High-content, targeted RNA-seq screening in organoids for drug discovery in colorectal cancer

Organoids allow the recapitulation of intestinal homeostasis and cancerogenesis in vitro; however, RNA sequencing (RNA-seq)-based methods for drug screens are missing. We develop targeted organoid sequencing (TORNADO-seq), a high-throughput, high-content drug discovery platform that uses targeted RNA-seq to monitor the expression of large gene signatures for the detailed evaluation of cellular phenotypes in organoids. TORNADO-seq is a fast, highly reproducible time- and cost-effective ($5 per sample) method that can probe cell mixtures and their differentiation state in the intestinal system. We apply this method to isolate drugs that enrich for differentiated cell phenotypes and show that these drugs are highly efficacious against cancer compared to wild-type organoids. Furthermore, TORNADO-seq facilitates in-depth insight into the mode of action of these drugs. Our technology can easily be adapted to many other systems and will allow for more systematic, large-scale, and quantitative approaches to study the biology of complex cellular systems.

Intestinal stem cells differentiation and its application in cancer therapy

Maxim Norkin

Colorectal cancer (CRC) is one of the deadliest cancers worldwide. Despite extensive research in the field, new therapies targeting CRC are in urgent need. CRC is a highly heterogeneous disease with high levels of intra- and inter-patient heterogeneity. Cancer stem cells are the driving force of tumor development and metastasis. One of the approaches to treat CRC is differentiation therapy thereby induction of cancer stem cell differentiation may lead to tumor regression. Intestinal normal and cancer organoids represent an ideal model for studying the differentiation process ex vivo. Gene expression profiling of drug-treated organoids can be used for quantification of the differentiation process with high precision. RNA-seq based drug-screening platforms in organoids are not yet established. Here we developed a high-throughput high-content targeted RNA-seq-based platform (TORNADO-seq) for monitoring gene expression of large gene signatures in intestinal organoids which allowed us to quantify and evaluate complex cellular phenotypes and cellular alterations upon drug treatment. We applied TORNADO-seq in drug screening in wt and cancer intestinal organoids. We found many differentiation-inducing drugs in wt organoids, including some drugs specifically enriching for certain cell phenotypes, e.g. enteroendocrine cells. Further, we applied identified differentiation-inducing drugs to cancer organoids (APClof;KRASG12D;TP53lof) and discovered that the majority of those differentiation-inducing drugs also targeted cancer organoids. Differentiation induction seems to be a common mode of action of cancer-targeting drugs. Based on gene expression profiles of treated cancer organoids, we were able to propose and confirm the mechanism of action of several drugs indicating that cancer cells may to be sensitive to alterations in the cholesterol biosynthesis pathway. In general, utilization of TORNADO-seq allows predicting connections between cellular phenotypes and signaling pathways in organoids which can be used for hypothesis generation in a variety of biological systems. The second part of the thesis is devoted to the investigation of mechanisms underlying the accumulation of genetic alterations in CRC tumors. It is not known whether cancer driver genes affect the rate of mutation acquisition. Here we performed exome sequencing of single cell-derived organoids obtained from tumors with either APClof, APClof;KRASG12D or APClof;KRASG12D;TP53lof mutation phenotypes that reflect early stages of CRC development. We discovered that presence of KRAS and P53 mutations didn't affect the number of single nucleotide substitutions (SNSs) in tumors, while the latter was increased in APC mutated tumors compared to wt cells and was highly correlated with the tumor age. P53 inactivation was highly correlated with the presence of large-scale (>10 Mb) copy number alterations (CNAs).

EPFL2021

The Bcl9-beta-catenin interface as a potential therapeutic target for colorectal cancer

Andreas Eduard Moor

Aberrantly active Wnt signaling is the hallmark driver of colorectal cancer (CRC). Bcl9/9l proteins are components of the main transcriptional activation complex of canonical Wnt signaling and interact with beta-catenin via a highly conserved homology domain (HD2). Intestinal loss of Bcl9 affects wound healing and Lgr5+-cancer stem cell (CSC) maintenance, but does not perturb normal tissue homeostasis. The aim of the present thesis was to validate the Bcl9-Î²-catenin interaction as a potential target for modulating Wnt signaling and abolishing intestinal stemness traits in colorectal cancer. We investigated the role of the Bcl9-beta-catenin interaction in the mouse AOM-DSS chemical carcinogenesis CRC model. Subsequently, we validated our observations in an independent transgenic APC-Kras CRC model (truncated APC tumor suppressor and mutated Kras expressed in the intestine). The expression of Bcl9 proteins was abolished exclusively in the gastrointestinal epithelium using constitutive or inducible Cre-recombinase expressed under the control of the Villin promoter. To corroborate that the loss of Bcl9 phenotype was due to the abrogation of the Bcl9-beta-catenin interaction and not to a Î²-catenin-independent function of Bcl9, we made use of an additional Bcl9 mutant mouse strain in which only the HD2 of both Bcl9 proteins was deleted, selectively preventing the Bcl9-beta-catenin interaction. The inducible deletion of Bcl9 in established AOM-DSS tumors led to a loss in stem cell, Wnt and EMT gene expression signatures, recapitulating the phenotype that we previously described when Bcl9 was deleted constitutively. A 14d partial decrease of Bcl9 proved sufficient to suppress the expression of mesenchymal marker Vimentin in a cell autonomous fashion. Importantly, AOM-DSS tumors with Bcl9 proteins selectively lacking the HD2 beta-catenin interaction domain displayed the same phenotype as observed in tumors that entirely lacked Bcl9 proteins. Bcl9-knockout tumors induced in the Wnt active APC-Kras tumor model were compared with their wild-type counterparts. Wild-type APC-Kras mutant tumors displayed fewer mesenchymal traits when compared to AOM-DSS induced tumors. Global gene expression analysis by RNA sequencing confirmed a strong positive correlation between Bcl9/9l-dependent transcriptional changes in both independent tumor models. Genes differentially expressed between wild-type and Bcl9/9l ablated tumors were used to probe CRC databases for correlation with patient outcome and revealed a strong association with vastly improved disease-free and overall survival, independent of clinical and molecular parameters. We have shown that preventing the interaction of Bcl9 with beta-catenin in established tumors in Wnt-activated CRC models rapidly resulted in the loss of stemness and mesenchymal traits and concomitant induction of a more differentiated phenotype. Importantly, these loss-of-Bcl9 traits are prognostic for favorable outcome in CRC patients and we have not observed any overt anomalies upon intestinal ablation of Bcl9 in adult mice. Overall, our observations indicate that targeting the Bcl9-beta-catenin interaction in Wnt-activated tumors might be well tolerated and a generally applicable pharmacological strategy to target CSCs to potentially prevent tumor recurrence and improve progression free survival.

EPFL2014

Bioprinting the Cancer Microenvironment

Margaux Catherine Marie Duchamp

Cancer is intrinsically complex, comprising both heterogeneous cellular compositions and microenvironmental cues. During the various stages of cancer initiation, development, and metastasis, cell-cell interactions (involving vascular and immune cells besides cancerous cells) as well as cell-extracellular matrix (ECM) interactions (e.g., alteration in stiffness and composition of the surrounding matrix) play major roles. Conventional cancer models both two- and three-dimensional (2D and 3D) present numerous limitations as they lack good vascularization and cannot mimic the complexity of tumors, thereby restricting their use as biomimetic models for applications such as drug screening and fundamental cancer biology studies. Bioprinting as an emerging biofabrication platform enables the creation of high-resolution 3D structures and has been extensively used in the past decade to model multiple organs and diseases. More recently, this versatile technique has further found its application in studying cancer genesis, growth, metastasis, and drug responses through creation of accurate models that recreate the complexity of the cancer microenvironment. In this review we will focus first on cancer biology and limitations with current cancer models. We then detail the current bioprinting strategies including the selection of bioinks for capturing the properties of the tumor matrices, after which we discuss bioprinting of vascular structures that are critical toward construction of complex 3D cancer organoids. We finally conclude with current literature on bioprinted cancer models and propose future perspectives.