Integration of organoids in peptide drug discovery: Rise of the high‐throughput screening

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 high 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 from small molecular screens, we explore the difficulties and potential avenues for creating new platforms to identify peptide agents using organoid models.


INTRODUCTION
Peptides consist of 5-50 amino acids and have a molecular weight between small molecules (500 Da) and antibodies (150 kDa). 1,2Unlike 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.4][5][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. 7For example, peptide-conjugated radionuclides efficiently deliver local radiation to cancer targets and kill tumor cells. 1,8,9n addition, cell-penetrating peptides (CPPs) are potential carriers to send drugs across the blood-brain barrier (BBB) into the brain.1][12] Multiple peptide-based drugs have been approved for clinical practice, highlighting the need for further studies to develop more efficient peptide drugs. 6owever, one of the largest gaps between basic research and clinical application for screening peptide drugs as potential treatments derive from the differences between existing preclinical models and humans. 135][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 selfrenewal. 131][22][23][24][25][26][27][28][29] Importantly, they can be used to study the mechanism of human diseases that are difficult to model in animals. 25,30,31Although human cells can be maintained under 2D conditions, they almost lose the features of native organs due to the lack of a similar microenvironment. 24,32,33The 3D-cultured organoids exhibit near-physiologic cellular composition and behaviors, and maintain genome stability even after long-term expansion. 18][36][37] A wide range of studies has 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. 13,38,39Although organoids have several applications 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 highthroughput peptide-screening platforms (See Table 1).

OVERVIEW OF ORGANOID-BASED DRUG SCREEN PLATFORM
The successful establishment of multicellular organoids simulating the structure and function of native organs has highlighted their remarkable advantages in highthroughput drug screening.At first, studies focused on investigating whether organoids can respond to drugs, and whether the drug response was similar to traditional model systems and humans.Recently, small molecular drugs have been broadly used in organoid-based drug screens.In this part, we will discuss the current progress and conclude the common procedure of organoid-based drug screens based on the study of small molecular drugs.
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 patientderived rectal organoids provided evidence for selecting VX-770 as a treatment for patients carrying rare CFTR mutations. 40Human 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, TA B L E 1 Overview of recent studies applying organoids in peptide research.

Applications
Organoid systems Peptide drugs Characteristics Ref.

RPT NET cell lines-derived spheroids
177 Lu-DOTATATE Evaluation of radiosensitizer drugs 93,97,98   Mouse-derived tumor spheroids 68 Ga-DOTATATE Demonstrate the utility of radiotracers in measuring therapeutic effect of RPT

F I G U R E 1
The generation procedure and application of patient-derived organoids.
suggesting the feasibility of primary liver cancer organoid models in drug screening and basic research. 41In addition, Vlachogiannis et al. reported a living biobank of colorectal and gastroesophageal cancer patient-derived organoids, and 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. 424][45][46][47] Mills et al. described a high-throughput multicellular human cardiac organoid platform and used this system to identify pro-proliferative candidates (a 5000compound library was screened) with minimized side effects on cardiac contractility and rhythm. 48,49Notably, the combination of this platform with contractile assays was the key point for rapidly assessing 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. 50n addition, the organoids were used to screen a library of Food and Drug Administration (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. 36,51-54Yuan et al. established patient-derived gallbladder carcinoma (CBC) organoid lines recapitulating the original in vivo tissues. 55Two 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 accurately investigating the sensitivity of large quantities of compounds.The major challenges in developing anti-cancer drugs include genetic heterogeneity, progressive growth, and metastasis of tumor cells.Patientderived tumor organoids thus are valuable model systems for drug discovery and precision oncology. 16An FDAapproved drug with therapeutic potential was selected after high-throughput drug screening using treatmentresistant and metastatic breast tumor organoids from patients' tissues. 56This 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. 57Tumor 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 tumorspecific CD8 + cytotoxic T lymphocytes was developed to discover potential drugs for improving neoantigen presentation and T-cell-mediated cytotoxicity. 58he prerequisite for establishing an organoid-based drug-screening platform is optimal organoid culture conditions. 13Organoids with high 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 and 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. 53econd, the protocol of high-throughput drug screening suitable for organoids is the core. 36,59Despite differences between the reported protocols, the main procedures are generally similar.The procedures are briefly described below. 56,58,59The steady-state organoids (or dissociated single cells) are seeded in multiwell plates, such as 384well plates and 96-well plates.Chemotherapeutic agents are easily dispensed to the multiwell plates using a drug dispenser.The corresponding software can generate the component distribution layout, including a series of drug candidates with different concentrations, positive control and negative control.After incubation with drugs for several days (depending on the features of the original tissues and compounds), 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 50 , area under the curve (AUC), or growth rate inhibition (GR) metrics, which indicate the effect of drugs, could be measured using the readout.The key point for drug screening is the drug screen layout.A positive control that contains a drug that can kill all the cells is critical.It is also necessary to include a negative control treatment with the relevant vehicle.
Third, 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, therapeutic drugs aim 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.

Peptide-based radiopharmaceutical therapy
1][62][63][64][65][66][67][68][69][70][71] RPT has advantages over existing therapeutic modalities, making it a safe, precise, and effective option to treat many diseases, especially cancers. 72,73Unlike traditional radiotherapy, the radiation administered from radionuclides inside the tumor microenvironments minimizes the injury of normal tissues. 60][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][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 investigated the therapeutic efficacy of combination with radionuclide-conjugated drugs and potential radiosensitizers.For example, Rae et al. investigated whether disulfiram could promote the anti-cancer effect of 131 I-metaiodobenzylguanidine ( 131 I-MIBG) using tumor spheroids derived from human neuroblastoma and glioma cells.The results suggested that disulfiram facilitated the killing function of 131 I-MIBG to noradrenaline transporter-positive tumor spheroids, which could serve as a radiosensitizer. 91ecently, 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). 65,95,96eanwhile, 68 Ga-DOTATATE, 64 Cu-DOTATATE (US), and 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. 60 177 u-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 177 Lu-DOTATATE.They demonstrated a significantly increased cytotoxicity after combined treatment, indicated by the reduced tumor spheroid growth. 97Similarly, 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 177 Lu-DOTATATE. 93The results indicated that the combination of onalespib and 177 Lu-DOTATATE significantly reduced spheroid growth compared to monotherapies.A p53-stabilizing peptide VIP116 was a radiosensitizer to inhibit neuroblastoma growth and enhanced 177 Lu-DOTATATE treatment.These results were also observed in tumor spheroid model systems. 98Radiotracers can also be used to monitor the status of tumor spheroids.Seifert et al. incubated 68 Ga-DOTATATE with mCherry-transgenic mouse pheochromocytoma (MPC mCherry )-derived tumor spheroids to study the morphologic and metabolic characteristics after HIF2α expression. 84The reduced cellular uptake of 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.
Nowadays, radiopharmaceuticals have been considered as a promising therapy for cancer patients.There is a substantial urgency to develop more RPT agents to meet the increasing clinical needs.Therefore, organoid models are suitable for screening large-scale RPT agents in a high-throughput way.However, multiple potential peptide-conjugated radionuclides are undergoing preclinical tests, such as 177 Lu-FAP-2286.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.

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, and 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. 99eoantigens are peptides with 8-10 amino acids produced by the degradation proteasome of intracellular proteins, which are the translational products of mutant oncogenesis. 100These 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 + 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. 101urrently, a wide range of studies focuses on identifying tumor neoantigens, which is critical for developing new treatment modalities for cancers, such as peptide-based tumor vaccines and personalized drugs to kill tumor cells. 102Mass spectrometry (MS)-based immunopeptidomics and computational predictions are the commonly used approaches to identify possible antigenic peptides. 99The latter is based on computergenerated algorithms to identify peptides that could be produced by mutated genes in tumors and are likely to associate with MHC molecules. 103The selected antigenic peptides are synthesized and used to activate T lymphocytes.The activated cytotoxic T lymphocytes (CTL, CD8 + T lymphocytes) are then co-cultured with tumor cells to evaluate the CTL response for neoantigen screens. 104,105wing 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. 106Wang 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. 107This study provides evidence for applying 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. 108,109atient-derived tumor organoids could avoid these issues and achieve personalized immunotherapy in the future.As 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. 110They also indicated that highly conserved antigenic peptides in HLA presentation could 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.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 low-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).Moreover, patient-derived organoids allow us to identify specific neoantigens and serve as personalized medicine platforms.Therefore, we propose that the utility of patient-derived organoids as preclinical models to identify tumor neoantigens high-throughput could be a powerful approach to developing precision therapies for cancer patients.

Cell-penetrating peptides
CPPs can cross tissues and cell surfaces without causing lethal injury to the membranes.However, 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. 111Given the characteristics of CPPs to cross cell membranes, more and more researchers focus on whether they can pass the BBB. 112The 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. 113The 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 Pglycoprotein (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 in 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. 114][117] Microfluidics could introduce blood flow to stimulate BBB more dynamically. 118However, these model systems require advanced equipment to establish the platform, increasing the experimental complexity of performing high-throughput drug screens.
Researchers recently reported 3D multicellular structures through self-organization arrangement of brain ECs, pericytes, and astrocytes. 119The in vitro spheroids can recapitulate the complex interactions between each cell type, which is critical cell behavior to maintain the essential function of BBB.Cho's group modified the method and established BBB organoids that could mimic the essential function of BBB. 120 They, for the first time, investigated whether the BBB organoids are suitable for screening BBBpenetrating drugs.BBB organoids were used to identify several CPPs that could cross the BBB, demonstrating their feasibility and utility as models for cost-effective, highthroughput drug screens.Many peptides are susceptible to being degraded by proteolysis, and show a relatively low ability to cross the BBB.The same group found that peptide macrocyclization could increase the cellular uptake of CPPs and found that one macrocyclic analog of transpotan-10 displays improved capacity to deliver across the BBB organoids. 121ogether these studies demonstrated that 3D 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. 114Furthermore, 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.

Host defense peptides
The primary biological functions of the naturally produced peptides, host defense peptides (HDPs, also known as antimicrobial peptides), are immunomodulatory, antiinflammatory, and anti-bacterial. 122HDP 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. 123,124ncreasing antimicrobial resistance (AMR) of organisms has become a severe issue for treating inflammatory diseases due to the excessive use of antibiotics. 125,126The naturally produced HDPs are promising candidates for developing treatments against the global threat caused by AMR organisms. 126Similarly, there is also an urgent need to establish novel model systems to screen HDPs as potential drug candidates.An organoid system called air-liquid interface (ALI) construct is widely used in investigating HDPs. 126ALI 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. 127Patients and healthy lung ALI models were used to examine the HDP expressions.9][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 Pseudomonas aeruginosa. 131Ritter 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. 132This study also indicated the sensitivity of ALI models compared to submerged cultures.Current studies introducing HDPs to organoid systems use ALI models, which involve complex manipulations and equipment, making them unsuitable for high-throughput screens.We believe that real 3D organoid models (infected with microbes) could avoid these issues and are a potential approach to discovering effective HDPs and their derivations in a high-throughput way.

Other therapeutic peptides
A wide range of therapeutic peptides showed potential effects on various diseases, 5,133 and some of them were investigated using organoid systems.For example, the two Axin-derived staple peptides SAHPA1 and xStAx that target β-catenin were reported to promote the activity of Wnt/β-catenin signaling pathway. 134The 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. 135,136The Frizzled 7 receptors (FZD7) are highly expressed in LGR5 + 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. 137The 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.Despite the therapeutic peptides, peptide-drug conjugates (PDCs) are also promising agents because of the target delivery of drugs to tumors.Therefore, given the advantages of peptides, a large scale of peptide-based drugs needs to be screened for the development of more effective agents for cancer therapy, which could be achieved by high-throughput screens of agents using tumor organoid models.

PERSPECTIVE AND CHALLENGES OF ORGANOID-BASED HIGH-THROUGHPUT PEPTIDE-SCREENING PLATFORMS
Nowadays, there is great progress in the establishment of organoid-based drug screen platforms.However, some challenges still need to be addressed.Patient-derived tumor organoids provide an ideal platform for determining personalized treatment for cancer patients.The success rate of patient-derived organoids needs to be improved.Not all tumor samples can generate healthy organoids, which will hamper the clinical application of organoid models to identify personalized regimens.Moreover, long-term expansion of patient-derived organoids is an important issue to establish an organoid biobank for drug screening and characterization.The proliferation capacity varies from patient to patient and different tumor types.Some organoids cannot maintain more than 1-week expansion in vitro.To this end, studies have optimized the culture method involving sample processing methods and the culture medium to promote the proliferation of tumor cells in organoids.Moreover, organoid heterogeneity is one of the major issues caused by the heterogeneity of tumor cells between each individual and within one tumor tissue.On one hand, patient-derived tumor organoids can be used as a model to determine each patient's personalized treatment.Although multiple cell subclusters exist in tumor tissues, the highly conserved neoantigens can be identified using the single cell-derived clonal organoids. 110Moreover, some patients show drug resistance even when applied with the regimens guided by molecular sequencing.Therefore, the mutation is not the only accurate guidance for determining personalized treatment.Recently, more and more studies have confirmed that patient-derived organoids can accurately predict patients' drug response, as the organoid drug response is consistent with their clinical results.
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.
9][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.This is a promising direction for developing more effective peptide-based drugs. 3Moreover, 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 unsuitable for assessing peptide candidates' binding affinity.Multiple approaches, such as fluorescent-labeled peptides, are used for traditional specific peptide discovery.The future direction is to modify the existing methods and develop appropriate assays that match the high-throughput organoid culture technology to directly assess peptide affinity.In addition, due to the difference in the emission range of radionuclides, [141][142][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. 50][151][152][153][154][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.

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 employing organoids to assess the effects of peptide candidates, exploring the utility and challenges of high-throughput peptide screening 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 advancing organoid technology.Consequently, organoids will be a captivating and novel preclinical model for discovering peptide drugs in the future.

A C K N O W L E D G M E N T S
This study was supported by the CAMS Innovation Fund for Medical Sciences (nos.2022-I2M-1-026-1, 2021-I2M-3-001, and 2022-I2M-2-002-2), the Nonprofit Central Research Institute Fund of Chinese Academy of Medical Sciences (no. 2022-RC350-04), and Beijing Nova Program to Kuan Hu.C O N F L I C T O F I N T E R E S T S TAT E M E N TThe authors declare no conflict of interest.O R C I DKuan Hu https://orcid.org/0000-0003-2448-2254RE F E R E N C E S