Studying colon cancer using intestinal organoid models for anticancer drug and gene functional analysis

Colorectal tumours demonstrate broad genetic and phenotypic heterogeneity in comparison to their matched normal tissue. To shed light into this diversity in depth, an advanced in-vitro 3D model capable of self-renewal, self-organisation and exhibit organ functionality is required. Self-renewal of the small intestine and colonic epithelium is mediated by the proliferation of stem cells and their progenitors in the crypt base. Recently, a long-term and stable 3D culture system maintaining basic crypt physiology known as organoid became available from both normal and matched-tumour tissues of the intestine. Organoids faithfully reproduce functional genetic, pharmacological and morphological alterations exist in their parental tissue. Patient-derived organoids (PDO) can be generated and expanded indefinitely in-vitro from a small biopsy sample of CRC patients (from both tumour and adjacent normal tissue), providing a promising platform for personalised medicine, drug discovery and targeted gene editing research studies. Therefore, in this study, we have initially adapted patient-derived organoids culture from 17 CRC patients in our laboratory and further investigated the phenotypical alteration of both tumour, and normal adjacent derived organoids. Based on our findings, tumour organoids displayed different phenotypical traits from one patient to another, highlighting their distinct molecular subtyping. The growth and morphological alterations of tumour organoids also significantly differed from normal derived organoids in culture. We further evaluated the effect of Noggin, a BMP antagonist on PDO and colon cancer cell lines. Our findings demonstrate that despite the significant role of Noggin in the homeostasis of normal colonic derived organoids, the administration of Noggin does not influence colon cancer cells and PDOs behaviour in culture. Furthermore, organoids have opened a new window toward drug discovery and personalised medicine. To address this, the anticancer therapeutic value of Rifabutin antibiotic, recently screened on our laboratory were also assessed in PDO and colon cancer cell lines. Rifabutin drastically hindered the growth of PDOs and colon cancer cell lines in culture and impeded Wnt/β-catenin signalling mediated cancer cell growth. With CRISPR/Cas9 induced-accurate mutations and 3D organoids cultures, it is now possible to model how individual genes may function in multiple tissue types and without the cost and ethical considerations of in-vivo alternatives. Hence, we aimed to combine the organoid system with genome editing technology, CRISPR/Cas9, to generate knockout organoids for rapid identification of genes with unknown biological function in the intestinal epithelium. Here, we evaluated the role of F-box genes as substrate recognition subunit of Skp, Cullin, F-box containing complex (SCF) E3-ligases in intestinal organoids. Knockout F-box intestinal organoids demonstrated differential phenotypical alterations that were associated with aberrant proliferation and differentiation and led the identification of different F-box genes including Fbxl5, Fbxo31, Fbxl18, Fbxo17 and Fbxl17. In conclusion, we have shown that organoids are invaluable and flexible tools to investigate multiple aspects of cellular events for homeostasis and disease condition and they may represent a significant step forward in reducing and replacing the use of animal models in research

Determinism and Divine Agency According to Allamah Tabatabaei

The way God intervenes in the natural world is considered a major and long-standing issue in the realm of theology and philosophy of religion where efforts to resolve it has led to proposing various and, at times, opposing views, spanning from determinism to delegation. By employing a novel criterion to distinguish between various divine actions in the world, the current paper attempts to present and assess Allamah Tabatabaei’s approach as an Islamic determinist philosopher in regard to the topic and seeks to lay the groundwork for offering solutions in both deterministic and indeterministic models by revisiting the causal homogeneity principle in order to find a way out of dominant intellectual impediments. To that end, the paper serves to show that by extending the concept of causal homogeneity within a deterministic framework, one can attest to the direct interference of immaterial entities in the material world, whereas by resting upon the ‘principle of causal necessity’ and a particular attitude towards the principle of homogeneity within an indeterministic framework, one can defend the interference of immaterial causes in order to realize one of the possible probabilities

Morphological alterations of cultured human colorectal matched tumour and healthy organoids.

Organoids have extensive applications in many fields ranging from modelling human development and disease, personalised medicine, drug screening, etc. Moreover, in the last few years, several studies have evaluated the capacity of organoids as transplantation sources for therapeutic approaches and regenerative medicine. Nevertheless, depending on the origin of the cells and anatomical complications, an organoid transplant may make tissue regeneration difficult. However, some essential aspects of organoids including the morphological alterations and the growth pattern of the matched tumour and their healthy derived organoids have received less attention. Therefore, the current work focused on culturing matched healthy and tumour organoids from the same patient with colorectal cancer (CRC) and assessed their timed growth and structural differences on a daily basis. The healthy organoids underwent proliferation and branching morphogenesis, while the tumour organoids did not follow the same pattern, and the majority of them developed cystic structures instead. However, the number and size of tumour organoids were different from one patient to another. The differential morphological changes of the healthy versus human colonic tumour organoids likely linked to distinct molecular and cellular events during each day. Thus, while their specific structural features provide valuable in vitro models to study various aspects of human intestinal/colon tissue homeostasis and CRC which avoid or replace the use of animals in research, this model may also hold a great promise for the transplantation and regenerative medicine applications

FLYWCH1, a novel suppressor of nuclear b-catenin, regulates migration and morphology in colorectal cancer

© 2018 American Association for Cancer Research. Wnt/b-catenin signaling plays a critical role during development of both normal and malignant colorectal cancer tissues. Phosphorylation of b-catenin protein alters its trafficking and function. Such conventional allosteric regulation usually involves a highly specialized set of molecular interactions, which may specifically turn on a particular cell phenotype. This study identifies a novel transcription modulator with an FLYWCH/Zn-finger DNA-binding domain, called "FLYWCH1." Using a modified yeast-2-hybrid based Ras-Recruitment system, it is demonstrated that FLYWCH1 directly binds to unphosphorylated (nuclear) b-catenin efficiently suppressing the transcriptional activity of Wnt/ b-catenin signaling that cannot be rescued by TCF4. FLYWCH1 rearranges the transcriptional activity of b-catenin/TCF4 to selectively block the expression of specific downstream genes associated with colorectal cancer cell migration and morphology, including ZEB1, EPHA4, and E-cadherin. Accordingly, overexpression of FLYWCH1 reduces cell motility and increases cell attachment. The expression of FLYWCH1 negatively correlates with the expression level of ZEB1 and EPHA4 in normal versus primary and metastatic colorectal cancer tissues in patients. Thus, FLYWCH1 antagonizes b-catenin/TCF4 signaling during cell polarity/migration in colorectal cancer. Implications: This study uncovers a new molecular mechanism by which FLYWCH1 with a possible tumor suppressive role represses b-catenin-induced ZEB1 and increases cadherin-mediated cell attachment preventing colorectal cancer metastasis

Low Level of Microsatellite Instability Correlates with Poor Clinical Prognosis in Stage II Colorectal Cancer Patients

The influence of microsatellite instability (MSI) on the prognosis of colorectal cancer (CRC) requires more investigation. We assessed the role of MSI status in survival of individuals diagnosed with primary colorectal cancer. In this retrospective crosssectional study the MSI status was determined in 158 formalin-fixed paraffin-embedded tumors and their matched normal tissues from patients who underwent curative surgery. Cox proportional hazard modeling was performed to assess the clinical prognostic significance. In this study we found that MSI-H tumors were predominantly located in the colon versus rectum ( = 0.03), associated with poorer differentiation ( = 0.003) and TNM stage II/III of tumors ( = 0.02). In CRC patients with stage II, MSI-L cases showed significantly poorer survival compared with patients who had MSI-H or MSS tumors ( = 0.04). This study indicates that MSI-L tumors correlate with poorer clinical outcome in patients with stage II tumors ( = 0.04) or in tumors located in the colon ( = 0.02). MSI-L characterizes a distinct subgroup of CRC patients who have a poorer outcome. This study suggests that MSI status in CRC, as a clinical prognostic marker, is dependent on other factors, such as tumor stage and location

Studying colon cancer using intestinal organoid models for anticancer drug and gene functional analysis

Colorectal tumours demonstrate broad genetic and phenotypic heterogeneity in comparison to their matched normal tissue. To shed light into this diversity in depth, an advanced in-vitro 3D model capable of self-renewal, self-organisation and exhibit organ functionality is required. Self-renewal of the small intestine and colonic epithelium is mediated by the proliferation of stem cells and their progenitors in the crypt base. Recently, a long-term and stable 3D culture system maintaining basic crypt physiology known as organoid became available from both normal and matched-tumour tissues of the intestine. Organoids faithfully reproduce functional genetic, pharmacological and morphological alterations exist in their parental tissue. Patient-derived organoids (PDO) can be generated and expanded indefinitely in-vitro from a small biopsy sample of CRC patients (from both tumour and adjacent normal tissue), providing a promising platform for personalised medicine, drug discovery and targeted gene editing research studies. Therefore, in this study, we have initially adapted patient-derived organoids culture from 17 CRC patients in our laboratory and further investigated the phenotypical alteration of both tumour, and normal adjacent derived organoids. Based on our findings, tumour organoids displayed different phenotypical traits from one patient to another, highlighting their distinct molecular subtyping. The growth and morphological alterations of tumour organoids also significantly differed from normal derived organoids in culture. We further evaluated the effect of Noggin, a BMP antagonist on PDO and colon cancer cell lines. Our findings demonstrate that despite the significant role of Noggin in the homeostasis of normal colonic derived organoids, the administration of Noggin does not influence colon cancer cells and PDOs behaviour in culture. Furthermore, organoids have opened a new window toward drug discovery and personalised medicine. To address this, the anticancer therapeutic value of Rifabutin antibiotic, recently screened on our laboratory were also assessed in PDO and colon cancer cell lines. Rifabutin drastically hindered the growth of PDOs and colon cancer cell lines in culture and impeded Wnt/β-catenin signalling mediated cancer cell growth. With CRISPR/Cas9 induced-accurate mutations and 3D organoids cultures, it is now possible to model how individual genes may function in multiple tissue types and without the cost and ethical considerations of in-vivo alternatives. Hence, we aimed to combine the organoid system with genome editing technology, CRISPR/Cas9, to generate knockout organoids for rapid identification of genes with unknown biological function in the intestinal epithelium. Here, we evaluated the role of F-box genes as substrate recognition subunit of Skp, Cullin, F-box containing complex (SCF) E3-ligases in intestinal organoids. Knockout F-box intestinal organoids demonstrated differential phenotypical alterations that were associated with aberrant proliferation and differentiation and led the identification of different F-box genes including Fbxl5, Fbxo31, Fbxl18, Fbxo17 and Fbxl17. In conclusion, we have shown that organoids are invaluable and flexible tools to investigate multiple aspects of cellular events for homeostasis and disease condition and they may represent a significant step forward in reducing and replacing the use of animal models in research