Organoid-based small molecular drug screen platform
The successful establishment of multicellular organoids simulating the structure and function of native organs has highlighted their remarkable advantages in high-throughput drug screening. At first, studies were focused on investigating whether organoids are capable of responding to drugs, and whether the drug response was similar to traditional model systems and humans. Dekkers et al. used cystic fibrosis (CF) patient-derived rectal organoids expressing different cystic fibrosis transmembrane conductance regulator (CFTR) mutations to investigate their responses to two drugs, VX-770 and VX-809. The drug responses in the organoids matched with the clinical trial data, and the data from patient-derived rectal organoids provided evidence for selecting VX-770 as a treatment for patients carrying rare CFTR mutations40. Human primary liver cancer organoids were established and shown to preserve the histological architecture, gene expression pattern and native tumorigenesis of their parental tumors. Using the organoid cultures, ERK (extracellular regulated protein kinases) inhibitor was identified as a potential drug to treat primary liver cancer, suggesting the feasibility of primary liver cancer organoid models in drug screening and basic research41. In addition, Georgios et al. reported a living biobank of colorectal and gastroesophageal cancer patient-derived organoids, the drug response of these organoid models was matched to the tumor genotypes. They also compared the drug responses in organoids and organoid-derived tumor xenograft models with patients in clinical trials, demonstrating the viability of organoid models for modeling patient responses in clinical trials 42.
As progress in organoid culture technology has been made, establishing an organoid-based high-throughput drug screening platform has become the focus of organoid research43-47. Mills et al. described a high-throughput multicellular human cardiac organoid platform and used this system to identify pro-proliferative candidates (a 5000-compound library was screened) with minimized side effects on cardiac contractility and rhythm48, 49. Notably, the combination of this platform with contractile assays was the key point for the rapid assessment of the drug response in organoids. Human pluripotent stem cell-derived lung organoids and colonic organoids were used to establish infected models of COVID-19, confirming the applicability of both organoids in SARS-CoV-2 infection research.50. In addition, the organoids were used to screen a library of FDA-approved drug candidates. Several drugs were found to inhibit SARS-CoV-2 entry in organoids specifically. This study was reported in 2020, when the COVID-19 pandemic was just starting to spread worldwide, which thus further highlights the advantages of human organoid model systems for effective and rapid drug discovery, especially for severe acute infectious diseases.
Patient-derived tumor organoid-based high-throughput screening platforms are also widely used for discovering anti-cancer drugs36, 51-54. Yuan et al. established patient-derived gallbladder carcinoma (CBC) organoid lines recapitulating the original in vivo tissues55. Two effective anti-tumor compounds that suppress CBC organoids growth were identified by screening a panel of compounds targeting CBC-specific signaling pathways. The immunohistochemistry results from patients and healthy individuals suggested the therapeutic value of these anti-tumor drugs. This study proves that patient-derived organoids are amenable to investigating the sensitivity of a large quantities of compounds accurately. The major challenges in developing anti-cancer drugs include genetic heterogeneity, progressive growth, and metastasis of tumor cells. Patient-derived tumor organoids thus are valuable model systems for drug discovery and precision oncology 16. A Food and Drug Administration (FDA)-approved drug with therapeutic potential was selected after high-throughput drug screening using treatment-resistant and metastatic breast tumor organoids from patients’ tissues56. This study indicated the feasibility of patient-derived tumor organoid model systems to uncover treatment drugs for cancer patients with different tumor phenotypes, including rare ones. Similarly, Toshimitsu et al. reported a robust drug screening platform applicable to a wide range of patient-derived colorectal organoids. They used suspension culture with agitation, allowing for the efficient expansion of organoids, which substantially facilitates the implementation of fast, personalized, tumor-type-agnostic drug testing in a clinically relevant timeframe57. Tumor microenvironment plays an essential role in modulating tumor progressive growth and metastasis. Patient-derived tumor organoids can also be used to screen drugs through interaction with critical factors in microenvironments, such as immune cells. Tumor immunology has become a crucial aspect of targeted cancer therapy, which mainly relies on the activation and killing function of cytotoxic T cells. A high throughput drug screening platform based on the co-culture of patient-derived tumor organoids with tumor-specific CD8+ cytotoxic T lymphocytes was developed to discover potential drugs for improving neoantigen presentation, and T-cell mediated cytotoxicity58.
The prerequisite for establishing an organoid-based drug screen platform is optimal organoid culture conditions 13. Organoids with a high degree of cell proliferation in vitro and without excessive cell death are suitable for living biobanking and high-throughput screens. Moreover, the organoids preserve the gene expression profile, genomic stability and histopathology features of their original parent tumors could serve as potential preclinical models for drug discovery. In addition, the fewest supplements in culture medium for maintenance of organoids were recommended to avoid alterations in tumor biology53.
Secondly, the protocol of high-throughput drug screening suitable for organoids is the core 36, 59. Despite there are differences between the reported protocols, the main procedures are generally similar. The procedures are briefly described below. The steady-state organoids (or dissociated single cells) are seeded in multiwell plates, such as 384-well plates and 96-well plates. Chemotherapeutic agents are easily dispensed to the multiwell plates using a drug dispenser. The component distribution layout including a series of drug candidates with different concentrations, positive control and negative control can be generated using the corresponding software. After incubation with drugs for several days (depending on the features of the original tissues and drugs), a CellTiter-Glo assay is used to detect cell viability as indicated by intracellular ATP levels. The luminescence signal from each well represents the cell viability readout. The IC50, area under the curve (AUC) or growth rate inhibition (GR) metrics, which indicated the effect of drugs, could be measured using the readout.
Thirdly, despite cell viability, the corresponding function analysis of organoids after treatment with drug candidates is also critical to select effective therapies. Whether additional functional analysis is needed and how to perform the experiments depend on the biology of the disease. For cancers, the aim of therapeutic drugs is to kill the tumor cells, thus, cell viability is usually sufficient to assess the drug effects. However, additional assays are necessary for diseases for which the treatment aims to alter the cell behaviors, such as other kinds of readout reflecting the cell function or custom-designed luciferase reporter systems indicating the interaction of downstream pathways that are activated by drug application.