The promises and challenges of patient‐derived tumor organoids in drug development and precision oncology

Abstract In the era of precision medicine, cancer researchers and oncologists are eagerly searching for more realistic, cost effective, and timely tumor models to aid drug development and precision oncology. Tumor models that can faithfully recapitulate the histological and molecular characteristics of various human tumors will be extremely valuable in increasing the successful rate of oncology drug development and discovering the most efficacious treatment regimen for cancer patients. Two‐dimensional (2D) cultured cancer cell lines, genetically engineered mouse tumor (GEMT) models, and patient‐derived tumor xenograft (PDTX) models have been widely used to investigate the biology of various types of cancers and test the efficacy of oncology drug candidates. However, due to either the failure to faithfully recapitulate the complexity of patient tumors in the case of 2D cultured cancer cells, or high cost and untimely for drug screening and testing in the case of GEMT and PDTX, new tumor models are urgently needed. The recently developed patient‐derived tumor organoids (PDTO) offer great potentials in uncovering novel biology of cancer development, accelerating the discovery of oncology drugs, and individualizing the treatment of cancers. In this review, we will summarize the recent progress in utilizing PDTO for oncology drug discovery. In addition, we will discuss the potentials and limitations of the current PDTO tumor models.


| Cancer cell lines as tumor models
Ever since the establishment of the HeLa cell line, which was derived from an African-American woman with cervical adenocarcinoma and cultured in the 1950s, 1 2D cultured cancer cell lines have been instrumental in basic cancer research as well as the development of oncology drugs. However, there are certain limitations of cancer cell lines that must be taken into consideration for both basic cancer research as well as drug discovery. 2 First, cancer cell lines lack the heterogeneity of primary tumors ( Figure 1). 3 One possible explanation is that only a few types of cells are able to survive the long-term in vitro 2D culture conditions; therefore, the survived cells are relatively homogeneous in nature. 4 Second, in vitro 2D culture condition may induce certain genetic alterations, most of which may

| Patient-derived tumor xenograft models
Patient-derived tumor xenografts (PDTX) can be generated by implanting surgically removed tumors from patients directly into immunodeficient mice. In this model, tumors can be either implanted orthotopically, that is, in the anatomic location of the parental tumor, or heterotopically, that is, in a location unrelated to that of the parental tumor. 8 This method has certain advantages over the aforementioned cancer cell lines and GEMT. First, the tumor cells can be passaged without the in vitro culture step, thus avoiding the in vitro culture-induced genetic changes and clonal selection. 9 Second, the tumors can be implanted alongside their stroma, which may more faithfully mimic the microenvironment of the parental tumors. 10 While extremely beneficial, the time to develop the PDX model can be long, sometimes taking up to 8 months to develop a single model. 3 When considering personalized therapeutics, many patients do not have the luxury of such an extended period of time. Lastly, the cost for the development of the PDTX models can be high because immunodeficient mice that the PDTX model requires are very expensive. 5

| In vitro organoids as disease models
Despite the advances that have been made using in vitro cultured cancer cell lines, GEMT, and PDTX, the need remains for more accurate, timely, and less resource-intensive cancer models. Patientderived tumor organoids (PDTO) may be well suited to fit this need.
An organoid is often described as an in vitro generated 3D cellular structure that architecturally and functionally mimics a particular organ/tissue. 11,12 It can be defined by a few key characteristics, including: (a) self-organization from stem cells/organ progenitor cells to resemble the 3D in vivo structure; (b) composing of multiple organ-specific cell types; (c) recapitulating at least some functions of the organ. While the formal use of organoids in research is relatively recent, it is an extension of the continuous efforts to create more accurate representations of in vivo biological processes by modifying in vitro culture conditions. 13 For example, in 1987, mammary epithelial cells were grown on reconstituted basement membranes instead of on plastic. This new method greatly enhanced the cells' in vivo-like morphology and functionality in producing milk proteins. 14 Despite some advancement, those models are not yet complete enough to be considered in vitro grown organs. Nonetheless, the use of organoids in research has been formalized recently, and research conducted as recently as 2009 investigated organoid development from murine intestinal stem cells. 15 Sato et al sought to create a culture system, which maintains the physiology of specific in vivo structures within the gut. The system involved stimulating the Lgr5 + intestinal cells with relevant gut growth factors in a 3D environment. Such factors included R-spondin-1, which enhances Wnt signaling, EGF, noggin, BMP inhibitor and 3D-Matrigel, which is an artificial laminin-rich extracellular matrix. The organoids maintained the architecture similar to small intestine, that is, structures that resembled villi and crypts.
The genome of these organoids remained remarkably stable over time and through serial passaging, as determined by whole-genome sequencing of both early and late passaged organoids.
Based on these successes, organoid usage has expanded and this method has been further developed to model various human diseases, in an effort to better understand their molecular mechanisms and to develop therapeutics. One example of this is the use of organoids as models for cystic fibrosis (CF). Dekkers et al 16 16 The CF organoids have also allowed investigators to measure levels of CFTR functionality to develop patient-specific therapies. Patient-derived organoids have also been utilized as models for a variety of other genetic disorders, including two liver disorders, alpha 1-antitrypsin deficiency (A1ATD) and Alagile syndrome. 18 In both instances, the histological and mutational characteristics of the organoids closely mirrored those of the patients, rendering this an effective model for elucidating specific mechanistic details and developing novel treatment modalities.
Another important application of patient-derived organoids, and the primary focus of this review, is in their utility within oncology research and drug discovery. Furthering the advances made in growing organoids from healthy intestinal tissue and from patients with various genetic disorders, researchers were also able to successfully establish organoids from patients with many different types of cancer, that is, the PDTOs. We have thoroughly searched the literature using the key words "organoids", "drugs", and "drug testing". The utility of PDTOs in modeling various types of cancers and in serving as drug screening tools will be the focus of this review. We will discuss the utility of PDTOs in drug testing in the following malignancies:

| Breast carcinoma-derived tumor organoids
Breast cancer (BC) is the most common malignancy among females in the United States, and the second leading cause of cancer-related mortality in this cohort. 33

| Pancreatic adenocarcinoma-derived tumor organoids
Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers in the US. 33 Patients with pancreatic cancer have very short mean survival times, partly due to the fact that many patients present without symptoms, or may only present with symptoms at a very late stage. Therefore, methods that allow for the rapid determination of individualized therapies are crucial in improving the survival rate of these patients. showed concordance with high purity; however, most primary tumor specimens had insufficient purity to reveal copy number alterations.
The PDAC organoids were able to recapitulate patient response to the most commonly used chemotherapeutic agents in this cohort.
Of the six patients who had progression-free survival longer than the mean survival time, five of them were treated with at least one drug to which the matched PDAC organoids were also particularly sensitive. Of the three patients who rapidly progressed, two were treated with a chemotherapeutic agent to which their PDAC organoids were markedly resistant.
The PDAC organoids were also able to reflect the temporal evo-

| Gastric adenocarcinoma-derived tumor organoids
There are two main types of gastric adenocarcinoma: the intestinal subtype, typically associated with Helicobacter pylori infections, and the diffuse subtype. Due to late presentation and vague symptoms, gastric adenocarcinoma is often diagnosed at late stages, and the survival rates are typically low. 34 As a result, timely and effective models for gastric adenocarcinoma are urgently needed.
Gao et al 23

| Metastatic gastrointestinal carcinoma-derived tumor organoids
Colorectal carcinoma (CRC) is the third most common malignancy in the United States among both females and males, and is also the third most common cause of malignancy-associated deaths. 33 The most common site for CRC to metastasize to is the liver, and liver metastases are a poor prognostic factor. 35   To test the stability of the EAC organoids, 4 of the 10 organoids were propagated and subjected to whole-genome sequencing at multiple passages over a 6-month period. The organoid genomes were relatively stable, showing less than 25% increase in the total number of mutations, none of which influenced the various cancer drivers.

| Esophageal adenocarcinoma-derived tumor organoids
Because of the relative stability of the EAC organoids, drug testing was performed using these organoids to standard EAC chemotherapy agents: 5-FU, epirubicin, and cisplatin. Six of the eight organoids tested were resistant to chemotherapy, which was consistent with the patients' poor response in the clinic.

| Urothelial carcinoma-derived tumor organoids
Bladder cancer is the fourth most prevalent cancer in men in the United States; however, it is relatively understudied. 33 The most common form of bladder cancer is urothelial carcinoma, which can be further subcategorized into nonmuscle-invasive and muscle-invasive carcinomas. While nonmuscle invasive bladder cancer has a relatively favorable prognosis, muscle-invasive bladder cancer generally has poor survival rates. Muscle-invasive bladder cancer can be further categorized into a luminal-like subtype, and a more aggressive subtype, with basal-like features. 38  Subsequently, 83% of these organoids were orthotopically implanted into immunodeficient mice as bladder cancer xenografts. showed that indeed the drug response of the xenograft largely recapitulates that of the cultured organoid.
Finally, the authors examined the drug response of the organoid lines established from the same patient at different times of their disease progression. The drug sensitivity of the organoids seems to track the drug sensitivity of the patient in the clinic, suggesting that the organoids could potentially be used for selecting the best treatment regimen for patients in the clinic.

| Endometrial adenocarcinoma-derived tumor organoids
The most common form of uterine cancer is adenocarcinoma of the endometrium, or the innermost lining of the uterus. The majority of cases of endometrial carcinoma are due to prolonged and high levels of estrogen exposure, and are considered endometrioid in morphology. 39 Girda et al 30  Despite the utility of these findings, the authors did not conduct genomic sequencing to confirm the mutational profile of the organoids in comparison to their parental tumors. Future studies should examine the genetic makeup of these models, and investigate their utility in targeted therapy research. In addition, the organoids were analyzed at their first passage only, and this may limit their ability to recapitulate the tumor heterogeneity that results from multiple passages. Finally, patient data would have been helpful to consider alongside the aforementioned results, to determine if the limited results seen with progestins were also seen in the clinical setting.

| Mesothelioma-derived tumor organoids
Mesothelioma is a malignancy of mesothelial surfaces, including the pleura, which is the most common location, peritoneum, pericardium, or tunica vaginalis of the testes. 40  LGA tumors did not respond to any therapy, likely due to their slow cycling. This finding is consistent with the clinical response of patients with LGA tumors. This trend was further confirmed using the mitochondrial quantification assays, and HGA organoids displayed variable responses and LGA organoids completely lacked susceptibility.
The organoids were also subjected to immunotherapy testing, to determine if this modeling system can support immunotherapybased drug screens. The research team injected cells from a dissociated lymph node, which was obtained from the same patient at the same time the LGA tumor specimen was obtained, into LGA organoids.
Screening was conducted with pembrolizumab and nivolumab, inhibitors of the programmed cell death receptor of lymphocytes. It was found that the organoids that were enriched with lymph nodes displayed signs of activation of T cells, indicating that these models may potentially be used for immunotherapy screening as well. These models were subjected to screening with various chemotherapeutic and targeted therapy agents, and organoid response often mirrored that of the parental tumor.

| CON CLUS ION
On the other hand, more research still needs to be done on the PTDOs. For example, many researchers noted that establishing organoids from low-grade tumors is often harder than from high-grade tumors. Developing new biomaterials that more closely mimic the extracellular matrix of different tissues/organs may increase the successful rate of establishing PTDOs from low-grade tumors. Incorporating blood vessel network may also increase the successful rate of establishing PTDOs from low-grade tumors. In addition, coculturing PTDOs with patient immune cells will make it possible to test the immuno-oncology drugs using the PDTOs. Finally, the drug testing results using the PTDOs still need to be further validated using animal models and clinical trials.
Nonetheless, the prospect of utilizing organoids in cancer drug development is an exciting one. The idea that tumor organoids can be established from tumor biopsies or surgically removed tumor tissues, and then used for prescreening to find optimal treatment regimens for cancer patients (Figure 2), may be realized in the near future.