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\begin{document}

\title{Integration of organoids in peptide drug discovery: rise of the
high-throughput screening}



\author[1]{Siqi Zhang}%
\author[1]{Jieting Shen}%
\author[1]{Xingkai Wang}%
\author[1]{Xiaona Sun}%
\author[1]{Yuxuan Wu}%
\author[2]{Ming-Rong Zhang}%
\author[1]{Rui Wang}%
\author[1]{hu kuan}%
\affil[1]{Chinese Academy of Medical Sciences \& Peking Union Medical College Institute of Materia Medica}%
\affil[2]{National Institutes for Quantum and Technology Quantum Medical Science Directorate Institute for Quantum Medical Science}%


\vspace{-1em}



  
  \date{May 15, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Organoids are three-dimensional cell aggregates with near-physiologic
cell behaviors and can undergo long-term expansion in vitro. They are
amenable to high-throughput drug screening processes, which renders them
a viable preclinical model for drug development. The procedure of
organoid-based high-throughput screening has been extensively employed
to discover small molecule drugs, encompassing the steps of generating
organoids, examining efficient drugs in organoid cultures, and data
assessment. Compared to small molecules, peptides are more
straightforward to synthesize, can be modified chemically, and
demonstrate a high degree of target specificity and low cytotoxicity.
Therefore, they have emerged as promising carriers to deliver drugs to
disease-associated targets, and could be efficient therapeutic drugs for
various diseases. To date, organoids have been used to evaluate the
efficacy of certain peptide agents; however, no organoid-based
high-throughput screening of peptide drugs has been reported. Given the
advantages of peptide drugs, there is an urgent need to establish
organoid-based peptide high-throughput screening platforms. In this
review, we discuss the typical approach of screening small-molecular
drugs with the use of organoid cultures, as well as provide an overview
of the studies that have incorporated organoids in peptide research.
Drawing on the knowledge gained from small molecular screens, we explore
the difficulties and potential avenues for creating new platforms to
identify peptide agents using organoid models.%
\end{abstract}%



\sloppy


\textbf{Integration of organoids in peptide drug discovery: rise of the
high-throughput screening}

\textbf{Running title: Organoid-based high-throughput peptide screening}

Siqi Zhang\textsuperscript{a}, Jieting Shen\textsuperscript{a}, Xingkai
Wang\textsuperscript{a}, Xiaona Sun\textsuperscript{a}, Yuxuan
Wu\textsuperscript{a}, Ming-Rong Zhang\textsuperscript{b}, Rui
Wang\textsuperscript{a,*}, Kuan Hu\textsuperscript{a,*}

\textsuperscript{a} State Key Laboratory of Bioactive Substance and
Function of Natural Medicines, Institute of Materia Medica, Chinese
Academy of Medical Sciences and Peking Union Medical College, Beijing,
China

\textsuperscript{b} Department of Advanced Nuclear Medicine Sciences,
Institute of Quantum Medical Science, National Institutes for Quantum
Science and Technology, Chiba, Japan.

\textbf{Corresponding Author}

Kuan Hu, State Key Laboratory of Bioactive Substance and Function of
Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing,
China\textbf{.} Email:

\emph{hukuan@imm.ac.cn.}

Rui Wang, State Key Laboratory of Bioactive Substance and Function of
Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing,
China\textbf{.} Email:\emph{wangrui@lzu.edu.cn}.

\textbf{Acknowledgments}

This study was supported by the CAMS Innovation Fund for Medical
Sciences (nos. 2021-I2M-1-026, 2022-I2M-1-026-1, 02120101, 02130101 and
2022-I2M-2-002), the Nonprofit Central Research Institute Fund of
Chinese Academy of Medical Sciences (no. 2022-RC350-04), and Beijing
Nova Program to K. H.

\textbf{Abstract}

Organoids are three-dimensional cell aggregates with near-physiologic
cell behaviors and can undergo long-term expansion in vitro. They are
amenable to high-throughput drug screening processes, which renders them
a viable preclinical model for drug development. The procedure of
organoid-based high-throughput screening has been extensively employed
to discover small molecule drugs, encompassing the steps of generating
organoids, examining efficient drugs in organoid cultures, and data
assessment. Compared to small molecules, peptides are more
straightforward to synthesize, can be modified chemically, and
demonstrate a high degree of target specificity and low cytotoxicity.
Therefore, they have emerged as promising carriers to deliver drugs to
disease-associated targets, and could be efficient therapeutic drugs for
various diseases. To date, organoids have been used to evaluate the
efficacy of certain peptide agents; however, no organoid-based
high-throughput screening of peptide drugs has been reported. Given the
advantages of peptide drugs, there is an urgent need to establish
organoid-based peptide high-throughput screening platforms. In this
review, we discuss the typical approach of screening small-molecular
drugs with the use of organoid cultures, as well as provide an overview
of the studies that have incorporated organoids in peptide research.
Drawing on the knowledge gained from small molecular screens, we explore
the difficulties and potential avenues for creating new platforms to
identify peptide agents using organoid models.

\textbf{Key words}

organoids, high-throughput screening, peptide-drug conjugates,
therapeutic peptides, peptide-based radiopharmaceuticals

\textbf{Introduction}

Peptides consist of 5-50 amino acids and have a molecular weight between
small molecules (500 Da) and antibodies (5000 Da)\textsuperscript{1, 2}.
Unlike small molecules, they are also not susceptible to degradation in
the human body, and thus display longer tumor retention. Moreover,
peptides can be rationally designed based on the sequence of protein
targets or interaction binding sites, affording them higher specificity
and selectivity than small molecules. Compared to antibodies, the
smaller peptides have a shorter clearance time from healthy organs and
can be easily synthesized or chemically modified \textsuperscript{3-6}.
Therefore, peptides are emerging as efficient treatment modalities for
various diseases. They can be used as carriers for delivery of drugs to
disease-associated targets\textsuperscript{7}. For example,
peptide-conjugated radionuclides are an efficient approach to delivering
local radiation to cancer targets and killing tumor
cells\textsuperscript{1, 8, 9}. Cell-penetrating peptides are potential
carriers to send drugs across the blood-brain barrier into the brain.
Many peptides also show remarkable therapeutic efficacy, which could
also serve as a treatment modality\textsuperscript{10-12}. Multiple
peptide-based drugs have been approved for clinical practice,
highlighting the need for further studies to develop more efficient
peptide drugs \textsuperscript{6}.

However, one of the largest gaps between basic research and clinical
application for screening peptide drugs as potential treatments derives
from the differences between existing preclinical models and
human\textsuperscript{13}. The animal models and two-dimensional (2D)
cell lines cannot fully preserve characteristics of in vivo human cells,
contributing to the high failure rate of tested drugs in phase I--III
clinical trials, resulting in a significant waste of medical
resources\textsuperscript{14-16}. The emergence of organoids provides
new insights into establishing a novel model system. Organoids are
three-dimensional (3D) multicellular constructs primarily generated from
pluripotent stem cells (PSCs) and adult stem cells (ASCs) through
self-organization and self-renewal\textsuperscript{13}. They can
recapitulate the structural and functional features of their in vivo
counterparts\textsuperscript{17-19}. Compared to time-consuming animal
models, organoids are easy to establish and
manipulate\textsuperscript{20-29}. Importantly, they can be used to
study the mechanism of human diseases that are difficult to model in
animals\textsuperscript{25, 30, 31}. Although human cells can be
maintained under two-dimensional conditions, they almost lose the
features of native organs due to the lack of a similar
microenvironment\textsuperscript{24, 32, 33}. The three-dimensional
cultured organoids exhibit near-physiologic cellular composition and
behaviors, and maintain genome stability even after long-term
expansion\textsuperscript{18}. These advantages make organoids suitable
preclinical models for high-throughput drug screening, which help to
exclude a series of resistant drugs in the human body, which thus
provides an effective approach to develop new drugs (Figure
1)\textsuperscript{34-37}.

A wide range of studies have focused on evaluating the feasibility and
utility of organoids in small molecular drug screens, from investigating
drug response to establishing organoid-based high-throughput screening
platforms\textsuperscript{13, 38, 39}. Although there are several
applications of organoids in studying peptide-based drugs, a
high-throughput organoid-based platform for screening peptides has not
been reported. Given the advantages of peptides, we believe the organoid
models will facilitate the development and clinical application of
peptide-conjugated drugs and therapeutic peptides. This review discusses
the advantages of organoids in peptide drug screens and how we learn
from organoid-based small molecular drug screening platforms based on
the feature of peptides. In the first part, the typical studies
involving small molecular drug application in organoid models are
introduced, and an overview of the methodology and theory of these
studies are provided. The second part discusses the recent attempts to
introduce peptide drugs in organoid models and presents the perspective
and challenges of organoid-based high throughput peptide screening
platforms.

\textbf{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
mutations\textsuperscript{40}. 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
research\textsuperscript{41}. 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 \textsuperscript{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 research\textsuperscript{43-47}. 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 rhythm\textsuperscript{48, 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.\textsuperscript{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
drugs\textsuperscript{36, 51-54}. Yuan et al. established
patient-derived gallbladder carcinoma (CBC) organoid lines
recapitulating the original in vivo tissues\textsuperscript{55}. 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 \textsuperscript{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'
tissues\textsuperscript{56}. 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 timeframe\textsuperscript{57}. 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\textsuperscript{+} cytotoxic T
lymphocytes was developed to discover potential drugs for improving
neoantigen presentation, and T-cell mediated
cytotoxicity\textsuperscript{58}.

The prerequisite for establishing an organoid-based drug screen platform
is optimal organoid culture conditions \textsuperscript{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
biology\textsuperscript{53}.

Secondly, the protocol of high-throughput drug screening suitable for
organoids is the core \textsuperscript{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 IC\textsubscript{50}, 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.

\textbf{Applications of organoid technology in peptide drug screening}

\textbf{Peptide-based radiopharmaceutical therapy}

Radiopharmaceutical therapy (RPT) is a novel treatment for cancer
patients, involving the targeted delivery of radionuclides to tumors by
conjugating to small molecules, peptides or
antibodies\textsuperscript{60-71}. RPT has advantages over existing
therapeutic modalities, which makes it a safe, precise and effective
option to treat many kinds of diseases, especially
cancers\textsuperscript{72, 73}. Unlike traditional radiotherapy, the
radiation administered from radionuclides inside the tumor
microenvironments minimizes the injury of normal
tissues\textsuperscript{60}. Importantly, by combining with molecular
imaging, including positron emission tomography (PET) and single-photon
emission computed tomography (SPECT), RPT allows diagnosis of the
whole-body tumor status and effects of therapy in a non-invasive way,
which is rapid and precise\textsuperscript{74-76}. To our knowledge,
organoid models used in RPT were mostly generated from three-dimensional
cultures of tumor cell lines, which are mostly like tumor
spheroids\textsuperscript{77-94}. Due to the limited cell types, the
cell line-derived tumor spheroids cannot present the characteristics of
the corresponding tumors. Despite this, they also display some spatial
features compared to monolayer cultured cell lines. Most of these
studies focused on investigating the therapeutic efficacy on combination
with radionuclide-conjugated drugs and potential radiosensitizers. For
example, Rea et al. investigated whether disulfiram could promote the
anti-cancer effect of \textsuperscript{131}I-metaiodobenzylguanidine
(\textsuperscript{131}I-MIBG) using tumor spheroids derived from human
neuroblastoma and glioma cells. The results suggested that disulfiram
facilitated the killing function of \textsuperscript{131}I-MIBG to
noradrenaline transporter-positive tumor spheroids, which could serve as
a radiosensitizer\textsuperscript{91}.

Recently, \textsuperscript{177}Lu-DOTATATE, a peptide receptor
radionuclide therapy (PRRT), was approved by USA and European Union (EU)
for the treatment of somatostatin-receptor (SSTR)-positive
neuroendocrine tumors (NETs)\textsuperscript{65, 95, 96}.
Meanwhile,\textsuperscript{68}Ga-DOTATATE,
\textsuperscript{64}Cu-DOTATATE (US) and \textsuperscript{68}Ga-DOTATOC
(EU) were also approved as companion diagnostic agents for PET imaging
of tumors in patients with SSTR-positive NETs, enabling the combination
of diagnostic imaging with targeted therapy\textsuperscript{60}.
\textsuperscript{177}Lu-DOTATATE is the only peptide-conjugated
radionuclide applied in tumor spheroid study. Tesson et al. used tumor
spheroids to evaluate the effectiveness of radiosensitizer drugs when
combined with\textsuperscript{177}Lu-DOTATATE, and demonstrated a
significantly increased cytotoxicity after combined treatment, indicated
by the reduced tumor spheroid growth\textsuperscript{97}. Similarly,
Lundsten et al. cultured NET cell line-derived tumor spheroids to
examine whether the heat shock protein 90 (HSP90) inhibitor onalespib
could enhance the efficacy of
\textsuperscript{177}Lu-DOTATATE\textsuperscript{93}. The results
indicated that the combination of onalespib
and\textsuperscript{177}Lu-DOTATATE significantly reduced spheroid
growth compared to monotherapies. A p53-stabilizing peptide VIP116 was
defined as a radiosensitizer to inhibit neuroblastoma growth and
enhanced\textsuperscript{177}Lu-DOTATATE treatment. These results were
also observed in tumor spheroid model systems\textsuperscript{98}.
Radiotracers can also be used to monitor the status of tumor spheroids.
Seifert et al. incubated \textsuperscript{68}Ga-DOTATATE with
mCherry-transgenic mouse pheochromocytoma (MPC\textsuperscript{mCherry})
derived tumor spheroids to study the morphologic and metabolic
characteristics after HIF2\selectlanguage{greek}α \selectlanguage{english}expression\textsuperscript{84}. The reduced
cellular uptake of \textsuperscript{68}Ga-DOTATATE demonstrated the
decreased expression level of SSTR2, suggesting that radiotracers can
also be used to measure therapeutic effects in organoid-based drug
screens.

Multiple potential peptide-conjugated radionuclides are undergoing
preclinical tests, such as \textsuperscript{177}Lu-FAP-2286. Thus, there
is a huge gap in introducing patient-derived organoids to RPT, which may
enable the discovery of more efficient modalities for the treatment of
refractory diseases.

\par\null

\textbf{Tumor neoantigens}

We have mentioned cancer immunotherapy in the section on small molecular
drug screening. This part will overview the detailed mechanism of tumor
neoantigens and cancer immunity, then discuss the application of
organoids in identifying tumor antigenic peptides.

Tumor neoantigens (also known as tumor-specific antigens) are
specifically present on tumor cells but not on normal cells and can be
recognized by T lymphocytes \textsuperscript{99}. Neoantigens are
peptides with eight to ten amino acids produced by the degradation
proteasome of intracellular proteins which are the translational
products of mutant oncogenesis\textsuperscript{100}. These antigenic
peptides are then associated with major histocompatibility complex (MHC)
class I molecules (human leukocytes antigen (HLA)) in the endoplasmic
reticulum and migrate to the cell membrane. Activated
CD8\textsuperscript{+} T lymphocytes recognize and bind to cancer cells
and kill their target cells. This process is a cycle in which the tumor
cell death results in the release of more antigens and improves the
immune response to tumor cells. Notably, tumor neoantigens are captured
by dendritic cells (DCs), then present to T lymphocytes for activating
them. Therefore, the core of cancer immunotherapy is to activate the
killing function of activated T lymphocytes or promote the presentation
of neoantigens\textsuperscript{101}.

Currently, a wide range of studies focuses on identifying tumor
neoantigens, which is critical for the development of new treatment
modalities for cancers, such as peptide-based tumor vaccines and
personalized drugs to kill tumor cells\textsuperscript{102}. Mass
spectrometry (MS)-based immunopeptidomics and computational predictions
are the commonly used approaches to identify possible antigenic
peptides\textsuperscript{99}. The latter is based on computer-generated
algorithms to identify peptides that could be produced by mutated genes
in tumors and are likely to associate with MHC
molecules\textsuperscript{103}. The selected antigenic peptides are
synthesized and used to activate T lymphocytes. The activated cytotoxic
T lymphocyte (CTL, CD8\textsuperscript{+} T lymphocytes) are then
co-cultured with tumor cells to evaluate the CTL response for neoantigen
screens\textsuperscript{104, 105}. Owing to the limited source of
patient-derived tumor tissues, studies have focused on using
patient-derived organoids to find out the mutated oncogenes and
investigate whether the activated CTLs can kill cancer cells. Newey et
al. expanded patient-derived colorectal cancer (CRC) organoids and
demonstrated the feasibility of MS-based immunopeptidomics of CRC
organoids in investigating neoantigen presentation in
vitro\textsuperscript{106}. Wang et al. generated patient-derived
hepatobiliary tumor organoids and found that they preserve most of the
characteristics of their parental tissues, such as genetic features and
neoantigen landscape. They used organoids as preclinical models to
identify the predicted-peptide activated CTLs that exhibited anti-tumor
activity\textsuperscript{107}. This study provides evidence for the
application of tumor organoids as preclinical models for rapid antigenic
peptide validation through a prediction-based approach. Few cancer
patients share the same neoantigens, and more than 99.95\% of
neoantigens are present in only one patient resulting from tumor
heterogeneity\textsuperscript{108, 109}. Patient-derived tumor organoids
could avoid these issues and achieve personalized immunotherapy in the
future. Since tumor heterogeneity is also characterized by different
cell subgroups within the same tumor tissue, Demmers et al. cultured
single cell-derived CRC organoid clones from the same patient and
demonstrated that the HLA class I peptide presentation landscape was
heterogeneous even within one individual\textsuperscript{110}. They also
indicated that highly conserved antigenic peptides in HLA presentation
could be identified using the single-cell derived clonal organoids,
which may be a suitable choice for designing anti-tumor vaccines.

The current studies that used organoids to perform tumor antigenic
peptide screens are 1ow-throughput. Patient-derived organoids enable
high-throughput screening of a large number of computationally-predicted
peptides. Furthermore, organoids can undergo extensive expansion,
allowing large quantities of material for MS-based immunopeptidomics
analysis (Figure 2). Therefore, we propose that the utility of
patient-derived organoids as preclinical models to high-throughput
identify tumor neoantigens could be a powerful approach to the
development of precision therapies for cancer patients.

\textbf{Cell-penetrating peptides}

Cell-penetrating peptides (CPPs) can cross tissues and cell surfaces
without causing lethal injury to the membranes. The mechanism of the
penetration process remains controversial. Most CPPs are endogenously
produced proteins and peptides, including heparin-binding proteins,
DNA-binding proteins, antimicrobial peptides and viral
peptides\textsuperscript{111}. Given the characteristics of CPPs to
cross cell membranes, more and more researchers focus on whether they
can pass the blood-brain barrier (BBB)\textsuperscript{112}. The BBB is
a complex microvasculature system mainly consisting of brain endothelial
cells (ECs) that are tightly lined in the cerebral vascular lumens. The
main function of BBB is to protect the brain. In addition to ECs,
astrocytic glia and neurons together are organized into well-structured
neurovascular units\textsuperscript{113}. The brain ECs express high
levels of tight junction proteins, efflux pumps, and specific
transporters. The tight junctions between ECs prevent molecules in the
blood from entering the central nervous system. The efflux pumps,
including P-glycoprotein (PgP), exclude foreign substances from the
brain. Specific transporters deliver essential nutrients to the brain,
such as glucose and amino acids. Therefore, BBB is the main obstacle to
delivering drugs to neural cells and developing effective treatments for
central nervous system diseases. Although CPPs are promising vectors to
deliver drugs across the BBB, no CPP-based treatment is currently used
clinical practice, partly due to the lack of suitable preclinical models
that can accurately mimic the features of BBB to discover potential CPPs
in a high throughput way\textsuperscript{114}. The widely used in vitro
models for BBB are co-culturing of brain ECs (top), astrocytes and
pericytes (bottom) in a transwell system\textsuperscript{115-117}.
Microfluidics could introduce blood flow to stimulate BBB more
dynamically\textsuperscript{118}. However, these model systems require
advanced equipment to establish the platform, increasing the
experimental complexity of performing high-throughput drug screens.

Researchers recently reported three-dimensional multicellular structures
through self-organization arrangement of brain ECs, pericytes and
astrocytes\textsuperscript{119}. The in vitro spheroids can recapitulate
the complex interactions between each cell type, which is critical cell
behavior to maintain the essential function of BBB. Lawler's group
modified the method and established BBB organoids that could mimic the
essential function of BBB\textsuperscript{120}. They, for the first
time, investigate whether the BBB organoids are suitable for screening
BBB-penetrating drugs. BBB organoids were used to identify several CPPs
that could cross the BBB, demonstrating their feasibility and utility as
models for cost-effective and high-throughput drug screens. Many
peptides are susceptible to being degraded by proteolytic, and show a
relatively low ability to cross the BBB. The same group found that
peptide macrocyclization could increase the cell uptake of CPPs and
found that one macrocyclic analog of transpotan-10 displays improved
capacity to deliver across the BBB organoids\textsuperscript{121}.

Together these studies demonstrated that three-dimensional multicellular
BBB organoids can capitulate the complex interactions and arrangements
of each cell type, and can reduce experimental complexity (Figure 2).
These advantages make BBB organoids ideal preclinical models for
high-throughput screening of CPPs that can cross
BBB\textsuperscript{114}. Furthermore, CPPs are also promising carriers
to transfer therapeutic drugs into tumor cells. Therefore, it is
necessary to establish tumor organoids that are amenable to the
discovery of efficient CPPs with high stability, internalization ability
and specificity.

\textbf{Host defense peptides}

The primary biological functions of the naturally-produced peptides,
host defense peptides (HDPs, also known as antimicrobial peptides), are
immunomodulatory, anti-inflammatory, and
anti-bacterial\textsuperscript{122}. HDP are peptides with 12--50 amino
acids composed of cationic and hydrophobic amino acids that adopt an
amphipathic conformation upon folding, usually after contact with
membranes\textsuperscript{123, 124}. Increasing antimicrobial resistance
(AMR) organisms has become a severe issue for the treatment of
inflammatory diseases due to the excessive use of
antibiotics\textsuperscript{125, 126}. The naturally-produced HDPs are
promising candidates for developing treatments against the global threat
caused by AMR organisms\textsuperscript{126}. Similarly, there is also
an urgent need to establish novel model systems to screen HDPs as
potential drug candidates. Currently, an organoid system called
air-liquid interface (ALI) construct are widely used in investigating
HDPs\textsuperscript{126}. ALI systems are comprised of a porous filter
separating the apical and basolateral compartments. Cells cultured on
the top chambers grow to multilayers and across the basal-apical
threshold, where the medium remains on the bottom of the cell layers and
the apical interface is surrounded by air. This system is ideal for
studying the biology of tissues interacting with liquid and air in
vivo\textsuperscript{127}. Patients and healthy lung ALI models were
used to examine the HDP expressions. The results demonstrated that they
are suitable models to study the essential function of HDPs in
respiratory diseases and enable the identification of drug
candidates\textsuperscript{128-130}. One study using primary ALI models
reported that the frog skin-derived HDPs Esc (1-21) and its synthetic
derivation both could protect the epithelial integrity when infected by
P. aeruginosa\textsuperscript{131}. Ritter et al. used ALI cultures with
an aerosol delivery system to predict acute local lung toxicity through
the assessment of various combinations of HDPs and
nanocarriers\textsuperscript{132}. This study also indicated the
sensitivity of ALI models compared to submerged cultures.

Current studies introducing HDPs to organoid systems are all using ALI
models, which involve complex manipulations and equipment, making them
unsuitable for high-throughput screens. We believe that real
three-dimensional organoid models (infected with microbes) are a
potential approach to discovering effective HDPs and their derivations
in a high-throughput way.

\textbf{Other therapeutic peptides}

A wide range of therapeutic peptides showed potential effects on various
diseases \textsuperscript{5, 133}, and some of them were investigated
using organoid systems. For example, the two Axin-derived staple
peptides SAHPA1 and xStAx that target \selectlanguage{greek}β-\selectlanguage{english}catenin were reported to promote
the activity of Wnt/\selectlanguage{greek}β-\selectlanguage{english}catenin signaling pathway\textsuperscript{134}.
The investigation of patient-derived tumor organoids showed that xStAx
binds to the VHL ligand to promote intestinal tumor death, highlighting
its potential as a novel anti-cancer drug.

Leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5)
positive intestinal adult stem cells are widely used to generate
intestinal organoids. The culture technique is mature, and the derived
organoids can undergo long-term expansion without changing the genetic
features\textsuperscript{135, 136}. The Frizzled 7 receptors (FZD7) are
highly expressed in LGR5\textsuperscript{+} intestinal stem cells and
are critical in regulating self-renewal. The development of targeted
drugs that bind to FZD7 is a potential approach to investigating FZD7
functions in cancer biology and developing novel regenerative therapy
for intestinal epithelium. Nile et al. identified a potent peptide
(dFz7-21) that specifically targets FZD7 and changes the conformation of
the FZD domain\textsuperscript{137}. The treatment of LGR5+ mice
intestinal stem cells derived organoids with FZD7 suppressed stem cell
function and disrupted the bud formation by impairing Wnt signaling,
proving the utility and feasibility of organoids as preclinical models.

\textbf{Perspective and challenges of organoid-based high-throughput
peptide screening platforms}

According to the methods of organoid-based small molecule drug screening
platforms, we believe that organoids can also be applied in the
high-throughput screening of peptides. This section presents the
perspective and challenges of applying organoids in peptide screens
based on their characteristics.

Owing to their remarkable binding affinity and specificity, peptides are
suitable for transporting cytotoxic drugs to disease
lesions\textsuperscript{138-140}. However, there is still a wide gap
between research and clinical application of peptide drugs. For example,
the discovery of organ-specific targeted peptides may further decrease
the side effect of this treatment modality. Given that peptides can be
rapidly synthesized and easily modified, researchers can generate
peptide libraries and perform high-throughput screening of the optimal
candidates using organoid models, which is a promising direction for
developing more effective peptide-based drugs\textsuperscript{3}.
Moreover, for peptides that deliver drugs in vivo, although the
interactions are present on the cell surface, they may not alter the
cell phenotype and cause a therapeutical effect. In RPT, the ``cold''
radionuclides without radioactivity are often used to label the peptide
and prescreen the peptides with high binding affinity to reduce the
experimental cost. The commonly used method, CellTiter-Glo assay,
determines drug efficacy by measuring cell viability, which is not
suitable for assessing the binding affinity of peptide candidates.
Multiple approaches are used for traditional specific peptide discovery,
such as using fluorescent labeled peptides. The future direction is to
modify the existing methods and develop appropriate assays that match
the high-throughput organoid culture technology, for direct assessment
of peptide affinity. In addition, due to the difference in the emission
range of radionuclides\textsuperscript{141-143}, the
peptide-radionuclide conjugates not bound to the cell surface but
located near the cells should be investigated to determine whether they
can kill the targeted cells, which may cause false-positive results
during screening.

Peptides hold high biological and chemical diversity; and most targeting
peptides exhibit therapeutic effects on diseases\textsuperscript{5}. For
example, the expression of immune checkpoint proteins (PD-1 and PD-L1)
can be suppressed by targeted peptides for increasing the immune
response to kill tumor cells \textsuperscript{144-148}. However, the
high diversity of nature product peptides poses a challenge to the
identification of the above-mentioned tumor neoantigens and bioactive
peptides derived from venoms, which are produced by many kinds of
organisms, such as plants, snakes, spiders and insects, and are found to
interact with tumors and modulate the proliferation, migration, immune
response of tumor cells, suggesting potential therapeutic agents for
cancers\textsuperscript{149-155}. Therapeutic peptides are promising
candidates for treating various diseases, and more effective peptide
drugs are required to address clinical demands. Thus, high-throughput
screening of therapeutic peptides using organoid technology may
significantly improve the efficiency for identifying and uncovering the
optimal peptides with high binding affinity and ideal therapeutic
effects. Furthermore, although peptides are low-cytotoxic drugs compared
to small molecules and antibodies, healthy organoid models can also be
used to examine their side effects on normal cells, which is one of the
advantages of organoid technology.

\textbf{Conclusion}

Owing to the straightforward synthesis and chemical modification, high
target specificity, low toxicity, and broad range of targets, peptides
have become a viable option for treating various diseases. This review
provides an overview of the studies that employ organoids to assess the
effects of peptide candidates, exploring the utility and challenges of
high-throughput screening of peptide using organoids in light of the
experiences from the organoid-based small molecule screens that have
been extensively examined in recent years. Despite the limitations and
difficulties in developing an organoid-based peptide high-throughput
platform, various techniques can be employed to overcome these issues,
including the advancement of organoid technology. Consequently,
organoids will be a captivating and novel preclinical model for
discovering peptide drugs in the future.

\textbf{Conflict of Interest}

All authors declare no conflict of interest.

Figure 1. The generation procedure and application of patient-derived
organoids.

Figure 2. The application of patient-derived organoids in
high-throughput screening of peptide conjugated drugs and therapeutic
drugs. PDO, patient-derived organoids; CPP, Cell-penetrating peptides.

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Table 1. Overview of recent studies applying organoids in peptide
research.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Applications & Organoid systems & Peptide drugs & Characteristics &
Ref.\tabularnewline
\midrule
\endhead
RPT & NET cell lines derived spheroids &
\textsuperscript{177}Lu-DOTATATE & Evaluation of radiosensitizer drugs.
& \textsuperscript{93, 97, 98}\tabularnewline
& mouse derived tumor spheroids & \textsuperscript{68}Ga-DOTATATE &
Demonstrate the utility of radiotracers in measure therapeutic effect of
RPT. & \textsuperscript{84}\tabularnewline
Tumor neoantigens & Patient-derived CRC organoids & -- & Demonstrate
that the organoids are feasible for investigating neoantigen
presentation in vitro. & \textsuperscript{106}\tabularnewline
& Patient-derived hepatobiliary tumor organoids & -- & Identify the
predicted antigenic peptides that can activate T lymphocytes using
organoids. & \textsuperscript{107}\tabularnewline
& Patient-derived single cell CRC organoids & -- & Demonstrate that the
HLA class I peptide presentation landscape was heterogeneous even within
one individual. & \textsuperscript{110}\tabularnewline
Cell-penetrating peptides & Human primary cell derived multicellular BBB
organoids & SynB1, L-2, PreS2-TLM Transportan 10, SAP, SAP(E), SVM3,
SVM4, Bip(1), Bip(2), (PPR)3, (PPR)5, DPV1047, DPV15, HIV-1 Tat,
Penetratin, Engrailed-2, HoxA-13, Polyarginine & Demonstrate the
feasibility of BBB organoid models for cost-effective and
high-throughput CPP screening. & \textsuperscript{114,
120}\tabularnewline
& Human primary cell derived multicellular BBB organoids &
Perfluoroarene-based, macrocyclic analogues of transportan-10 &
Demonstrate that abiotic peptide macrocycles exhibit increased capacity
for penetrating to the brain using BBB organoids. &
\textsuperscript{121}\tabularnewline
Host defense peptides & Patients and healthy lung ALI organoids & -- &
Demonstrate the utility of ALI organoids to study the function of HDPs
in respiratory diseases. & \textsuperscript{128-130}\tabularnewline
& Human primary cell derived lung ALI organoids & Esc (1-21) &
Demonstrate strong potential of Esc (1-21) in protecting the P.
aeruginosa infection using ALI organoids. &
\textsuperscript{131}\tabularnewline
& Human alveolar lung cell line derived ALI organoids & AA139, M33 &
Demonstrate that ALI organoids are promising models to predict acute
lung toxicity through assessment of various combinations of HDPs and
nanocarriers. & \textsuperscript{132}\tabularnewline
Other therapeutic peptides & Patient-derived CRC organoids & staple
peptides SAHPA1 and xStAx & Demonstrate the potential of these peptides
as a novel anti-cancer drug using organoids. &
\textsuperscript{134}\tabularnewline
& Adult mice intestinal epithelium derived organoids & dFz7-21 (a
peptide specifically binding to FZD7) & Demonstrate that dFz7-21 inhibit
stem cell function in intestinal organoids. &
\textsuperscript{137}\tabularnewline
\bottomrule
\end{longtable}

Abbreviations: RPT, Radiopharmaceutical therapy; CRC, Colorectal cancer;
HLA, human leukocytes antigen; BBB, blood-brain barrier; CPP,
Cell-penetrating peptides; ALI, air-liquid interface; HDP, Host defense
peptides; FZD, Frizzled.\selectlanguage{english}
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