Predicting tumour resistance to paclitaxel and carboplatin utilising genome-wide screening in haploid human embryonic stem cells.

Taxanes and platinum molecules, specifically paclitaxel and carboplatin, are widely used anticancer drugs that induce cell death and serve as first-line chemotherapy for various cancer types. Despite the efficient effect of both drugs on cancer cell proliferation, many tumours have innate resistance against paclitaxel and carboplatin, which leads to inefficient treatment and poor survival rates. Haploid human embryonic stem cells (hESCs) are a novel and robust platform for genetic screening.

Targeted inhibition of Wnt signaling with a Clostridioides difficile toxin B fragment suppresses breast cancer tumor growth.

Wnt signaling pathways are transmitted via 10 homologous frizzled receptors (FZD1-10) in humans. Reagents broadly inhibiting Wnt signaling pathways reduce growth and metastasis of many tumors, but their therapeutic development has been hampered by the side effect. Inhibitors targeting specific Wnt-FZD pair(s) enriched in cancer cells may reduce side effect, but the therapeutic effect of narrow-spectrum Wnt-FZD inhibitors remains to be established in vivo.

Genome-wide screen for anticancer drug resistance in haploid human embryonic stem cells.

Anticancer drugs are at the frontline of cancer therapy. However, innate resistance to these drugs occurs in one-third to one-half of patients, exposing them to the side effects of these drugs with no meaningful benefit. To identify the genes and pathways that confer resistance to such therapies, we performed a genome-wide screen in haploid human embryonic stem cells (hESCs).

Uncovering the mode of action of engineered T cells in patient cancer organoids.

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity.

Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids.

Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive).

Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsies.

Microscopy analysis of tumour samples is commonly performed on fixed, thinly sectioned and protein-labelled tissues. However, these examinations do not reveal the intricate three-dimensional structures of tumours, nor enable the detection of aberrant transcripts. Here, we report a method, which we name DIIFCO (for diagnosing in situ immunofluorescence-labelled cleared oncosamples), for the multimodal volumetric imaging of RNAs and proteins in intact tumour volumes and organoids.

Patient-Derived Ovarian Cancer Organoids Mimic Clinical Response and Exhibit Heterogeneous Inter- and Intrapatient Drug Responses.

There remains an unmet need for preclinical models to enable personalized therapy for ovarian cancer (OC) patients. Here we evaluate the capacity of patient-derived organoids (PDOs) to predict clinical drug response and functional consequences of tumor heterogeneity. We included 36 whole-genome-characterized PDOs from 23 OC patients with known clinical histories.

Assessing the origin of high-grade serous ovarian cancer using CRISPR-modification of mouse organoids.

High-grade serous ovarian cancer (HG-SOC)-often referred to as a "silent killer"-is the most lethal gynecological malignancy. The fallopian tube (murine oviduct) and ovarian surface epithelium (OSE) are considered the main candidate tissues of origin of this cancer. However, the relative contribution of each tissue to HG-SOC is not yet clear.

An organoid platform for ovarian cancer captures intra- and interpatient heterogeneity.

Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids.

Expanding the boundaries of embryonic stem cells.

The boundaries of embryonic stem cell (ESC) research have extended considerably in recent years in several important ways. Alongside a deeper understanding of the pluripotent state, ESCs have been successfully integrated into various fields, such as genomics, epigenetics, and disease modeling. Significant progress in cell fate control has pushed directed differentiation and tissue engineering further than ever before and promoted clinical trials.

Targeting pancreatic progenitor cells in human embryonic stem cell differentiation for the identification of novel cell surface markers.

New sources of beta cells are needed in order to develop cell therapies for patients with diabetes. An alternative to forced expansion of post-mitotic beta cells is the induction of differentiation of stem-cell derived progenitor cells that have a natural self-expansion capacity into insulin-producing cells. In order to learn more about these progenitor cells at different stages along the differentiation process in which they become progressively more committed to the final beta cell fate, we took the approach of identifying, isolating and characterizing stage specific progenitor cells.

Stepwise differentiation of human embryonic stem cells into early endoderm derivatives and their molecular characterization.

Human embryonic stem cells have the potential to differentiate into all human cell types and therefore hold a great therapeutic promise. Differentiation into the embryonic endoderm and its derivatives is of special interest since it can provide a cure for severe widespread clinical conditions such as diabetes and hepatic failure. In this work we established a unique experimental outline that enables the study of early human endoderm development and can help improve and create new differentiation protocols.

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage.

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups.

Characterization of gastrulation-stage progenitor cells and their inhibitory crosstalk in human embryoid bodies.

Human embryoid bodies (HEBs) are cell aggregates that are produced during the course of embryonic stem cell differentiation in suspension. Mature HEBs have been shown to contain derivatives of the three embryonic germ layers. In this study, using a combination of laser capture microscopy followed by DNA microarray analysis and cell sorting, we demonstrate that early HEBs are composed of three major cell populations.

Manipulation of the human genome in human embryonic stem cells.

Human embryonic stem cells (HESCs) have a tremendous clinical and scientific importance since they may serve as a cell source for transplantation and as a system for the study of human development and disease. The genetic engineering of HESCs has become instrumental in achieving these goals. Here we discuss various methodologies to genetically manipulate HESCs and propose a variety of applications of the modified cells in basic and applied research.