocytic pathway and intracellular trafficking in cancer cells. After loading a plasmid DNA (pDNA) encoding the apoptotic Fas Ligand (FasL), we proved their therapeutic activity by measuring the cell death rate after treatment of MDA-MB-231 cells. We have also used xenografted zebrafish embryos as a first in vivo approach to demonstrate the efficacy of the proposed PSN-pDNA formulation in a more complex model. Finally, intratumoral and intraperitoneal administration to mice-bearing MDA-MB-231 xenografts resulted in a significant decrease in tumour cell growth, highlighting the potential of the developed gene therapy nanoformulation for the treatment of triple negative breast cancer.

Magnetically propelled chained nanocomposites for biologically relevant media exploration.

Elongated nanostructures to be remotely and magnetically propelled in biologically relevant media, have gained attention as offering themselves as effective tools or carriers in theragnostics applications. However, the magnetic actuation associated remains challenging due to the lack of mechanical information in the media of interest, taking into account biophysical or biomedical purposes. In this study, we detail the magnetic actuation of magnetically propelled chained nanocomposites considering their dynamics, in which their velocity can be modulated in terms of the viscosity of the medium considered, given a magnetic field gradient. Simpler cases of distilled water, a water/glycerol mixture and a fluid made of cell extracts (imitating the cytosol of cells) of known viscosity are the basis experiments for the study of more complex media inside HeLa cells, murine NIH-3T3 fibroblasts and zebrafish larvae, offering the mechanical information required. The experimental results indicate that the magnetically propelled performance of the chained nanostructures can be precisely controlled in potentially changing scenarios, where drug and heat delivery, magnetic separation, or microfluidic technologies are demanded, using a magnetic field gradient and providing good estimations of the dynamical parameters involved.

Red Blood Cells Protein Profile Is Modified in Breast Cancer Patients.

Metastasis is the primary cause of death for most breast cancer (BC) patients who succumb to the disease. During the hematogenous dissemination, circulating tumor cells interact with different blood components. Thus, there are microenvironmental and systemic processes contributing to cancer regulation. We have recently published that red blood cells (RBCs) that accompany circulating tumor cells have prognostic value in metastatic BC patients. RBC alterations are related to several diseases. Although the principal known role is gas transport, it has been recently assigned additional functions as regulatory cells on circulation. Hence, to explore their potential contribution to tumor progression, we characterized the proteomic composition of RBCs from 53 BC patients from stages I to III and IV, compared with 33 cancer-free controls. In this work, we observed that RBCs from BC patients showed a different proteomic profile compared to cancer-free controls and between different tumor stages. The differential proteins were mainly related to extracellular components, proteasome, and metabolism. Embryonic hemoglobins, not expected in adults' RBCs, were detected in BC patients. Besides, lysosome-associated membrane glycoprotein 2 emerge as a new RBCs marker with diagnostic and prognostic potential for metastatic BC patients. Seemingly, RBCs are acquiring modifications in their proteomic composition that probably represents the systemic cancer disease, conditioned by the tumor microenvironment.

Zebrafish as a platform to evaluate the potential of lipidic nanoemulsions for gene therapy in cancer.

Gene therapy is a promising therapeutic approach that has experienced significant groth in recent decades, with gene nanomedicines reaching the clinics. However, it is still necessary to continue developing novel vectors able to carry, protect, and release the nucleic acids into the target cells, to respond to the widespread demand for new gene therapies to address current unmet clinical needs. We propose here the use of zebrafish embryos as an in vivo platform to evaluate the potential of newly developed nanosystems for gene therapy applications in cancer treatment. Zebrafish embryos have several advantages such as low maintenance costs, transparency, robustness, and a high homology with the human genome. In this work, a new type of putrescine-sphingomyelin nanosystems (PSN), specifically designed for cancer gene therapy applications, was successfully characterized and demonstrated its potential for delivery of plasmid DNA (pDNA) and miRNA (miR). On one hand, we were able to validate a regulatory effect of the PSN/miR on gene expression after injection in embryos of 0 hpf. Additionally, experiments proved the potential of the model to study the transport of the associated nucleic acids (pDNA and miR) upon incubation in zebrafish water. The biodistribution of PSN/pDNA and PSN/miR in vivo was also assessed after microinjection into the zebrafish vasculature, demonstrating that the nucleic acids remained associated with the PSN in an in vivo environment, and could successfully reach disseminated cancer cells in zebrafish xenografts. Altogether, these results demonstrate the potential of zebrafish as an in vivo model to evaluate nanotechnology-based gene therapies for cancer treatment, as well as the capacity of the developed versatile PSN formulation for gene therapy applications.

Comparative analysis of capture methods for genomic profiling of circulating tumor cells in colorectal cancer.

The genomic profiling of circulating tumor cells (CTCs) in the bloodstream should provide clinically relevant information on therapeutic efficacy and help predict cancer survival. Here, we contrasted the genomic profiles of CTC pools recovered from metastatic colorectal cancer (mCRC) patients using different enrichment strategies (CellSearch, Parsortix, and FACS). Mutations inferred in the CTC pools differed depending on the enrichment strategy and, in all cases, represented a subset of the mutations detected in the matched primary tumor samples. However, the CTC pools from Parsortix, and in part, CellSearch, showed diversity estimates, mutational signatures, and drug-suitability scores remarkably close to those found in matching primary tumor samples. In addition, FACS CTC pools were enriched in apparent sequencing artifacts, leading to much higher genomic diversity estimates. Our results highlight the utility of CTCs to assess the genomic heterogeneity of individual tumors and help clinicians prioritize drugs in mCRC.

What Zebrafish and Nanotechnology Can Offer for Cancer Treatments in the Age of Personalized Medicine.

Cancer causes millions of deaths each year and thus urgently requires the development of new therapeutic strategies. Nanotechnology-based anticancer therapies are a promising approach, with several formulations already approved and in clinical use. The evaluation of these therapies requires efficient in vivo models to study their behavior and interaction with cancer cells, and to optimize their properties to ensure maximum efficacy and safety. In this way, zebrafish is an important candidate due to its high homology with the human genoma, its large offspring, and the ease in developing specific cancer models. The role of zebrafish as a model for anticancer therapy studies has been highly evidenced, allowing researchers not only to perform drug screenings but also to evaluate novel therapies such as immunotherapies and nanotherapies. Beyond that, zebrafish can be used as an "avatar" model for performing patient-derived xenografts for personalized medicine. These characteristics place zebrafish in an attractive position as a role model for evaluating novel therapies for cancer treatment, such as nanomedicine.

Intratumor genetic heterogeneity and clonal evolution to decode endometrial cancer progression.

Analyzing different tumor regions by next generation sequencing allows the assessment of intratumor genetic heterogeneity (ITGH), a phenomenon that has been studied widely in some tumor types but has been less well explored in endometrial carcinoma (EC). In this study, we sought to characterize the spatial and temporal heterogeneity of 9 different ECs using whole-exome sequencing, and by performing targeted sequencing validation of the 42 primary tumor regions and 30 metastatic samples analyzed. In addition, copy number alterations of serous carcinomas were assessed by comparative genomic hybridization arrays. From the somatic mutations, identified by whole-exome sequencing, 532 were validated by targeted sequencing. Based on these data, the phylogenetic tree reconstructed for each case allowed us to establish the tumors' evolution and correlate this to tumor progression, prognosis, and the presence of recurrent disease. Moreover, we studied the genetic landscape of an ambiguous EC and the molecular profile obtained was used to guide the selection of a potential personalized therapy for this patient, which was subsequently validated by preclinical testing in patient-derived xenograft models. Overall, our study reveals the impact of analyzing different tumor regions to decipher the ITGH in ECs, which could help make the best treatment decision.

Dissecting Breast Cancer Circulating Tumor Cells Competence via Modelling Metastasis in Zebrafish.

BACKGROUND: Cancer metastasis is a deathly process, and a better understanding of the different steps is needed. The shedding of circulating tumor cells (CTCs) and CTC-cluster from the primary tumor, its survival in circulation, and homing are key events of the metastasis cascade. In vitro models of CTCs and in vivo models of metastasis represent an excellent opportunity to delve into the behavior of metastatic cells, to gain understanding on how secondary tumors appear. METHODS: Using the zebrafish embryo, in combination with the mouse and in vitro assays, as an in vivo model of the spatiotemporal development of metastases, we study the metastatic competency of breast cancer CTCs and CTC-clusters and the molecular mechanisms. RESULTS: CTC-clusters disseminated at a lower frequency than single CTCs in the zebrafish and showed a reduced capacity to invade. A temporal follow-up of the behavior of disseminated CTCs showed a higher survival and proliferation capacity of CTC-clusters, supported by their increased resistance to fluid shear stress. These data were corroborated in mouse studies. In addition, a differential gene signature was observed, with CTC-clusters upregulating cell cycle and stemness related genes. CONCLUSIONS: The zebrafish embryo is a valuable model system to understand the biology of breast cancer CTCs and CTC-clusters.

Dangerous Liaisons: Circulating Tumor Cells (CTCs) and Cancer-Associated Fibroblasts (CAFs).

The crosstalk between cancer cells and the tumor microenvironment (TME) is a key determinant of cancer metastasis. Cancer-associated fibroblasts (CAFs), one of the main cellular components of TME, promote cancer cell invasion and dissemination through mechanisms including cell-cell interactions and the paracrine secretion of growth factors, cytokines and chemokines. During metastasis, circulating tumor cells (CTCs) are shed from the primary tumor to the bloodstream, where they can be detected as single cells or clusters. The current knowledge about the biology of CTC clusters positions them as key actors in metastasis formation. It also indicates that CTCs do not act alone and that they may be aided by stromal and immune cells, which seem to shape their metastatic potential. Among these cells, CAFs are found associated with CTCs in heterotypic CTC clusters, and their presence seems to increase their metastatic efficiency. In this review, we summarize the current knowledge on the role that CAFs play on metastasis and we discuss their implication on the biogenesis, metastasis-initiating capacity of CTC clusters, and clinical implications. Moreover, we speculate about possible therapeutic strategies aimed to limit the metastatic potential of CTC clusters involving the targeting of CAFs as well as their difficulties and limitations.

Analysis of a Real-World Cohort of Metastatic Breast Cancer Patients Shows Circulating Tumor Cell Clusters (CTC-clusters) as Predictors of Patient Outcomes.

Circulating tumor cell (CTC) enumeration has emerged as a powerful biomarker for the assessment of prognosis and the response to treatment in metastatic breast cancer (MBC). Moreover, clinical evidences show that CTC-cluster counts add prognostic information to CTC enumeration, however, their significance is not well understood, and more clinical evidences are needed. We aim to evaluate the prognostic value of longitudinally collected single CTCs and CTC-clusters in a heterogeneous real-world cohort of 54 MBC patients. Blood samples were longitudinally collected at baseline and follow up. CTC and CTC-cluster enumeration was performed using the CellSearch® system. Associations with progression-free survival (PFS) and overall survival (OS) were evaluated using Cox proportional hazards modelling. Elevated CTC counts and CTC-clusters at baseline were significantly associated with a shorter survival time. In joint analysis, patients with high CTC counts and CTC-cluster at baseline were at a higher risk of progression and death, and longitudinal analysis showed that patients with CTC-clusters had significantly shorter survival compared to patients without clusters. Moreover, patients with CTC-cluster of a larger size were at a higher risk of death. A longitudinal analysis of a real-world cohort of MBC patients indicates that CTC-clusters analysis provides additional prognostic value to single CTC enumeration, and that CTC-cluster size correlates with patient outcome.

Introduction - Biology of Breast Cancer Metastasis and Importance of the Analysis of CTCs.

Breast cancer metastasis is a complex multistep process during which tumor cells undergo structural and functional changes that allow them to move away from the primary tumor and disseminate to distant organs and tissues. Despite the inefficiency of this process, some populations of circulating tumor cells (CTCs), which are those cells responsible of metastases formation, are able to survive in blood circulation and grow into secondary tumors. Metastatic breast cancer remains an incurable disease, and the phenomenon of metastasis represents the larger cause of death in these patients. The application of liquid biopsy techniques and the advancements in the field have shown the prognostic value of CTCs, suggesting the importance that CTCs analyses may have in the clinic. However, their implementation in routine clinic has not been yet achieved due to the yet small body of evidence showing their clinical utility. This introductory chapter will revise the key aspects of breast cancer metastasis and discuss the importance of CTC analyses in the management of breast cancer patients.

Relevance of CTC Clusters in Breast Cancer Metastasis.

Metastasis is the major cause of mortality in patients with breast cancer; however, the mechanisms of tumor cell dissemination and metastasis formation are not well established yet. The study of circulating tumour cells (CTCs), the metastatic precursors of distant disease, may help in this search. CTCs can be found in the blood of cancer patients as single cells or as tumor cell aggregates, known as CTC clusters. CTC clusters have differential biological features such as an enhanced survival and metastatic potential, and they hold great promises for the evaluation of prognosis, diagnosis and therapy of the metastatic cancer. The analysis of CTC clusters offers new insights into the mechanism of metastasis and can guide towards the development of new diagnostic and therapeutic strategies to suppress cancer metastasis. This has become possible thanks to the development of improved technologies for detection of CTCs and CTC clusters. However, more efficient methods are needed in order to address important questions regarding the metastatic potential of CTC and future clinical applications. In this chapter, we explore the current knowledge on the role of CTC clusters in breast cancer metastasis, their origin, metastatic advantages and clinical importance.