Patient-derived tumor organoids: a new avenue for preclinical research and precision medicine in oncology.

Over the past decade, the emergence of patient-derived tumor organoids (PDTOs) has broadened the repertoire of preclinical models and progressively revolutionized three-dimensional cell culture in oncology. PDTO can be grown from patient tumor samples with high efficiency and faithfully recapitulates the histological and molecular characteristics of the original tumor. Therefore, PDTOs can serve as invaluable tools in oncology research, and their translation to clinical practice is exciting for the future of precision medicine in oncology. In this review, we provide an overview of methods for establishing PDTOs and their various applications in cancer research, starting with basic research and ending with the identification of new targets and preclinical validation of new anticancer compounds and precision medicine. Finally, we highlight the challenges associated with the clinical implementation of PDTO, such as its representativeness, success rate, assay speed, and lack of a tumor microenvironment. Technological developments and autologous cocultures of PDTOs and stromal cells are currently ongoing to meet these challenges and optimally exploit the full potential of these models. The use of PDTOs as standard tools in clinical oncology could lead to a new era of precision oncology in the coming decade.

Length limitation of astral microtubules orients cell divisions in murine intestinal crypts.

Planar spindle orientation is critical for epithelial tissue organization and is generally instructed by the long cell-shape axis or cortical polarity domains. We introduced mouse intestinal organoids in order to study spindle orientation in a monolayered mammalian epithelium. Although spindles were planar, mitotic cells remained elongated along the apico-basal (A-B) axis, and polarity complexes were segregated to basal poles, so that spindles oriented in an unconventional manner, orthogonal to both polarity and geometric cues. Using high-resolution 3D imaging, simulations, and cell-shape and cytoskeleton manipulations, we show that planar divisions resulted from a length limitation in astral microtubules (MTs) which precludes them from interacting with basal polarity, and orient spindles from the local geometry of apical domains. Accordingly, lengthening MTs affected spindle planarity, cell positioning, and crypt arrangement. We conclude that MT length regulation may serve as a key mechanism for spindles to sense local cell shapes and tissue forces to preserve mammalian epithelial architecture.

ABSP: an automated R tool to efficiently analyze region-specific CpG methylation from bisulfite sequencing PCR.

MOTIVATION: Nowadays, epigenetic gene regulations are studied in each part of the biology, from embryonic development to diseases such as cancers and neurodegenerative disorders. Currently, to quantify and compare CpG methylation levels of a specific region of interest, the most accessible technique is the bisulfite sequencing PCR (BSP). However, no existing user-friendly tool is able to analyze data from all approaches of BSP. Therefore, the most convenient way to process results from the direct sequencing of PCR products (direct-BSP) is to manually analyze the chromatogram traces, which is a repetitive and prone to error task. RESULTS: Here, we implement a new R-based tool, called ABSP for analysis of bisulfite sequencing PCR, providing a complete analytic process of both direct-BSP and cloning-BSP data. It uses the raw sequencing trace files (.ab1) as input to compute and compare CpG methylation percentages. It is fully automated and includes a user-friendly interface as a built-in R shiny app, quality control steps and generates publication-ready graphics. AVAILABILITY AND IMPLEMENTATION: The ABSP tool and associated data are available on GitHub at https://github.com/ABSP-methylation-tool/ABSP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

[Patient-derived tumor organoids (or tumoroid), a growing preclinical model for oncology]. / Les tumoroïdes, modèles précliniques en plein essor pour l'oncologie.

The recent emergence of tumor organoid cultures, or tumoroids, has enriched the repertoire of preclinical models in oncology. These microtumors are obtained in vitro by including cells from patient tumor samples in an extracellular matrix and cultured in specific media. Very close to the tumor of origin, tumoroids can be amplified fairly rapidly from a small quantity of tissue, established with high success rate for most tumor types, easily genetically engineered, and stored in biobanks. Tumoroids thus offer numerous possibilities in terms of basic research, such as the study of carcinogenesis or mechanisms of chemoresistance, but also the identification of new targets and preclinical validation of new anti-cancer compounds or personalized medicine. Technological developments and enrichment of tumoroids with other cell types are currently ongoing to optimally exploit the full potential of these models.

Title: Les tumoroïdes, modèles précliniques en plein essor pour l'oncologie. Abstract: La récente émergence des cultures d'organoïdes tumoraux, ou tumoroïdes, a permis d'enrichir le répertoire des modèles précliniques en oncologie. Très proches de la tumeur dont elles dérivent, ces microtumeurs offrent de nombreuses possibilités en termes de recherche fondamentale, telles que l'étude de la carcinogenèse ou de la chimioré-sistance, de validation préclinique de nouvelles molécules à visée anticancéreuse, ou encore de personnalisation des traitements. Divers développements techniques et l'enrichissement des tumoroïdes par l'addition d'autres types cellulaires sont actuellement en cours pour améliorer la pertinence de ces modèles et exploiter de façon optimale leur remarquable potentiel.

[Patient-derived tumor organoids (or tumoroid) as valuable precision medicine tools]. / Les organoïdes dérivés de tumeurs (ou tumoroïdes), des outils de choix pour la médecine de précision en oncologie.

Review of literature shows that it is possible to establish tumor-derived organoids, or tumoroids, from almost any type of tumor, and that these "micro-tumors" could be used to develop functional assays allowing the prediction of the patient response to treatments and/or the identification of predictive molecular signatures associated with the development of these therapies. Although it is still essential to optimize culture conditions to promote and accelerate the establishment of tumoroids, or to recapitulate tumor microenvironment, many applications are now possible in the field of prediction of response to treatments and in guiding therapeutic decision-making. Using tumoroids as standard tools in clinical oncology could make precision oncology enter a new era in the coming decade. Numerous ongoing research and clinical trials conducted throughout the world aim to validate the interest of this approach.

Title: Les organoïdes dérivés de tumeurs (ou tumoroïdes), des outils de choix pour la médecine de précision en oncologie. Abstract: Il est désormais possible d'établir des tumoroïdes à partir de presque tout type de tumeur, notamment en vue de la mise en place de tests fonctionnels prédictifs et/ou de l'identification de signatures moléculaires prédictives. Bien que l'optimisation des conditions de culture ou la complexification du micro-environnement des tumoroïdes soit encore nécessaire, de nombreuses applications sont déjà envisageables dans le domaine de la prédiction de la réponse aux traitements et de l'orientation de la décision thérapeutique. Par l'introduction de leur utilisation en clinique, l'oncologie de précision pourrait bien entrer dans une nouvelle ère dans le courant de la décennie à venir.

A new pancreatic adenocarcinoma-derived organoid model of acquired chemoresistance to FOLFIRINOX: First insight of the underlying mechanisms.

BACKGROUND INFORMATION: Although improvements have been made in the management of pancreatic adenocarcinoma (PDAC) during the past 20 years, the prognosis of this deadly disease remains poor with an overall 5-year survival under 10%. Treatment with FOLFIRINOX, a combined regimen of 5-fluorouracil, irinotecan (SN-38) and oxaliplatin, is nonetheless associated with an excellent initial tumour response and its use has allowed numerous patients to go through surgery while their tumour was initially considered unresectable. These discrepancies between initial tumour response and very low long-term survival are the consequences of rapidly acquired chemoresistance and represent a major therapeutic frontier. To our knowledge, a model of resistance to the combined three drugs has never been described due to the difficulty of modelling the FOLFIRINOX protocol both in vitro and in vivo. Patient-derived tumour organoids (PDO) are the missing link that has long been lacking in the wide range of epithelial cancer models between 2D adherent cultures and in vivo xenografts. In this work we sought to set up a model of PDO with resistance to FOLFIRINOX regimen that we could compare to the paired naive PDO. RESULTS: We first extrapolated physiological concentrations of the three drugs using previous pharmacodynamics studies and bi-compartmental elimination models of oxaliplatin and SN-38. We then treated PaTa-1818x naive PDAC organoids with six cycles of 72 h-FOLFIRINOX treatment followed by 96 h interruption. Thereafter, we systematically compared treated organoids to PaTa-1818x naive organoids in terms of growth, proliferation, viability and expression of genes involved in cancer stemness and aggressiveness. CONCLUSIONS: We reproductively obtained resistant organoids FoxR that significantly showed less sensitivity to FOLFORINOX treatment than the PaTa-1818x naive organoids from which they were derived. Our resistant model is representative of the sequential steps of chemoresistance observed in patients in terms of growth arrest (proliferation blockade), residual disease (cell quiescence/dormancy) and relapse. SIGNIFICANCE: To our knowledge, this is the first genuine in vitro model of resistance to the three drugs in combined therapy. This new PDO model will be a great asset for the discovery of acquired chemoresistance mechanisms, knowledge that is mandatory before offering new therapeutic strategies for pancreatic cancer.

H3.3K27M Mutation Controls Cell Growth and Resistance to Therapies in Pediatric Glioma Cell Lines.

High-grade gliomas represent the most lethal class of pediatric tumors, and their resistance to both radio- and chemotherapy is associated with a poor prognosis. Recurrent mutations affecting histone genes drive the tumorigenesis of some pediatric high-grade gliomas, and H3K27M mutations are notably characteristic of a subtype of gliomas called DMG (Diffuse Midline Gliomas). This dominant negative mutation impairs H3K27 trimethylation, leading to profound epigenetic modifications of genes expression. Even though this mutation was described as a driver event in tumorigenesis, its role in tumor cell resistance to treatments has not been deciphered so far. To tackle this issue, we expressed the H3.3K27M mutated histone in three initially H3K27-unmutated pediatric glioma cell lines, Res259, SF188, and KNS42. First, we validated these new H3.3K27M-expressing models at the molecular level and showed that K27M expression is associated with pleiotropic effects on the transcriptomic signature, largely dependent on cell context. We observed that the mutation triggered an increase in cell growth in Res259 and SF188 cells, associated with higher clonogenic capacities. Interestingly, we evidenced that the mutation confers an increased resistance to ionizing radiations in Res259 and KNS42 cells. Moreover, we showed that H3.3K27M mutation impacts the sensitivity of Res259 cells to specific drugs among a library of 80 anticancerous compounds. Altogether, these data highlight that, beyond its tumorigenic role, H3.3K27M mutation is strongly involved in pediatric glioma cells' resistance to therapies, likely through transcriptomic reprogramming.

Analysis of the proximal promoter of the human colon-specific B4GALNT2 (Sda synthase) gene: B4GALNT2 is transcriptionally regulated by ETS1.

BACKGROUND: The Sda antigen and corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in normal colonic mucosa and are down-regulated to a variable degree in colon cancer tissues. Although their expression profile is well studied, little is known about the underlying regulatory mechanisms. METHODS: To clarify the molecular basis of Sda expression in the human gastrointestinal tract, we investigated the transcriptional regulation of the human B4GALNT2 gene. The proximal promoter region was delineated using luciferase assays and essential trans-acting factors were identified through transient overexpression and silencing of several transcription factors. RESULTS: A short cis-regulatory region restricted to the -72 to +12 area upstream of the B4GALNT2 short-type transcript variant contained the essential promoter activity that drives the expression of the human B4GALNT2 regardless of the cell type. We further showed that B4GALNT2 transcriptional activation mostly requires ETS1 and to a lesser extent SP1. CONCLUSIONS: Results presented herein are expected to provide clues to better understand B4GALNT2 regulatory mechanisms.

Mucin expression, epigenetic regulation and patient survival: A toolkit of prognostic biomarkers in epithelial cancers.

Twenty mucin genes have been identified and classified in two groups (encoding secreted and membrane-bound proteins). Secreted mucins participate in mucus formation by assembling a 3-dimensional network via oligomerization, whereas membrane-bound mucins are anchored to the outer membrane mediating extracellular interactions and cell signaling. Both groups have been associated with carcinogenesis progression in epithelial cancers, and are therefore considered as potential therapeutic targets. In the present review, we discuss the link between mucin expression patterns and patient survival and propose mucins as prognosis biomarkers of epithelial cancers (esophagus, gastric, pancreatic, colorectal, lung, breast or ovarian cancers). We also investigate the relationship between mucin expression and overall survival in the TCGA dataset. In particular, epigenetic mechanisms regulating mucin gene expression, such as aberrant DNA methylation and histone modification, are interesting as they are also associated with diagnosis or prognosis significance. Indeed, mucin hypomethylation has been shown to be associated with carcinogenesis progression and was linked to prognosis in colon cancer or pancreatic cancer patients. Finally we describe the relationship between mucin expression and non-coding RNAs that also may serve as biomarkers. Altogether the concomitant knowledge of specific mucin-pattern expression and epigenetic regulation could be translated as biomarkers with a better specificity/sensitivity performance in several epithelial cancers.

Cross-talk between YAP and RAR-RXR Drives Expression of Stemness Genes to Promote 5-FU Resistance and Self-Renewal in Colorectal Cancer Cells.

The mechanisms whereby the Hippo pathway effector YAP regulates cancer cell stemness, plasticity, and chemoresistance are not fully understood. We previously showed that in 5-fluorouracil (5-FU)-resistant colorectal cancer cells, the transcriptional coactivator YAP is differentially regulated at critical transitions connected with reversible quiescence/dormancy to promote metastasis. Here, we found that experimental YAP activation in 5-FU-sensitive and 5-FU-resistant HT29 colorectal cancer cells enhanced nuclear YAP localization and the transcript levels of the retinoic acid (RA) receptors RARα/γ and RAR target genes CYP26A1, ALDH1A3, and LGR5 through RA Response Elements (RARE). In these two cell models, constitutive YAP activation reinforced the expression of the stemness biomarkers and regulators ALDH1A3, LGR5, and OCT4. Conversely, YAP silencing, RAR/RXR inhibition by the pan-RAR antagonist BMS493, and vitamin A depletion downregulated stemness traits and self-renewal. Regarding the mechanisms engaged, proximity-dependent labeling, nuclear YAP pulldown coupled with mass spectrometry, and chromatin immunoprecipitation (ChIP)/re-ChIP experiments revealed: (i) the nuclear colocalization/interaction of YAP with RARγ and RXRs; and (ii) combined genomic co-occupancy of YAP, RARα/γ, and RXRα interactomes at proximal RAREs of LGR5 and ALDH1A3 promoters. Moreover, activation of the YAP/RAR-RXR cross-talk in colorectal cancer cells promoted RAR self-activation loops via vitamin A metabolism, RA, and active RAR ligands generated by ALDH1A3. Together, our data identify YAP as a bona fide RAR-RXR transcriptional coactivator that acts through RARE-activated stemness genes. IMPLICATIONS: Targeting the newly identified YAP/RAR-RXR cross-talk implicated in cancer cell stemness maintenance may lead to multitarget combination therapies for patients with colorectal cancer.

Long non-coding RNAs: the tentacles of chromatin remodeler complexes.

Chromatin remodeler complexes regulate gene transcription, DNA replication and DNA repair by changing both nucleosome position and post-translational modifications. The chromatin remodeler complexes are categorized into four families: the SWI/SNF, INO80/SWR1, ISWI and CHD family. In this review, we describe the subunits of these chromatin remodeler complexes, in particular, the recently identified members of the ISWI family and novelties of the CHD family. Long non-coding (lnc) RNAs regulate gene expression through different epigenetic mechanisms, including interaction with chromatin remodelers. For example, interaction of lncBRM with BRM inhibits the SWI/SNF complex associated with a differentiated phenotype and favors assembly of a stem cell-related SWI/SNF complex. Today, over 50 lncRNAs have been shown to affect chromatin remodeler complexes and we here discuss the mechanisms involved.

Unsupervised Hierarchical Clustering of Pancreatic Adenocarcinoma Dataset from TCGA Defines a Mucin Expression Profile that Impacts Overall Survival.

Mucins are commonly associated with pancreatic ductal adenocarcinoma (PDAC) that is a deadly disease because of the lack of early diagnosis and efficient therapies. There are 22 mucin genes encoding large O-glycoproteins divided into two major subgroups: membrane-bound and secreted mucins. We investigated mucin expression and their impact on patient survival in the PDAC dataset from The Cancer Genome Atlas (PAAD-TCGA). We observed a statistically significant increased messenger RNA (mRNA) relative level of most of the membrane-bound mucins (MUC1/3A/4/12/13/16/17/20), secreted mucins (MUC5AC/5B), and atypical mucins (MUC14/18) compared to normal pancreas. We show that MUC1/4/5B/14/17/20/21 mRNA levels are associated with poorer survival in the high-expression group compared to the low-expression group. Using unsupervised clustering analysis of mucin gene expression patterns, we identified two major clusters of patients. Cluster #1 harbors a higher expression of MUC15 and atypical MUC14/MUC18, whereas cluster #2 is characterized by a global overexpression of membrane-bound mucins (MUC1/4/16/17/20/21). Cluster #2 is associated with shorter overall survival. The patient stratification appears to be independent of usual clinical features (tumor stage, differentiation grade, lymph node invasion) suggesting that the pattern of membrane-bound mucin expression could be a new prognostic marker for PDAC patients.

Galectin-3 modulates epithelial cell adaptation to stress at the ER-mitochondria interface.

Cellular stress response contributes to epithelial defense in adaptation to environment changes. Galectins play a pivotal role in the regulation of this response in malignant cells. However, precise underlying mechanisms are largely unknown. Here we demonstrate that Galectin-3, a pro and anti-apoptotic lectin, is required for setting up a correct cellular response to stress by orchestrating several effects. First, Galectin-3 constitutes a key post-transcriptional regulator of stress-related mRNA regulons coordinating the cell metabolism, the mTORC1 complex or the unfolded protein response (UPR). Moreover, we demonstrated the presence of Galectin-3 with mitochondria-associated membranes (MAM), and its interaction with proteins located at the ER or mitochondrial membranes. There Galectin-3 prevents the activation and recruitment at the mitochondria of the regulator of mitochondria fission DRP-1. Accordingly, loss of Galectin-3 impairs mitochondrial morphology, with more fragmented and round mitochondria, and dynamics both in normal and cancer epithelial cells in basal conditions. Importantly, Galectin-3 deficient cells also display changes of the activity of the mitochondrial respiratory chain complexes, of the mTORC1/S6RP/4EBP1 translation pathway and reactive oxygen species levels. Regarding the ER, Galectin-3 did not modify the activities of the 3 branches of the UPR in basal conditions. However, Galectin-3 favours an adaptative UPR following ER stress induction by Thapsigargin treatment. Altogether, at the ER-mitochondria interface, Galectin-3 coordinates the functioning of the ER and mitochondria, preserves the integrity of mitochondrial network and modulates the ER stress response.

Chemotherapy-induced ileal crypt apoptosis and the ileal microbiome shape immunosurveillance and prognosis of proximal colon cancer.

The prognosis of colon cancer (CC) is dictated by tumor-infiltrating lymphocytes, including follicular helper T (TFH) cells and the efficacy of chemotherapy-induced immune responses. It remains unclear whether gut microbes contribute to the elicitation of TFH cell-driven responses. Here, we show that the ileal microbiota dictates tolerogenic versus immunogenic cell death of ileal intestinal epithelial cells (IECs) and the accumulation of TFH cells in patients with CC and mice. Suppression of IEC apoptosis led to compromised chemotherapy-induced immunosurveillance against CC in mice. Protective immune responses against CC were associated with residence of Bacteroides fragilis and Erysipelotrichaceae in the ileum. In the presence of these commensals, apoptotic ileal IECs elicited PD-1+ TFH cells in an interleukin-1R1- and interleukin-12-dependent manner. The ileal microbiome governed the efficacy of chemotherapy and PD-1 blockade in CC independently of microsatellite instability. These findings demonstrate that immunogenic ileal apoptosis contributes to the prognosis of chemotherapy-treated CC.

Colon cancer stemness as a reversible epigenetic state: Implications for anticancer therapies.

The recent discovery of cancer cell plasticity, i.e. their ability to reprogram into cancer stem cells (CSCs) either naturally or under chemotherapy and/or radiotherapy, has changed, once again, the way we consider cancer treatment. If cancer stemness is a reversible epigenetic state rather than a genetic identity, opportunities will arise for therapeutic strategies that remodel epigenetic landscapes of CSCs. However, the systematic use of DNA methyltransferase and histone deacetylase inhibitors, alone or in combination, in advanced solid tumors including colorectal cancers, regardless of their molecular subtypes, does not seem to be the best strategy. In this review, we first summarize the knowledge researchers have gathered on the epigenetic signatures of CSCs with the difficulty of isolating rare populations of cells. We raise questions about the relevant use of currently available epigenetic inhibitors (epidrugs) while the expression of numerous cancer stem cell markers are often repressed by epigenetic mechanisms. These markers include the three cluster of differentiation CD133, CD44 and CD166 that have been extensively used for the isolation of colon CSCs.and . Finally, we describe current treatment strategies using epidrugs, and we hypothesize that, using correlation tools comparing associations of relevant CSC markers with chromatin modifier expression, we could identify better candidates for epienzyme targeting.

Gemcitabine-induced epithelial-mesenchymal transition-like changes sustain chemoresistance of pancreatic cancer cells of mesenchymal-like phenotype.

Growing body of evidence suggests that epithelial-mesenchymal transition (EMT) is a critical process in tumor progression and chemoresistance in pancreatic cancer (PC). The aim of this study was to analyze the role of EMT-like changes in acquisition of resistance to gemcitabine in pancreatic cells of the mesenchymal or epithelial phenotype. Therefore, chemoresistant BxPC-3, Capan-2, Panc-1, and MiaPaca-2 cells were selected by chronic exposure to increasing concentrations of gemcitabine. We show that gemcitabine-resistant Panc-1 and MiaPaca-2 cells of mesenchymal-like phenotype undergo further EMT-like molecular changes mediated by ERK-ZEB-1 pathway, and that inhibition of ERK1/2 phosphorylation or ZEB-1 expression resulted in a decrease in chemoresistance. Conversely, gemcitabine-resistant BxPC-3 and Capan-2 cells of epithelial-like phenotype did not show such typical EMT-like molecular changes although the expression of the tight junction marker occludin could be found decreased. In pancreatic cancer patients, high ZEB-1 expression was associated with tumor invasion and tumor budding. In addition, tumor budding was essentially observed in patients treated with neoadjuvant chemotherapy. These findings support the notion that gemcitabine treatment induces EMT-like changes that sustain invasion and chemoresistance in PC cells.

Epigenetic Regulation by lncRNAs: An Overview Focused on UCA1 in Colorectal Cancer.

Colorectal cancers have become the second leading cause of cancer-related deaths. In particular, acquired chemoresistance and metastatic lesions occurring in colorectal cancer are a major challenge for chemotherapy treatment. Accumulating evidence shows that long non-coding (lncRNAs) are involved in the initiation, progression, and metastasis of cancer. We here discuss the epigenetic mechanisms through which lncRNAs regulate gene expression in cancer cells. In the second part of this review, we focus on the role of lncRNA Urothelial Cancer Associated 1 (UCA1) to integrate research in different types of cancer in order to decipher its putative function and mechanism of regulation in colorectal cancer cells. UCA1 is highly expressed in cancer cells and mediates transcriptional regulation on an epigenetic level through the interaction with chromatin modifiers, by direct regulation via chromatin looping and/or by sponging the action of a diversity of miRNAs. Furthermore, we discuss the role of UCA1 in the regulation of cell cycle progression and its relation to chemoresistance in colorectal cancer cells.

Comparison of two bone markers with growth evolution in 74 girls with central precocious puberty.

BACKGROUND: The bone markers bone alkaline phosphatase (BAP) and C-terminal telopeptide of type I collagen crosslinks (CTX) are correlated with growth rate during normal puberty. The objective of this study was to evaluate the relationship between the serum concentrations of BAP and CTX and growth evolution in girls with idiopathic central precocious puberty (CPP) to help predict adult height. METHODS: A retrospective single-center study was conducted in 74 girls with CPP for whom a serum sample at initial evaluation was available to retrospectively measure BAP and CTX concentrations; 66.2% of them were untreated. RESULTS: The serum BAP concentrations showed significant positive correlations with height in standard deviations (SDS) at the initial evaluation (n = 62; r = 0.31; p = 0.015) and with the difference between bone and chronological ages (n = 61; r = 0.39; p = 0.002). BAP was also positively correlated with adult height as measured in both cm and SDS in untreated patients (n = 19; r = 0.58; p = 0.009). The serum CTX concentrations showed significant positive correlations with growth rate the year before the initial evaluation as measured in both cm and SDS (n = 65; r = 0.34; p = 0.006). CONCLUSIONS: This study revealed significant correlations of serum BAP and CTX concentrations with growth evolution in girls with CPP. The high positive correlation between serum BAP and adult height in untreated girls suggests that BAP can possibly be used to optimize models of adult height prediction in girls with CPP.