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.