A colorectal cancer classification system that associates cellular phenotype and responses to therapy

Colorectal cancer (CRC) is a major cause of cancer mortality. Whereas some patients respond well to therapy, others do not, and thus more precise, individualized treatment strategies are needed. To that end, we analyzed gene expression profiles from 1,290 CRC tumors using consensus-based unsupervised clustering. The resultant clusters were then associated with therapeutic response data to the epidermal growth factor receptor–targeted drug cetuximab in 80 patients. The results of these studies define six clinically relevant CRC subtypes. Each subtype shares similarities to distinct cell types within the normal colon crypt and shows differing degrees of 'stemness' and Wnt signaling. Subtype-specific gene signatures are proposed to identify these subtypes. Three subtypes have markedly better disease-free survival (DFS) after surgical resection, suggesting these patients might be spared from the adverse effects of chemotherapy when they have localized disease. One of these three subtypes, identified by filamin A expression, does not respond to cetuximab but may respond to cMET receptor tyrosine kinase inhibitors in the metastatic setting. Two other subtypes, with poor and intermediate DFS, associate with improved response to the chemotherapy regimen FOLFIRI1 in adjuvant or metastatic settings. Development of clinically deployable assays for these subtypes and of subtype-specific therapies may contribute to more effective management of this challenging disease.

Defining new criteria for selection of cell-based intestinal models using publicly available databases

Michel Aguet, Pascale Anderle Pedone, Mauro Delorenzi, Thierry Sengstag

Background: The criteria for choosing relevant cell lines among a vast panel of available intestinal-derived lines exhibiting a wide range of functional properties are still ill-defined. The objective of this study was, therefore, to establish objective criteria for choosing relevant cell lines to assess their appropriateness as tumor models as well as for drug absorption studies. Results: We made use of publicly available expression signatures and cell based functional assays to delineate differences between various intestinal colon carcinoma cell lines and normal intestinal epithelium. We have compared a panel of intestinal cell lines with patient-derived normal and tumor epithelium and classified them according to traits relating to oncogenic pathway activity, epithelial-mesenchymal transition (EMT) and stemness, migratory properties, proliferative activity, transporter expression profiles and chemosensitivity. For example, SW480 represent an EMT-high, migratory phenotype and scored highest in terms of signatures associated to worse overall survival and higher risk of recurrence based on patient derived databases. On the other hand, differentiated HT29 and T84 cells showed gene expression patterns closest to tumor bulk derived cells. Regarding drug absorption, we confirmed that differentiated Caco-2 cells are the model of choice for active uptake studies in the small intestine. Regarding chemosensitivity we were unable to confirm a recently proposed association of chemo-resistance with EMT traits. However, a novel signature was identified through mining of NCI60 GI50 values that allowed to rank the panel of intestinal cell lines according to their drug responsiveness to commonly used chemotherapeutics. Conclusions: This study presents a straightforward strategy to exploit publicly available gene expression data to guide the choice of cell-based models. While this approach does not overcome the major limitations of such models, introducing a rank order of selected features may allow selecting model cell lines that are more adapted and pertinent to the addressed biological question.

BioMed Central2012

The Cell Differentiation Status Influences the Outcome of Notch-Induced Malignancy

Caroline Poisson

The Notch signaling cascade is a well conserved pathway that regulates many aspects of embryonic development and plays a crucial role in the differentiation process and tissue homeostasis in multiple organs of adult species. Several human pathological disorders, including cancer, arise as a consequence of the deregulation of the Notch signaling pathway. Notch deregulation, up to date, has been best characterized in acute T-cell lymphoblastic leukemia (T-ALL). Indeed, in T-ALL pediatric patients activating Notch1 mutations have been identified in more than 50% of the cases. Increased knowledge of the mechanisms underlying T-ALL development and maintenance is clearly needed as one out of 5 patients with T-ALL has a very poor prognosis. Moreover, the high toxicity of chemotherapeutic regimens has been associated with elevated risks of developing severe secondary health problems. In the present study, we characterized the role of Notch in T-ALL induction and maintenance. Using transgenic mouse models, we could demonstrate that the oncogenic potential of Notch is dependent on RBP-Jκ transcriptional activity. Indeed, the canonical Notch signaling pathway, mediated by the RBP-Jκ transcription factor, is essential for both Notch-mediated T-ALL induction and maintenance, as well as for the induction of a Notch-mediated lymphoproliferative disorder. Moreover, constitutive overexpression of Notch1 at discrete developmental stages within the hematopoietic system, led us to identify a correlation between the differentiation status of a cell, within the hierarchy of the hematopoietic tree, and its sensitivity to Notch1-induced transformation. We demonstrated that forced Notch1 expression in hematopoietic stem cells was not sufficient to induce T-ALL. Nevertheless, aberrant Notch1 expression in the bone marrow resulted in the development of a lethal lymphoproliferative disorder. Overexpression of Notch1 in thmus at the DN2 to DN3 stage of T-cell delvelopment generated an aggressive transplantable T-ALL with important metastatic potential. This suggested that progenitors already committed to the T-cell lineage are highly susceptible to Notch-mediated transformation, which is relevant to the human disease. Nevertheless, forced Notch1 expression after the β-selection checkpoint in the thymys, was no longer sufficient to induce transformation of late DN3 to DN4 thymocytes. We therefore established a murine genetic system that allowed us to dissect the mechanisms of aberrant Notch1 signaling in a lymphoproliferative disorder and a true T-ALL model. We were able to assess the genetic signature that is involved in the transplantability and metastatic potential of leukemic cells and generated a list of candidate genes implicated in these processes. In addition, we correlated genes identified in our murine models with genes associated as well in human T-ALL profiles to identify genes implicated in T-ALL pathogenesis in both mouse and human.

EPFL2011

Towards elucidating the structural and cellular determinants of a-synuclein seeding and toxicity

Somanath Jagannath

Although it has been nearly two and half decades since the discovery of alpha-synuclein (aSyn) as the major component of Lewy bodies (LBs), our understanding of the involvement of different aSyn species, their seeding, spreading and toxicity in Parkinson's disease (PD) is limited. One of the major causes underlying this knowledge gap is the unavailability of adequate tools, techniques, and model systems to capture, monitor, and evaluate the role of different aSyn species in the disease process. In addition, our understanding of cell-type specific contributions to disease pathogenesis is limited. In this thesis, we systematically address the current knowledge gaps and elucidate the structural and cellular determinants of aSyn seeding and toxicity using cellular and animal model systems of PD. In the first chapter of this thesis, we describe an antibody characterization and validation pipeline to assess the specificity of antibodies to well-characterized preparations of various aSyn species, including monomers, fibrils, and different type of oligomers. Using an array of techniques, we demonstrate that that: i) none of the antibodies tested are specific for one particular type of aSyn species, including monomers, oligomers or fibrils; ii) all antibodies that were reported to be oligomer-specific also recognized fibrillar aSyn; and iii) a few antibodies (e.g., the antibody clone 26F1, 5G4) showed high specificity for oligomers and fibrils but did not bind to monomers. These findings suggest that most aSyn aggregate-specific antibodies cannot be used to differentiate between oligomers and fibrils, thus highlighting the importance of exercising caution when interpreting results obtained using these antibodies. In the second chapter of this thesis, we systematically addressed the current knowledge gaps on the role of aSyn oligomers in the initiation, seeding and pathology spreading. Towards this goal, we used different biophysical approaches to investigate aSyn seeding in vitro and in neurons and animal models of aSyn seeding and pathology spreading. we observed that oligomeric forms do not form in the PFF-based neuronal seeding model and we report that different types of oligomers do not seed pathology in vitro and primary hippocampal neurons, and their presence slows rather than accelerates aSyn fibrillization. Altogether, our work points to fibrils as the most seeding competent species and suggests that they are the critical mediators of pathology spreading in PD and other synucleinopathies.In the third chapter of this thesis, we used single-nuclei RNA-sequencing to investigate transcriptional changes in different cell types of the amygdala brain region, which is highly susceptible to developing pS129 positive aggregates in the PD brains. We identified that most of the top 100 differentially expressed genes (DEGs) are of neuronal rather than non-neuronal cell types. In addition, we report that the gene expression profile of a handful of PD risk genes such as Sncaip, Park7, Maob, Hspa8, Scl2a3 and Hsf3 and some pathways that have been associated with PD are differentially altered in specific cell types. Our study highlights the involvement of unique gene expression changes and their associated pathways in different cell types in driving the disease process at an early stage in a PFF-based mouse model of PD.

EPFL2022