Functional Characterization of Circulating Tumour Cell (CTC) Clusters in Breast Cancer

Breast cancer (BC) is the most common malignancy in women and the second leading cause of cancer-related deaths. Most cancer-related deaths are due to metastasis, a process by which tumour cells spread to secondary sites. Circulating tumour cells (CTCs) are those tumour cells that are released into the bloodstream, and they are the responsible for the formation of metastases. CTCs can be found as individual cells, or as small groups of cells, called CTC clusters. CTC clusters have a higher metastatic potential than individual CTCs. However, there is little knowledge about the biology of CTC clusters due to their low frequency in the blood of BC patients. The objective of this thesis project is to conduct a comparative study between individual CTCs and CTC clusters, to further study the biology of CTC clusters and to determine the differential characteristics that provide them with greater metastatic potential. The isolation of CTC clusters from samples of BC patients not only allowed us to confirm the prognostic value of CTC clusters but also to optimize workflows that maximise the detection of CTC clusters. The development of in vitro models of CTC clusters and their later functional/molecular characterization showed that these models properly recapitulated the phenotypic features of the CTC clusters isolated from patient samples. The combination of the tools presented in this thesis can contribute to overcome the restrictions derived from the low frequency of CTC clusters in patient samples and allow a deeper understanding about the role of CTC clusters during metastasis

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

Cáncer de mama; Metástasis; Pez cebraCàncer de mama; Metàstasi; Peix zebraBreast cancer; Metastasis; ZebrafishBackground: 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.This work was supported by Roche-Chus Joint Unit (IN853B 2018/03) funded by Axencia Galega de Innovación (GAIN), Consellería de Economía, Emprego e Industria. I.M.-P. is funded by the Training Program for Academic Staff fellowship (FPU16/01018), from the Ministry of Education and Vocational Training, Spanish Government. P.H. is funded by a Predoctoral fellowship (IN606A-2018/019) from Axencia Galega de Innovación (GAIN, Xunta de Galicia). N.C.-U. is funded by Axudas Predoutorais do IDIS (Instituto de Investigación Sanitaria de Santiago)

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.This work was supported by Roche-Chus Joint Unit (IN853B 2018/03) funded by Axencia Galega de Innovación (GAIN), Consellería de Economía, Emprego e Industria. I.M.-P. is funded by the Training Program for Academic Staff fellowship (FPU16/01018), from the Ministry of Education and Vocational Training, Spanish Government. P.H. is funded by a Predoctoral fellowship (IN606A-2018/019) from Axencia Galega de Innovación (GAIN, Xunta de Galicia). N.C.-U. is funded by Axudas Predoutorais do IDIS (Instituto de Investigación Sanitaria de Santiago).S

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 outcomeThis research was supported by Roche-Chus Joint Unit (IN853B 2018/03), funded by Axencia Galega de Innovación (GAIN), Consellería de Economía, Emprego e Industria and by the Instituto de Salud Carlos III (ISCIII) and FEDER (PI13/01388). L.M.-R. is supported by Asociación Española Contra el Cáncer (AECC). I.M.-P. is funded by the Training Program for Academic Staff fellowship (FPU16/01018), from the Ministry of Education and Vocational Training, Spanish GovernmentS

Systems thinking from the One Health perspective in the learning of health issues in High School

Traballo Fin de Máster Universitario en Profesorado de Educación Secundaria Obrigatoria e Bacharelato, Formación Profesional e Ensinanzas de Idiomas. Especialidade de Ciencias Experimentais, Matemáticas, e Tecnoloxía e Informática. Curso 2021-2022A noción “Unha soa Saúde” (One Health) fai referencia a existencia de relacións interdependentes entre a saúde humana, a saúde animal e a dos ecosistemas. Esta perspectiva resulta clave para a comprensión integral de problemáticas de saúde complexas, como a pandemia de COVID-19. Malia a súa relevancia, existen escasas investigacións de aula que aborden este enfoque na ensinanza de problemas de saúde na aula de ciencias. Este TFM sitúase nesta liña de investigación emerxente na didáctica das ciencias, sobre pensamento sistémico e futuro para a acción socio-científica. Os participantes son unha aula de 3º de ESO (N=18) dun centro público. Os obxectivos de investigación son: 1) Examinar o pensamento sistémico do alumnado en relación á pandemia de COVID-19; 2) Analizar o pensamento temporal (retrospectivo e/ou prospectivo) que aplican neste contexto; 3) Identificar o tipo de accións que propoñen para previr futuras pandemias. A análise dos obxectivos mostra que a maioría dos participantes aplican o pensamento sistémico e temporal para identificar posibles causas da pandemia de COVID-19. Ademais, o alumnado é quen de propoñen accións colectivas para previr futuras pandemias. Da análise cualitativa derivan implicacións educativas relativas á importancia de incorporar a perspectiva “Unha soa Saúde” na ensinanza de problemáticas socio-científicasLa noción “Una sola Salud” hace referencia a relaciones interdependientes entre la salud humana, la animal y la de los ecosistemas. Esta perspectiva es clave para la comprensión integral de problemas sanitarios complejos, como la pandemia de COVID-19. A pesar de su relevancia, existen escasas investigaciones de aula que aborden este enfoque en la enseñanza de cuestiones de salud en el aula de ciencias. Este TFM se sitúa en esta línea de investigación emergente en la didáctica de las ciencias, sobre el pensamiento sistémico y futuro para la acción sociocientífica. Los participantes son un aula de 3º de ESO (N=18) de un colegio público. Los objetivos de investigación son: 1) Examinar el pensamiento sistémico en relación con la pandemia de COVID-19; 2) Analizar el pensamiento temporal (retrospectivo y/o prospectivo) aplicado en este contexto; 3) Identificar el tipo de acciones que proponen para prevenir futuras pandemias. El análisis muestra que la mayoría de los participantes aplican pensamiento sistémico y temporal para identificar posibles causas de la pandemia de COVID-19. Además, proponen acciones colectivas para prevenir futuras pandemias. Del análisis cualitativo derivan implicaciones educativas relacionadas con la importancia de incorporar la perspectiva “Una sola Salud” a la enseñanza de problemáticas sociocientíficasThe One Health approach involves the existence of interdependent relationships between human health, animal health and the health of ecosystems. This approach is essential for a suitable understanding of complex health issues, such as the COVID-19 pandemic. Despite its relevance, there is a limited number of research focused on this perspective related to health issues learning in science education. This TFM is related to this emerging research line in the science education, which is about systems thinking and future thinking for agency. The participants of this TFM were a 3rd year classroom of high school (N=18) of a public school. The research objectives are: 1) Assessing the kind of systems thinking regarding the COVID-19 pandemic; 2) Analysing students’ temporal thinking (retrospective and/or prospective) exhibited in this context; 3) Identifying the type of actions proposed by students to prevent future pandemics. The analysis of these objectives showed that the majority of participants were able to use systems thinking and temporal thinking to identify potential causes of the COVID-19 pandemic. In addition, students proposed collective actions to prevent future pandemics. This qualitative analysis has educational implications regarding the relevance of including the One Health perspective during teaching socio-scientific issue

¿Cuál es la noción de Resistencia a Antibióticos del profesorado en formación desde el enfoque “One Health”?

La resistencia a antibióticos (RA) es un problema que puede abordarse desde el enfoque One Health (OH). Sin embargo, su enseñanza en el aula de ciencias no ha sido todavía incorporada desde este enfoque integral. Este trabajo investiga cuál es la noción de RA que presenta el profesorado en formación de dos universidades de España y en qué medida incorporan la idea de contagio bajo un enfoque OH. Se analizan las respuestas individuales a un cuestionario sobre RA, siendo codificadas de acuerdo al agente y mecanismo de resistencia señalado, a las referencias que hacen a la idea de contagio, así como a las interacciones entre la salud humana, ambiental y animal que explican este problema. Los resultados muestran que los participantes tienen una noción de RA alejada de la científica y de una visión sistémica de acuerdo con el enfoque OH. Se derivan implicaciones para mejorar su adecuada comprensión y enseñanza en el aula.Este trabajo fue financiado por los proyectos de investigación PID2022-137010OB-I00 y PID2022-138166NB-C21, promovidos por MCIN/AEI/10.13039/501100011033/FEDER, U

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.We kindly thank Prof. Erik Sahai for providing the vCAF cells. We thank the personnel from the Experimental Biomedicine Centre (CEBEGA) of the University of Santiago de Compostela for their technical support. We thank Jose Antonio Trillo Franco for his technical assistance with the in vitro experiemnts.S

First results on use-wear analysis over several Ancient Neolithic context from Northwest Africa

This paper is a brief presentation about the functionality of lithic tools from several Early Neolithic sites in North-West Africa. In the last years, research teams have focused on this territory in order to study the origin of the first Neolithic communities. Data from the Northwest African coast are still scarce. Therefore this paper intends to fill the long existing gap between both shores of the West Mediterranean, an area that should be considered as a singular entity during the Early Holocene.Peer reviewe

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

Effects of IFIH1 rs1990760 variants on systemic inflammation and outcome in critically ill COVID-19 patients in an observational translational study

Variants in IFIH1, a gene coding the cytoplasmatic RNA sensor MDA5, regulate the response to viral infections. We hypothesized that IFIH1 rs199076 variants would modulate host response and outcome after severe COVID-19. Patients admitted to an intensive care unit (ICU) with confirmed COVID-19 were prospectively studied and rs1990760 variants determined. Peripheral blood gene expression, cell populations, and immune mediators were measured. Peripheral blood mononuclear cells from healthy volunteers were exposed to an MDA5 agonist and dexamethasone ex-vivo, and changes in gene expression assessed. ICU discharge and hospital death were modeled using rs1990760 variants and dexamethasone as factors in this cohort and in-silico clinical trials. About 227 patients were studied. Patients with the IFIH1 rs1990760 TT variant showed a lower expression of inflammation-related pathways, an anti-inflammatory cell profile, and lower concentrations of pro-inflammatory mediators. Cells with TT variant exposed to an MDA5 agonist showed an increase in IL6 expression after dexamethasone treatment. All patients with the TT variant not treated with steroids survived their ICU stay (hazard ratio [HR]: 2.49, 95% confidence interval [CI]: 1.29-4.79). Patients with a TT variant treated with dexamethasone showed an increased hospital mortality (HR: 2.19, 95% CI: 1.01-4.87) and serum IL-6. In-silico clinical trials supported these findings. COVID-19 patients with the IFIH1 rs1990760 TT variant show an attenuated inflammatory response and better outcomes. Dexamethasone may reverse this anti-inflammatory phenotype. Centro de Investigación Biomédica en Red (CB17/06/00021), Instituto de Salud Carlos III (PI19/00184 and PI20/01360), and Fundació La Marató de TV3 (413/C/2021). Patients with severe COVID-19 often need mechanical ventilation to help them breathe and other types of intensive care. The outcome for many of these patients depends on how their immune system reacts to the infection. If the inflammatory response triggered by the immune system is too strong, this can cause further harm to the patient. One gene that plays an important role in inflammation is IFIH1 which encodes a protein that helps the body to recognize viruses. There are multiple versions of this gene which each produce a slightly different protein. It is possible that this variation impacts how the immune system responds to the virus that causes COVID-19. To investigate, Amado-Rodríguez, Salgado del Riego et al. analyzed the IFIH1 gene in 227 patients admitted to an intensive care unit in Spain for severe COVID-19 between March and December 2020. They found that patients with a specific version of the gene called TT experienced less inflammation and were more likely to survive the infection. Physicians typically treat patients with moderate to severe COVID-19 with corticosteroid drugs that reduce the inflammatory response. However, Amado-Rodríguez, Salgado del Riego et al. found that patients with the TT version of the IFIH1 gene were at greater risk of dying if they received corticosteroids. The team then applied the distribution of IFIH1 variants among different ethnic ancestries to data from a previous clinical trial, and simulated the effects of corticosteroid treatment. This 'mock' clinical trial supported their findings from the patient-derived data, which were also validated by laboratory experiments on immune cells from individuals with the TT gene. The work by Amado-Rodríguez, Salgado del Riego et al. suggests that while corticosteroids benefit some patients, they may cause harm to others. However, a real-world clinical trial is needed to determine whether patients with the TT version of the IFIH1 gene would do better without steroids