Patient Derived Organoids

By Anjli Venkateswaran

Introduction to Patient Derived Organoids

Preclinical drug development relies on in vitro and in vivo models to screen and validate new therapeutic modalities and evaluate the efficacy of existing therapies for other diseases. Cell culture models are widely used research tools that typically involve a two-dimensional cell monolayer. While this approach is robust and easy to manipulate, these models have limited translational value. 2D culture systems do not fully recapitulate complex tissue architecture, including multiple cell types and vasculature. 3D models are being explored as a viable alternative to overcome these limitations. Organoids are defined as three-dimensional cell clusters that self-organize to form functional tissues and mini-organs (Corro et al. 2020). Organoids are typically derived from stem cells that have the ability to proliferate and differentiate into multiple cell types. Stem cells are derived from 3 sources – embryonic stem (ES) cells, adult stem (AS) cells or induce pluripotent stem (iPS) cells. ES cells raise ethical and regulatory issues, and AS cells are limited to specific tissues like the intestine. But the development of iPS cells has revolutionized the field of organoid development (McCauley and Wells 2017) and resulted in generating organoids from various organs as well as patient-derived organoids (Hockemeyer and Jaenisch 2016). Patient-derived organoids (PDOs) are referred to as “disease in the dish” and are grown from iPS cells derived from patient tissues, so they contain all the genetic drivers of the disease. PDOs are considered to be better models compared to organoids generated from healthy tissues that are manipulated or stimulated to induce the development of the disease phenotype. Additionally, PDOs allow researchers to study complex idiopathic diseases that cannot be fully recapitulated in animal models or cell-based models. PDOs facilitate the understanding of genetic and disease development differences in patient populations, which can be an advantage and a challenge. There can be multiple underlying mechanisms for a given disease, and PDOs allow granular analysis of the signaling and disease development changes in different segments of a specific patient populations. This can result in highly variable PDO populations that poses analytical and statistical challenges. Another important application of PDOs is to support the understanding of drug-gene interactions at the individual patient level. This gives information on whether a patient can metabolize and distribute a drug sufficiently or whether there are adverse interactions between two drugs in a specific patient (Busslinger et al. 2020). PDOs are amenable to gene editing methods, and there are reports of successful gene correction in PDOs (Hockemeyer and Jaenisch 2016).

As the use of PDOs becomes more widespread, there are several reports highlighting the development and applications of PDO across various disease areas. Some examples are briefly discussed below:

Cystic fibrosis (CF) is a genetic disease that currently affects about 90,000 people worldwide and is caused by mutations in the CFTR gene encoding a transmembrane conductance channel that bidirectionally transports chloride ions and water. Mutations in the CFTR gene lead to dysregulation of the CFTR protein causing the disease phenotype and about 2,000 mutations have been identified to date. Researchers at the University of Utrecht published the first report of patient derived organoids where intestinal organoids were developed from rectal biopsies of cystic fibrosis patients (Dekkers et al. 2013). This model has several advantages: rectal biopsy tissues are relatively easy to source, and the protocols for developing viable long-lasting intestinal organoids are available. Additionally, several studies have shown that intestinal organoids derived from CF patients are a robust model to screen novel therapies via well-defined endpoints (Dekkers et al. 2013, van Mourik et al. 2019). CF patient derived intestinal organoids have been shown to have high translational value. For example, ataluren, a new therapy for CF patients with nonsense mutations in the CFTR gene, which failed to meet the primary endpoint in a phase III trial, was shown to have no efficacy in CF patient derived organoids (Zomer-van Ommen et al. 2016). One of the most common mutations in the CFTR gene is F508del, a deletion of a phenylalanine amino acid that results in recycling of the CFTR protein. A study published in 2020 showed that organoids derived from induced pluripotent stem cells (iPSCs) of a CF patient with the F508del mutation could be corrected by TALENS gene-editing1. TALENS-mediated homologous recombination was used to remove the three-nucleotide defect in the CFTR gene—essentially correcting the disease-causing genetic defect (Fleischer et al. 2020).

Neurodegenerative diseases are challenging to model in animal models for several reasons including species differences and limited understanding of disease development, so there is significant interest in developing CNS organoids to study brain development and disease pathophysiology. The cell sources for brain organoid development are typically ES or iPS cells. Recently, several studies have shown that CNS organoids recapitulate several structural and functional characteristics including neurogenesis, cell migration, and neural circuitry. However, CNS organoids have certain limitations including variability in organoid architecture and cell composition and lack of vasculature. Microfluidic systems are being explored as a tool to better control the assembly of brain organoids. Organ-on-chips, microwell arrays, and droplet-based methods can be used to generate CNS organoids of specific size, shape, and cell composition.
One of the main issues with CNS organoids is the maintenance of long-term viable cultures especially for the study of neurodegenerative diseases such as Alzheimer’s disease (AD) or Parkinson’s disease (PD). One solution is to introduce vasculature in the grafting organoids in adult mouse brains, resulting in the development of vasculature via the recruitment of mouse endothelial cells. Another approach is to section organoids and expose the slices to an air-liquid interface to increase gas exchange and nutrient delivery. The development of vascular organoids (vOrganoids) is another method for introducing vasculature in organoid culture; in this approach, organoids are co-cultured with human vascular endothelial cells (HUVECs) (Shi et al. 2021). The co-culture approach was shown to induce the formation of a tubular network of blood vessels, allowing the long-term culture of organoids (up to 200 days).
Human midbrain organoids have been developed using PD patient derived iPS cells (reviewed in Galet et al. 2020), and these organoids have been shown to recapitulate key features of the PD phenotype including the expression of tyrosine hydroxylase, a marker of dopaminergic neurons, and neuromelanin granules, which are observed in the substantia nigra region of the brain. Several disease driver mutations have been identified in PD including mutations in LRRK2, PINK1 and the PARKIN gene family. To facilitate mechanism of action studies on how specific mutations induce diseases, PD patient derived organoids have been generated from patients carrying those mutations. PDOs can also be used to study the effect of environmental stress and xenobiotics on sporadic forms of PD (Galet et al. 2020). Similarly, PDOs derived from Alzheimer’s patients with mutations in the APP (amyloid precursor protein) or PSEN1 (presenilin) genes can recapitulate the hallmarks of AD including b-amyloid aggregates, hyperphosphorylated Tau protein, and enlarged endosomes (Raja et al. 2016). Interestingly, AD PDOs have reduced b-amyloid aggregates in response to b- and g-secretase inhibitors such as BACE-1. These data suggest that AD PDOs have the potential to be used to screen novel therapies for AD.

Gastrointestinal diseases have been modeled in intestinal organoids developed from human adult intestine stem cells co-cultured with fibroblasts, immune cells, enteric neurons, and vascular endothelial cells to model the intestinal stem cell niche. Intestinal organoids that are cultured as 2D monolayers on a membrane are very useful to study barrier function as these monolayers provide access to the apical and basolateral sides of intestinal epithelial cells. Noel et al. was one of the earlier groups to describe an enteroid monolayer model, with intestinal spheroids were seeded on the basolateral side of a membrane while macrophages were seeded under the membrane (Noel et al. 2017). This model was used to study host-pathogen interactions where macrophages develop projections that passed through the filter pores in response to enterotoxic E.coli bacteria that had infected the epithelial cells. The intestinal organoid monolayer model is increasingly being used to study a range of host-pathogen interactions including mechanisms of infection and transmission from one organoid to another. One of the most highly prevalent microbes in the GI tract is Helicobacter pylori, which has been shown to cause peptic ulcers and gastric cancers. Intestinal organoids derived from iPS cells have been used to study H. pylori infection in a more physiologically relevant cell model (McCracken et al. 2014). Recently, PDOs derived from colonic fibroblasts of ulcerative colitis (UC) patients were shown to recapitulate disease phenotype: low numbers of goblet cells and compromised tight junctions that impacted the integrity of the intestinal barrier (Sarvestani et al. 2021). The UC PDOs accelerated epithelial cell turnover and nonuniform cell proliferation, which are observed in UC patients. Additionally, E-cadherin and b-catenin expression in the UC PDOs were largely confined to the cell membrane instead of the tight junctions, resulting in increased permeability of the intestine barrier. Transcriptomic analysis of the UC PDOs showed a strong inflammation signature with increased expression of CXCL8 and its receptor CXCR1, suggesting that CXCL8 could be one of the main contributors to the inflammation seen in UC. Indeed, repertaxin, a small molecule inhibitor of CXCL8 can improve the UC phenotype in cultured PDOs as well as PDOs implanted in immunocompromised NSG mice (Sarvestani et al. 2021). This study demonstrates that PDOs can be a superior alternative to animal models for understanding inflammatory bowel disease (IBD) pathophysiology and identifying novel therapies.

Liver diseases such as FLD (fatty liver disease) or NASH (nonalcoholic steatohepatitis) are highly prevalent in the United States. It is estimated that about 10-46% of the US population has FLD (, whereas NASH is the most prevalent chronic liver disease worldwide (McCarron et al. 2019). Liver organoids can be developed from a few different sources such as adult and fetal liver tissues and iPS cells. Liver organoids can be either of the hepatocyte lineage or the cholangiocyte lineage, depending on the combination of growth factors that the cells are exposed to (Nuciforo et al. 2020), and transdifferentiation is possible between the two lineage organoids. Several monogenic metabolic diseases have been modeled in liver organoids including Wilson’s disease, Wolman’s disease, hepatitis, and alcohol-related liver disease where organoids derived from normal livers are exposed to ethanol to induce liver injury and stress. Ductal organoids derived from NASH liver biopsy samples have been developed (McCarron et al. 2019). These PDOs showed significant delay in growth and development, reduced LDL uptake, and increased lipid accumulation. Most interestingly, the PDO derived from NASH patients showed clinically relevant responses to therapies that are currently being tested and include activators of the Hippo/YAP pathway and anti-inflammatory/anti-fibrotic drugs. This initial study demonstrates that while NASH PDOs can be challenging to culture, they are a clinically relevant model to screen novel therapies for NASH.

Patient Derived Cultures in Cancer

Cancer indications are one of the leading causes of mortality worldwide. It is estimated that about 19.3 million new cancer cases and about 10 million cancer deaths occurred in 2020 (Sung et al. 2021). Due to the unmet clinical needs, there is active research and drug development ongoing in the area, and several in vitro and in vivo models have been extensively studied for different cancer indications. However, the success rate of new therapies in the clinic is low; it is estimated that only 3.4% of cancer therapies were approved between 2000-2015 (Wong et al. 2018). One of the main reasons for failure of many anti-cancer therapies is inter- and intra-patient tumor heterogeneity in morphology, gene expression pattern, metastatic potential, and mutational and epigenetic profiles (McGranahan and Swanton 2015). To understand these heterogeneities, discover, test new anticancer treatments, and help eliminate therapies that are likely to fail early in the pipeline, more physiologically relevant preclinical cancer models are needed. 3D cell culture models have been widely used in cancers due to availability of source tissues. Several tissue specific cancer cell lines that are commercially available have been used to form 3D constructs. Additionally, tumor cells taken from patient tumors have been xenografted into animal models subcutaneously or orthotopically (within the organ associated with the original tumor). The patient derived xenograft (PDX) mouse models are typically immunocompromised, so they have limited applicability to study immune-oncology therapies.

Patient Derived Explants (PDE)

Patient Derived Explants or PDEs are ex vivo models in which fresh tumors from biopsy or surgical resections are directly used for drug studies. Typically, tumor pieces are sectioned or minced and placed in culture before drug treatments. Once the drug exposure is complete, the tissues are fixed or homogenized for endpoint analysis. PDEs are generated using little to no tissue disruption and include tumor cells, stroma, immune cells, and vasculature, so they are an accurate microcosm of the native tumor environment (Powley et al. 2020). PDEs facilitate the interrogation of molecular and histological tumor characteristics in a single sample to construct a more complete picture of the tumor. PDEs have been developed from various tumor indications including prostate (Tieu et al, 2021) and endometrial (Collins et al. 2020) cancers. However, PDEs can be extremely fragile and are liable to disintegrate rapidly and degrade over time. Researchers have optimized culture conditions to increase the PDE viability. For example, Majumder et al. developed a novel ex vivo system that included autologous patient serum and immune cells as well as tumor indication and grade matched matrix proteins. This autologous PDE system supported the culture for about 7 days, allowing a window of opportunity to test several drug regimens and identify the optimal therapy for the patient (Majumder et al. 2015).

Endpoint analysis in PDEs after drug treatment can follow one of two paths – the explants can be formalin fixed and paraffin embedded (FFPE) for histological and immunohistochemical analyses or homogenized for DNA, RNA, and total protein analyses. FFPE sections can be stained using H&E (hematoxylin and eosin) staining to study changes in tumor and stroma compartments as well as understand the impact of culture conditions on tissue integrity. Sections can also be stained with specific antibodies to measure important parameters such as proliferation using Ki-67, apoptosis using cleaved caspase-3, or PARP and immune cell markers such as PD-L1 and CD3. DNA and RNA isolated from homogenized explants are largely used to identify tumor mutational burden or transcriptome profiling, whereas protein analysis is used to understand the overall changes in protein expression and post translational modifications. In addition to endpoint analysis of the explant tissues, secreted proteins such as cytokines can be measured in the culture media at various timepoints to evaluate early and delayed changes in cytokine expression after drug treatment.

PDEs have several advantages and limitations compared to other 3D cell models (reviewed in Powley et al. 2020). Explants maintain the individual patient specific tumor architecture and microenvironment including immune cells, such as tumor infiltrating lymphocytes (TILs) and macrophages, and stromal cells. The retention of the native environment facilitates studies on tumor stroma interactions and paracrine signaling and permits the comparison of tumor cells to matched normal cells. Since explants are generated from fresh tissue, they are more predictive of patient response, and the data generated from the explants can be correlated with the individual patient response. PDEs are a very useful model to study changes in immune cells in response to checkpoint inhibitors that are the primary drug targets for most tumor indications. PDEs have limitations primarily in terms of source tissue availability and the culture time frame. Since fresh tissues are needed in sufficient quantities, PDEs can be generated primarily from surgical resections, which requires access to hospital networks, surgeons, and pathologists. Building a network to reliably source fresh tumors is a major undertaking. PDEs are not suited for longitudinal studies as they tend to start degrading in about 3 days, so there is a tight timeline to generate as much data as possible. Due to the short culture time, it is difficult to measure direct tumor killing effects of immunotherapies that can take several weeks to induce cytotoxicity. The amount of fresh tissue that is available varies between indications, but the finite amount of tissue does limit the potential to screen multiple therapies. This is compounded by the fact that each timepoint and treatment data point should include multiple replicates to account for intra- and inter-explant variation of tumor tissues and immune cells. Despite these limitations, PDEs have a unique role in preclinical drug development of novel cancer therapies as they are the only model that truly represents the native tumor state.

Patient Derived Xenografts

Patient-derived tumor xenografting (PDTX) are developed by direct implantation of primary human tumor samples into immunocompromised mice. PDTX recapitulates the in vivo tumor better than 2D cell cultures or animal models (Sachs and Clevers 2014) and can be used to identify cancer biomarkers and improve the diagnosis. For instance, PDXs for pancreatic cancer has successfully modeled the disease when engrafted into the pancreas of the murine model and were used to study the mechanisms of resistance to therapies. Maykel et al grafted human colon cancer fragments into genetically modified (NSG and NRG) mouse models (Maykel et al. 2014). Those PDXs retained the tumor native architecture such as nuclear orientation, goblet cells, and distribution of stromal cells and were used for testing tumor response to 5-fluorouracil. One caveat is that the murine stroma cells infiltrate the human PDXs, which can affect the translation of the results to clinical trials (reviewed by Baker et al. 2016). In a study, patient tumor xenografts from resected tumors of 37 patients from 8 various cancer types, including ovary, kidney, and pancreatic were implanted into SCID mice. Although diseases were modeled, before the first passage, human stroma and blood vessels were replaced by those of murine origin (Hylander et al. 2013). In general, response of PDXs to therapies are predictive of patients’ response (reviewed by Hidalgo et al. 2014), but they have some limitations: large amount of biopsy or resected tissue is needed to generate PDXs; it takes several months to generate PDXs; the generation and maintenance of immunocompromised mice are costly; are not suitable for high-throughput screening; PDXs engrafted into immunocompromised mice may not be applicable to immunotherapies (reviewed by Baker et al. 2016; Sachs and Clevers 2014). Thus, there are strong needs for a better model system for studying cancer.

Patient Derived Organoids (PDOs)

PDOs derived from human tumors are steadily becoming an established platform for preclinical validation of cancer drug assets. Primary tumor cell lines have been used to develop organoids that can be grown in a matrix that mimics the in vivo basement membrane. They can be generated from small amount of patient tissues and can be grown to support drug screening and mechanism of action studies. Currently, PDOs are available for several tumor indications including liver, prostate, breast, colon, and pancreatic, and the list of indication specific PDOs is expected to grow. PDOs from tumor tissues start with the culture of small pieces of tumors in a hydrogel or scaffold and specialized media to support the growth of 3D constructs.
However, PDOs have some limitations in that they do not fully recapitulate the tumor microenvironment and lack the vasculature. To overcome those limitations primary tumor cells can be co-cultured with immune cells, resected cancer tissues, and cancer-associated fibroblasts (Kuen et al. 2013). Kuen et al generated a 3D co-culture model with pancreatic cancer cells, cancer associated fibroblasts, and monocytes to investigate effects of cell-cell interactions and soluble immune modulators such as cytokines on monocyte differentiation and function. Immunosuppressive cytokines like IL-10, which promote polarization of M2 like macrophages and myeloid derived suppressive cells, were detected in these organoids similar to patient tumors. The presence of these cytokines is an indicator of poor prognosis. This model can be used for modeling diseases and testing immunotherapies. Boj et al. (2015) developed organoids from murine pancreas tumor cells to study different stages of the cancer. Organoids that are cultured directly from patient samples can grow within days compared to PDX growth in animal models that can take several months. Additionally, PDOs are more efficient than PDXs implanted in mice in capturing the heterogeneity, polarity, cell-cell interactions, and structure of the native tumor (Tsai et al. 2018). Driehuis et al. generated PD pancreatic organoids from tissues obtained from biopsies or surgical resections that were characterized using genomic sequencing and histology (Driehuis et al. 2019). PDOs derived from different patient biopsies responded differently to the same drug, gemcitabine.

PDOs can be generated from stem cells, and the main types of stem cells that can be used for generating organoids are pluripotent stem cells (PSCs), embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), and organ-specific adult stem cells (ASCs) (Sato et al. 2009; 2011; Drost et al. 2015; Schwank et al. 2013; Camp et al. 2015, Lancaster et al. 2013). PSC derived organoids can also be genetically modified to model cancer development and progression (Drost et al. 2015; Huang et al. 2015). Organoids generated from adult stem cells have the capability to differentiate and regenerate and have been used to generate pancreatic organoids (Huch et al. 2013). Huang et al isolated human pluripotent stem cells from pancreatic tissues that formed progenitor cells in culture (Huang et al. 2015). The progenitor cells formed a construct with basement membrane and gene expression similar to that of the native progenitor cells. The progenitor cells formed adenocarcinomas representing the original tumor after being engrafted in mice. This model was used to screen Ezh2 inhibitors. Huch et al induced the Wnt-Lgr5-Rspo pathway in adult pancreatic duct cells and developed pancreatic organoids with the potential of long-term expansion of adult progenitors. This approach can be used for creating patient-derived organoids in vitro to model diseases and test new therapeutic (Huch et al. 2013).

Organoids can also be derived from patient biopsies and resections, and these patient-derived organoids recapitulate histological and physiological characteristics of the original tumor. This approach has been used to culture organoids from biopsy samples of several cancers including prostate cancer and human glioblastoma (Karthaus et al. 2014; Gao et al. 2014; Hubert et al. 2016). In vitro models of cancer are developed in the form of organoids grown from biopsy or resected tumor samples. For example, Gao et al used metastatic prostate cancer biopsy samples from 6 patients to generate PDOs (Gao et al. 2014). Those samples contained at least 10% cancer cells and were maintained for 1 to 2 months. They recapitulated the molecular diversity of native prostate cancer subtype: TMPRSS2-ERG fusion, SPOP mutation, SPINK1 overexpression, and CHD1 loss. Tsai et al used surgical specimens, rapid autopsy specimens, or ascites from 28 patients to culture patient-derived pancreatic cancer organoids (Tsai et al. 2018). Those organoids showed the architecture and cellular morphology of pancreatic adenocarcinoma and expressed the tumor markers maspin, Muc5ac, CRR9, and PDX1 in a similar pattern as the original tumor sample.

PDOs typically lack stromal cells, vasculature, and immune cells found in the native tumor microenvironment, which limits their application in immuno-oncology drug development. Additionally, they may not represent the genetic heterogeneity of tumors, and it is possible that one clonal cell population has a growth advantage.  Animal-based matrices, commonly used in generating organoids, are not mechanically dynamic and easily scalable. Matrices such as hybrid polyethylene glycol (PEG) hydrogels are more dynamic and can expand the applicability of organoids (Gjorevski et al. 2016). Despite limitations, patient-derived organoids are promising tools for disease modeling, gene therapy, understanding tumor growth and metastasis pathways, drug screening, and personalized and regenerative therapies, and evaluating the mechanism of action of single or combination therapies (Vivarelli et al. 2020).

Applications of Patient Derived Explants and Organoids in Drug Development

PDEs and PDOs have the potential to be widely used in various aspects of drug development and patient care. One of the most applicable uses is in personalized medicine. It is well known that there are individual differences among patients with a specific type of cancer indication, which accounts for variability in response to specific therapies. The efficacy and safety of single or combination therapies can be tested on PDOs or PDEs derived from individual patients to identify the optimal therapy for them. This will help maximize clinical success while managing adverse events. Additionally, the response to new therapies can be evaluated prior to administration to patients. PDOs can also be used in co-clinical trials where efficacy and dosing can be evaluated in patient tissue and animal models to understand the translatability of specific preclinical models (Liu et al. 2021).

Organoid biobanking is created from tissue resections collected after surgery, which are used to develop organoids. Tumor organoids from biobanking can be used for live culture or be kept frozen in liquid nitrogen for further use. This provides the opportunity for growing tumor organoids from different subtypes to develop and test personalized medicine as evidenced by the collection of different subtypes of colorectal cancer tumors (Delpu et al. 2011). In another study, a PDO biobank from surgically resected glioblastoma tumors was developed to investigate patient-specific responses to CAR-T cells in a co-culture model (Jacob et al. 2020). A living biobank of pancreatic intraductal papillary mucinous neoplasms (IPMNs) was reported recently where the genomic and transcriptomic signatures were identified using whole genome and RNA sequencing respectively (Huang et al. 2020). The development of PDO biobanks are very valuable resources to study tumor heterogeneity and better segment patient populations in a given tumor indication. They offer a physiologically relevant model to study tumor development and progression and can be used to screen new therapies as well as evaluate the efficacy and toxicity of available therapies.

As described in the previous sections, PDEs and PDOs are increasingly being used to study critical signaling pathways that result in disease development. Since PDEs and PDOs preserve the native tumor environment and clonality, they are the optimal preclinical model to evaluate the efficacy of new therapeutic candidates as monotherapies or in combination with currently available therapies.

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