Organoids and colorectal cancer

Organoids were first established as a three‐dimensional cell culture system from mouse small intestine. Subsequent development has made organoids a key system to study many human physiological and pathological processes that affect a variety of tissues and organs. In particular, organoids are becoming very useful tools to dissect colorectal cancer (CRC) by allowing the circumvention of classical problems and limitations, such as the impossibility of long‐term culture of normal intestinal epithelial cells and the lack of good animal models for CRC. In this review, we describe the features and current knowledge of intestinal organoids and how they are largely contributing to our better understanding of intestinal cell biology and CRC genetics. Moreover, recent data show that organoids are appropriate systems for antitumoral drug testing and for the personalized treatment of CRC patients.The work in the authors’ laboratory is funded by the Agencia Estatal de Investigación (PID2019‐104867RB‐I00/AEI/10.13039/501100011033), the Ministerio de Ciencia e Innovación (SAF2017‐90604‐REDT/NuRCaMeIn), and the Instituto de Salud Carlos III—Fondo Europeo de Desarrollo Regional (CIBERONC/CB16/12/00273, CIBERONC/CB16/12/00398 and ICI20/00057)

Vitamin D effects on human colon normal and tumour organoids

Trabajo presentado en FEBS Open Bio, celebrado en Lisboa (Portugal) del 09 al 14 de julio de 2022.Many studies indicate an association between vitamin D deficiency and increased colorectal cancer risk and, specially, mortality. Accordingly, the active vitamin D metabolite 1a,25-dihydroxyvitamin D3 (calcitriol) inhibits the proliferation and promotes the differentiation of colon carcinoma cells and of other tumour cell types, and also has antitumour effects in animal models of colon cancer. These results prompted us to analyse the effects of calcitriol on human colon normal and cancer stem cells. To this end, we established a living biobank of patient-derived colon organoids generated from the tumour mass and from the adjacent healthy tissue obtained from surgical biopsies. Organoids are a three-dimensional culture system of normal or cancer stem cells and their progeny with a self-organized multicellular structure. By immunohistochemistry and RNAscope in situ hybridization, we found that vitamin D receptor is expressed in LGR5+ colon stem cells in human tissue and in normal and tumour organoid cultures. RNA-sequencing assays showed that both organoid types respond differentially to calcitriol with profound and contrasting changes in their transcriptomic profiles. This was confirmed in an independent series of patient-derived organoids by RT-qPCR assays. In normal organoids, calcitriol upregulates stemness-related genes and inhibits cell proliferation. In contrast, in tumour organoids calcitriol has little effect on stemnessrelated genes, while it induces differentiation-associated genes, and variably reduces cell proliferation. Concordantly, electron microscopy analyses showed that calcitriol does not affect the blastic cell phenotype in normal organoids, but it induces a series of differentiated features in tumour organoids. These results indicate that calcitriol maintains the undifferentiated phenotype of human normal colon stem cells (homeostatic action), while it promotes the differentiation of colon cancer stem cells (anticancer action).

Sur8, a determinant protein in colorectal cancer tumor progression

Resumen del trabajo presentado en el 43rd Annual Meeting of the SEBBM, celebrado en Barcelona (España) del 19 al 21 de julio de 2021.Colorectal cancer (CRC) has the highest incidence rate in the Spanish population. The most important challenge consists on the discovery of efficient disease treatments, due to high mortality rates in highly developed stages. Sur8 is a scaffold protein that positively modulates ERK signaling pathway, which has a major role in the progression and metastasis in colorectal cancer. The main goals of our research are to determine the role that Sur8 plays in the development and progression of CRC and to analyze its possible therapeutic potential. For this purpose, our group has developed an inducible conditional mouse model msur8f/fVillinCreERT2. In order to determine Sur8 action in the colonic tissue, we have developed organoids from the colon epithelium of healthy mice and have analyzed gene expression pattern by an RNAseq approach. Sur8 KO affects oncogenic CRC transcription factors expression, as well as the modulation of some Wnt pathway regulators. In regard to miRNA data, we have observed deregulation of miRNAs related to CRC in Sur8 KO organoids. To determine the role that Sur8 plays in the development and progression of CRC, we have subjected our inducible conditional mice to chemical carcinogenesis and we have observed that Sur8 KO males display less and smaller tumors and do not present any adenocarcinoma. In addition, we have carried out Sur8 silencing in human CRC cell lines by infection with constitutive shRNA lentiviruses. We have observed that Sur8 silencing produces decreases of cell tumor proliferation, and reduction of p-ERK levels. Finally, we are evaluating the effects of putative therapeutic agents against Sur8 in human CRC cell lines. Concretely, we are testing Celastrol, which has been described that binds and blocks the action of Sur8 in vitro. We have observed that Celastrol treatment diminishes the cell tumor proliferation in this model. Altogether, our results indicate that Sur8 may have a determinant role in CRC progression and that Sur8 could be a potential molecular target for the design of novel strategies against CRC

Clinico-pathological characteristics and outcomes of patients with early-onset colorectal cancer

[Background]: The rising incidence of colorectal cancer (CRC) among young patients is alarming. We aim to characterize the clinico-pathological features and outcomes of patients with early-onset CRC (EOCRC). [Methods]: We included all of the patients with pathologically confirmed diagnosis of CRC at Hospital Universitario La Paz from October 2016 to September 2020. EOCRC age cut-off was 50 years. All statistical analyses were carried out using SPSS v.25. [Results]: A total of 1152 patients were diagnosed with CRC, fifty-nine (5,1%) of them were After a median follow-up of 24 months, 279 patients have died. Median overall survival (OS) was not reached in either group (p = 0,06). Three-year OS was 80% (95%CI: 73-87) and 67 (95%CI: 65-69) in the younger and older group, respectively. In patients with localized disease that underwent surgery or other antineoplastic treatment ( n = 856), 159 events for disease-free survival (DFS) were observed. Median DFS was [Conclusions]: Patients with EOCRC are diagnosed at a more advanced stage and display distinct biological features (more prevalence of dMMR and WT tumors among others). Studies focusing on screening in this population and deeper molecular profiling are needed

Clinico-pathological characteristics and outcomes of patients with early-onset colorectal cancer

[Background]: The rising incidence of colorectal cancer (CRC) among young patients is alarming. We aim to characterize the clinico-pathological features and outcomes of patients with early-onset CRC (EOCRC).[Methods]: We included all of the patients with pathologically confirmed diagnosis of CRC at Hospital Universitario La Paz from October 2016 to September 2020. EOCRC age cut-off was 50 years. All statistical analyses were carried out using SPSS v.25. [Results]: A total of 1152 patients were diagnosed with CRC, fifty-nine (5,1%) of them were After a median follow-up of 24 months, 279 patients have died. Median overall survival (OS) was not reached in either group (p ¼ 0,06). Three-year OS was 80% (95% CI: 73-87) and 67 (95%CI: 65-69) in the younger and older group, respectively. In patients with localized disease that underwent surgery or other antineoplastic treatment ( n ¼ 856), 159 events for disease-free survival (DFS) were observed. Median DFS was not reached in either group (p ¼0,144). Three-year DFS was 86% (95%CI: 79-93) and 73% (95%CI: 71-75, respectively). In patients with metastatic disease (n ¼ 332; synchronous or metachronic), median OS was not reach in the EOCRC group vs 18,1 (95%CI: 13,8-22,4), p ¼ 0,05). In those patients with metastatic EOCRC with mutational status assessed (n ¼23), no difference in OS according to RAS was observed (p ¼ 0,55).[Conclusions]: Patients with EOCRC are diagnosed at a more advanced stage and display distinct biological features (more prevalence of dMMR and WT tumors among others). Studies focusing on screening in this population and deeper molecular profiling are needed.Peer reviewe

Human colon organoids: a 3D system to study stem cell differentiation and vitamin D effects

Trabajo presentado en la Reunión de Jóvenes Investigadores del Programa de Mecanismos de Progresión Tumoral del CIBERONC, celebrada en modalidad virtual el 22 de septiembre de 2021

Establecimiento de un sistema de diferenciación celular en organoides colónicos humanos. Efecto de la vitamina D

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de Lectura: 11-11-2022Esta Tesis tiene embargado el acceso al texto completo hasta el 11-05-2024El epitelio intestinal es uno de los tejidos de más rápida renovación del organismo, consecuencia de la capacidad de proliferación y diferenciación de las células troncales/stem del fondo de las criptas de Lieberkühn a los distintos linajes celulares. Estudios en modelos genéticos de ratón y líneas humanas inmortalizadas han propuesto la participación en estos procesos de proliferación y diferenciación de varios tipos celulares del microambiente (estroma o nicho) y de las rutas de señalización WNT/β-catenina, Notch, BMP, EGF, Hedgehog e Hippo. En esta Tesis se ha puesto a punto un sistema de diferenciación celular en organoides colónicos humanos generados por las células stem de las criptas de tejido sano de pacientes con cáncer colorrectal (CCR). Los estudios realizados empleando medios de cultivo suplementados con agentes o inhibidores han permitido una completa diferenciación a los linajes celulares mayoritarios del epitelio colónico, enterocítico y mucosecretor, que finalmente conduce a la muerte celular. No se ha observado diferenciación al linaje enteroendocrino ni a células Tuft. Tanto los análisis de la expresión de marcadores genéticos mediante RT-qPCR, Western blot e inmunofluorescencia, como los ensayos transcriptómicos globales (RNA-seq) y los estudios ultraestructurales empleando microscopía electrónica han mostrado la importancia de las rutas BMP y Notch, y de la inactivación de la ruta WNT/β-catenina. El tratamiento combinado con BMP4 y DBZ (inhibidor de la ruta Notch) modifica profundamente el perfil de expresión génica de los organoides, de modo distinto según la edad de los pacientes de los que se generaron. Por el contrario, los organoides colónicos tumorales, que son generados por las células cancerosas stem (Cancer Stem Cells) de los CCR, son muy poco sensibles a los medios utilizados, y solo exhiben una mínima respuesta de diferenciación celular. Se ha estudiado también la acción de la vitamina D sobre estos procesos de diferenciación en organoides normales y tumorales. Su metabolito más activo, el calcitriol o 1α,25-dihidroxivitamina D3, reduce en organoides normales la diferenciación celular favoreciendo el mantenimiento del fenotipo indiferenciado de las células stem, lo que se traduce en una mayor viabilidad de los organoides, principalmente en los generados a partir de pacientes de más edad. Asimismo, tanto el número de genes regulados por calcitriol como las funciones en las que están implicados difieren según la edad. En organoides tumorales, por el contrario, y de acuerdo con su propuesta acción antitumoral, el calcitriol induce parcialmente características fenotípicas de diferenciación celula

Vitamin D effects on cell differentiation and stemness in cancer

This article belongs to the Special Issue Stemness and Differentiation in Cancer.Vitamin D3 is the precursor of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), a pleiotropic hormone that is a major regulator of the human genome. 1,25(OH)2D3 modulates the phenotype and physiology of many cell types by controlling the expression of hundreds of genes in a tissue- and cell-specific fashion. Vitamin D deficiency is common among cancer patients and numerous studies have reported that 1,25(OH)2D3 promotes the differentiation of a wide panel of cultured carcinoma cells, frequently associated with a reduction in cell proliferation and survival. A major mechanism of this action is inhibition of the epithelial–mesenchymal transition, which in turn is largely based on antagonism of the Wnt/β-catenin, TGF-β and EGF signaling pathways. In addition, 1,25(OH)2D3 controls the gene expression profile and phenotype of cancer-associated fibroblasts (CAFs), which are important players in the tumorigenic process. Moreover, recent data suggest a regulatory role of 1,25(OH)2D3 in the biology of normal and cancer stem cells (CSCs). Here, we revise the current knowledge of the molecular and genetic basis of the regulation by 1,25(OH)2D3 of the differentiation and stemness of human carcinoma cells, CAFs and CSCs. These effects support a homeostatic non-cytotoxic anticancer action of 1,25(OH)2D3 based on reprogramming of the phenotype of several cell types.The work in the authors’ laboratory is funded by the Agencia Estatal de Investigación (PID2019-104867RB-I00/AEI/10.13039/501100011033), the Agencia Estatal de Investigación-Fondo Europeo de Desarrollo Regional (SAF2016-76377-R, MINECO/AEI/FEDER, EU), the Ministerio de Economía y Competitividad (SAF2017-90604-REDT/NuRCaMeIn), and the Instituto de Salud Carlos III-Fondo Europeo de Desarrollo Regional (CIBERONC; CB16/12/00273).Peer reviewe

Colon cancer stem cells: organoids and target of therapeutic intervention

Trabajo presentado en el XVIII Congreso de la Sociedad Española de Biología Celular, celebrado en Badajoz (España) del 15 al 18 de octubre de 2019.In 2007, Hans Clevers' group identified a stem cell population in the gut that was able to renew the entire intestinal epithelium every few days. These cells expressed LgrS, a plasma membrane protein (Lgrs+ cells). Subsequent research led to demonstrate in mice that cancer stem cells (CSC) are the origin of cancer. Clevers's group also developed a new 3D culture technology to indefinitely grow this population of intestinal Lgrs+ stem cells and their progeny first from mouse intestine and later from human intestine. These cultures give rise to miniaturized complex structures that resemble the organ they derived from and were called organoids or mini-guts. An enormous work done in this field has allowed to obtain organoids from multiple organs (liver, pancreas, bladder, breast, lung, prostate...) of different species. Organoids reflect the genetic background, cellular heterogeneity and the structure and functionality of their tissue of origin, are genetically stable and can be obtained from a simple biopsy from patient-derived healthy and diseased tissues. Organoid models are now becoming a promising tool far medical research, disease modelling, drug development, personalized treatment and regenerative medicine, and constitute an alternative far animal models. Recent studies have demonstrated the presence of Lgrs+ stem cells populations in both tumors and organoids of human and mouse origin. They are considered as both the tumor initiating cells and the cell population responsable far tumor relapse after chemotherapeutic treatments. However, the existence of additional stem cell types in the gut r4 cells, Dlll+s cells) and the large plasticity of intestinal cells have obscured the possible therapeutic value of therapies directed against LGRs+ ese. We have established a living biobank of organoids from healthy and tumor colon tissue of 39 colon cancer patients. By means of global transcriptomic studies (RNA-sequencing and chromatin immunoprecipitation-sequencing), we have analyzed and compared the gene expression profiles of human colon normal and tumor organoids and their response to calcitriol, the active vitamin D metabolite, proposed to protect against colon cancer. Our data indicate that calcitriol contributes to maintain the stemness of normal colon stem cells while it induces cell differentiation in tumor colon organoids (Fernández-Barral et al., FEBS J., 2019). Thus, our results favor a protective effect of vitamin D acting over CSC