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Organoid culture is an approach that allows three-dimensional growth for stem cells to self-organize and develop multicellular structures. Intestinal organoids have been widely used to study cellular or molecular processes in stem cell and cancer research. These cultures possess the ability to maintain cellular complexity as well as recapitulate many properties of the human intestinal epithelium, thereby providing an ideal in vitro model to investigate cellular and molecular signaling pathways. These include, but are not limited to, the mechanisms required for maintaining balanced populations of epithelial cells. Notch signaling is one of the major pathways of regulating stem cell functions in the gut, driving proliferation and controlling cell fate determination. Notch also plays an important role in regulating tumor progression and metastasis. Understanding how Notch pathway regulates epithelial regeneration and differentiation by using intestinal organoids is critical for studying both homeostasis and pathogenesis of intestinal stem cells that can lead to discoveries of new targets for drug development to treat intestinal diseases. In addition, use of patient-derived organoids can provide effective personalized medicine. In this review, we summarize the current literature regarding epithelial Notch pathways regulating intestinal homeostasis and regeneration, highlighting the use of organoid cultures and their potential therapeutic applications.
Over the last few decades, one of the key advances in stem cell research is the rapid development of organoids, an ex vivo culture system of self-renewing stem cell populations that grow in a three-dimensional environment and can differentiate into organ-specific cell types that retain similar spatial organization and cellular functions as their parental organs. Organoids derived from the gut epithelium of animals or humans have increasingly become an efficient approach to model intestinal development and disease progression, allowing researchers to closely study both homeostatic and pathophysiological events of the intestinal niche. Intestinal organoid cultures have provided a highly physiologically and functionally relevant system, opening avenues for researchers to dissect important signaling pathways. One key pathway is the Notch signaling pathway that regulates cellular proliferation and differentiation processes. Because of the genomic stability of organoids,
their use has also been extensively applied to the field of Notch-driven cancer research. In this review, we will cover a spectrum of Notch signaling studies and applications, exploiting intestinal epithelium organoids. We will address the critical cross talk between Notch and other important developmental pathways. Last, we will address existing limitations and future directions for Notch studies using organoid-based technologies.
Intestinal Epithelial-Derived Organoids
The mammalian intestine is a tubular organ with an inner surface lined by an epithelium that is constantly and rapidly renewed, with complete regeneration accomplished in 3 to 7 days. The integrity of the intestinal epithelium relies on proliferation and differentiation of intestinal stem cells (ISCs) that are located at the base of the crypt and that contain crypt base columnar cells (CBCs) and quiescent intestinal stem cells (QISCs). CBCs, intercalated between Paneth cells, are actively dividing stem cells. The R-spondin receptor and the Wnt target gene, Lgr5, is identified as a critical CBC marker of stem cells in the adult small intestine and colon. It is well accepted that daughter cells of murine Lgr5+ CBCs rapidly divide until reaching the crypt/villus boundary and differentiate into all epithelial lineages.
Other than Lgr5, CBCs in the mouse express Olfm4, Ascl2, Tnfrsf19, Musashi-1, helix-loop-helix transcriptional factor (Hes1), Smoc2, and other stem cell markers.
Currently, they are defined as a population of slowly dividing stem cells, also referred as +4 cells, as they reside at position 4 above the crypt base (in a supra-Paneth cell position). Reported markers of QISCs include Bmi1, Hopx, Tert, and Lrig1.
reported that neither of those four proposed markers exhibited enrichment for the quiescent +4 cells. Some QISCs can express Lgr5 as well, and display extensive stem cell characteristics under conditions of regeneration.
who found that a population of Lgr5+ QISCs were able to reacquire stem cell functions after injury. However, the dividing pattern and microenvironment (the stem cell niche) of +4 cells remain unclear. Finally, a series of studies reported that lost Lgr5+ stem cells could be replenished by differentiated cells.
Therefore, debates remain regarding the precise identity of QISCs.
Because the Lgr5 marker gene was identified and more of the homeostasis-controlling signaling pathways were discovered, the stemness of ISCs is becoming more fully understood, leading to success in the application and development of mini–gut-like ex vivo models derived from human or animal ISC-containing intestinal crypts, referred to as intestinal organoids (Figure 1A). Organoids are long-term yet indefinite culture systems embedded in Matrigel, which is produced from the mouse extracellular matrix.
Although a single Lgr5+ stem cell is enough for full development into an organoid, other studies indicated that the Lgr5+ cell is not the sole source for organoid formation.
ISC-derived three-dimensional organoids mirror the cellular construction of the intestine by forming a polarized spherical structure with a central lumen. The lumen is enveloped by a simple villus epithelium, with the apical side facing inside the lumen, whereas the basal domain binds with the Matrigel and culture media mixture. Stem cell niche factors required to support crypt stem cells include R-spondin, epidermal growth factor (EGF), Noggins, Wnt ligands, and Notch ligands, among others.
Lgr5+ stem cells can be efficiently expanded with CHIR99021, a glycogen synthase kinase 3β inhibitor, and valproic acid, a histone deacetylase inhibitor that also activates Notch.
Under limited growth factors, these spheroid-like organoids start to bud and eventually appear as an asymmetrical, multi–crypt-shaped structure with apoptotic cells filling the lumen. The budding organoids contain major mature absorptive (enterocytes) and secretory intestinal epithelium lineages (Paneth, goblet, enteroendocrine, and tuft cells), and remain in a similar ratio as in an in vivo setting.
When Wnt factors interact with the complex made of the seven-pass transmembrane receptor, Frizzled, and the single-pass low-density lipoprotein receptor-related protein, Disheveled and AXIN are recruited, which, in turn, triggers the stabilization of β-catenin by inhibition of phosphorylation kinases glycogen synthase kinase 3 and CKIα. The unphosphorylated form of β-catenin can escape from proteasomal degradation and subsequently bind to the transcription factor, TCF/LEF, that targets Wnt-responsive genes.
In comparison, BMP signals are activated in the villus compartment and negatively regulate crypt stem cells by inhibiting the Wnt signaling pathway. After BMP binds to BMP receptors, it forms complexes with Smad1/5/8 and Smad4, which then inhibit stemness genes in the nucleus. BMP signaling is regulated by its antagonists, like Noggin, Gremlin1, Gremlin 2, and chordin-like 1. Their expressions are enriched in human colon basal crypts, likely serving as ISC niche factors.
indicating that inhibition of BMP through, for example, the addition of Noggin, can generate a crypt-permissive environment. Among the four signaling pathways, Notch has attracted much attention because of its highly conserved process that is based on direct cell-cell contact. Activation of Notch serves as an essential niche factor of maintaining stem cell homeostasis. Although intracellular transduction is somewhat simple, Notch signaling is remarkably intricate and regulates diverse developmental and disease processes.
In the canonical Notch signaling pathway of mammals, there are four Notch receptors, NOTCH1 to NOTCH4, and five Notch ligands, Jagged 1, Jagged 2, and Delta-like 1, Delta-like 3, and Delta-like 4. NOTCH receptors are single-pass (type I) heterodimeric transmembrane proteins that contain an extracellular domain and an intracellular domain (NICD) apart from the transmembrane portion.
Once the ligand from a signal-sending cell binds to specific EGF-like repeats located on the extracellular domain of a signal-receiving cell, proteolytic cleavage is initiated by a disintegrin and metalloprotease 10 at the cell surface, followed by release of the NICD fragment into the cytoplasm via γ-secretase–mediated cleavage. Subsequently, intracellular NICD translocates to the nucleus, where it forms a transcriptional activator complex with the DNA-binding protein, RBP-Jκ/CSL, and Mastermind-like coactivators, which activates transcription of its downstream target genes (Figure 1B). This is also a short-lived signal complex that is rapidly degraded because of the NICD PEST domain.
One of the key Notch effector proteins that is transcriptionally targeted by NICD is a basic Hes that is paramount for absorptive differentiation. Hes1 knockout embryos of newborn mice show an unusually large number of secretory cells.
More important, Hes1 suppresses atonal homolog 1 (ATOH1; or Math1 in mice) from activating secretory cell differentiation. Math1 is a key transcription factor for cells destined to a secretory phenotype
Another aspect of Notch signaling is cis-inhibition, which may occur when Notch receptors and ligands are expressed in the same cell to inhibit Notch activation in the signal-sending cell.
The molecular mechanism of cis-inhibition is still poorly defined. There is little evidence demonstrating their roles in the mammalian intestine and, thus, they will not be discussed herein.
Use of Organoids to Study Notch Signaling in Proliferation and Differentiation of ISCs
It is well established that Notch signaling acts as an important niche factor for maintaining intestinal stem cell population.
first reported that stem cell proliferation and function were lost when the Notch cascade was globally blocked with a γ-secretase inhibitor in crypts, whereas secretory lineage populations significantly increased. Lineage tracing analysis using tamoxifen-inducible Notch-creERT knockout mice confirmed that Notch1 and Notch2 expression levels are both associated with crypt stem cells and intestinal homeostasis.
Subsequent studies suggested that Notch1 expression may be predominant in the small intestine and regulates stem cell genes, such Olfm4, a direct Notch target in CBC. Carulli et al
similarly showed that deletion of Notch1, but not Notch2, resulted in goblet cell hyperplasia in the mouse small intestine. Consistently, Notch1 and Notch2 double-knockout mice generate a more severe phenotype with a remarkable loss of Olfm4 than single knockout of Notch1 or Notch2.
Conditional knockout mice of Rbp-Jκ, Adam10, or Dll11/DllL4 double-mutant strains display consistent phenotypes as the Notch1 and Notch2 double-knockout mice, including deficiency of proliferating crypt progenitors and induced postmitotic goblet cells in the intestine of adult mice.
Loss of intestinal crypt progenitor cells owing to inactivation of both Notch1 and Notch2 is accompanied by derepression of CDK inhibitors p27Kip1 and p57Kip2.
Collectively, these data strongly imply that Notch signaling pathway is required to maintain the ISC homeostasis in intestinal crypts. Notch regulation is also responsible for specifying epithelium cell fates functioning as an absorptive-secretory switch.
When proliferation and differentiation of the intestinal epithelium are balanced, Notch maintains the ISC population and promotes proliferation of a multipotent progenitor termed a transit-amplifying cell, with the abilities of differentiating into either an absorptive (eg, enterocytes) or secretory (eg, goblet cells) cell type. Transit-amplifying cells with inactive Notch signaling are promoted for secretory differentiation.
Despite the identical genetic background, the transit-amplifying cell fate is highly dependent on the population context, also known as niche factors, present in their environment. Niche cells, such as Paneth cells, express Notch1/2 ligands, Dll1, and Dll4. It is proposed that, as ISCs divide, filling vacant yet restricted niche space, daughter cells migrate upwards, moving away from Paneth cells. As such, they lose Notch and Wnt activity and reset as bipotential progenitors and eventually become differentiated cells.
Bipotential progenitor cells without active Notch signaling up-regulate Atoh1 expression, followed by a secretory cell-type differentiation. Their neighboring cells with active Notch signaling, on the other hand, receive signals from ligand-expressing cells and stay fated toward absorptive enterocytes driven by expression of Hes that represses Atoh1.
Activation of Notch signaling keeps neighboring cells from Notch activation through cell-autonomous transcriptional repression of Notch ligands, a process referred to as lateral inhibition,
As a result of this lateral inhibition process, homeostasis of absorptive cells and secretory cells is maintained, with secretory cells interspersed among the enterocytes.
To date, in addition to animal models, the process of Notch regulation in intestinal homeostasis and differentiation has been studied and visualized through organoid culture systems. Notch signaling is crucial as one of the niche factors for growing single-cell–derived organoid culture. Sato et al
showed that the addition of Notch ligand to the organoid culture growing from a single Lgr5+ stem cell significantly decreased cell death and increased organoid size. As the organoid develops from a single stem cell, a symmetrical, sphere-like multicellular structure first appears. Paneth cells emerge around 72 hours to serve as niche cells, supplying signals essential for stem-cell support, including EGF, Wnt, and Notch.
As transit-amplifying cells proliferate and push themselves toward the villus, a gradient of Wnt3 is generated along the crypt axis, inducing formation of a crypt, with Notch lateral inhibition acting as the enterocyte-secretory fate switch.
Under Notch inhibition via the direct action of dibenzazepine, a γ-secretase inhibitor, proliferating cells significantly decreased while most of those converted into goblet cells.
Notch inhibition by dibenzazepine decreased CBC stem cell numbers and progenitor cell proliferation, and profoundly reduced the organoid initiation efficiency.
Corroborating animal studies, a human colon organoid assay combined with CRISPR/Cas9 revealed a Notch positive feedback loop in the ISC niche, which is critical to ISC self-renewal and the spatiotemporal region of the stem cell niche.
Although mouse models and organoid culture clearly demonstrated the requirement of Notch signaling in intestinal stem cell homeostasis, recent work from Tsai et al
illustrated the increasing requirement of Notch signaling in ISC as human development progress. Fetal organoids showed less sensitivity to γ-secretase inhibitors than adult organoids, and that early fetal intestinal epithelium is not as sensitive to NOTCH inhibition as later fetal intestine, indicating that Notch pathway regulates human ISC compartment differently across early and late stages of fetal development as well as in the adult. Interestingly, the same study also reported the expression patterns of Notch receptors, where NOTCH2 and NOTCH3 expression levels are abundant in fetal intestinal stem cells, whereas a high level of NOTCH2 expression is seen in the adult.
Whether the high expression of NOTCH3 in early fetal epithelium, which then gradually decreases over time, is suggestive of a suppression of Notch signaling, as indicated by other studies, remains to be clarified.
Nevertheless, these data indicated the difference in intestinal stem cell niche between human and mice and suggested that mouse intestinal organoids may not accurately represent human gut. In addition, intestinal organoids are more reflective of a regenerative state than normal homeostasis in vivo. Therefore, translating data obtained from bench work using animal models or organoids to clinical use requires caution.
Use of Organoids to Study Notch Signaling in Regeneration of Intestinal Epithelial Cells
Notch signaling is actively involved in responding to intestinal cell injury for promoting ISC self-renewal. Loss of Notch-induced recovery, resulting from γ-secretase inhibition or intestine-specific Notch1/2 knockout, has been demonstrated in epithelial injury models generated by chemical agents
The advantages of utilizing organoid cultures for stem cell repair experiments have been recognized and applied to various disease conditions that previously were impossible to demonstrate in traditional in vitro culture systems. Compared with traditional in vivo animal experimentation, organoid systems allow visualization of the dynamic intestinal repair process, providing a novel platform to dissect recovery mechanisms through observing changes in their numbers, sizes, morphogenesis, or shifts in cell types and gene signatures.
There are two primary methods to evaluate intestinal recovery capabilities using organoid-based technology. One is the survival assay wherein organoids are directly exposed to in vitro damage with subsequent analysis. The other is referred to as the organoid-forming assay or reconstitution assay, wherein damaging-inducing agents are first introduced to mice before organoid culture and development. This allows the investigators to evaluate the persistence of ISCs or their abilities to regenerate. Both assays are applied in transgenic mice to understand the roles of each component in Notch signaling in response to epithelial damage. The latter method combined with linage-tracing analysis has been mainly used to assess Notch regenerative functions displayed by multipotency of a specific cell type in the mouse intestine after damage.
Analyses performed by these two organoid assays using different gastrointestinal injury models, including high-dose radiation, chemotherapeutic agents, and dextran sulphate sodium (DSS)–induced colitis, are discussed below.
Radiotherapy, one of the most effective and widely used treatments for cancer and other diseases, causes DNA damage and selectively targets rapidly proliferating cells, including ISCs. Exposure to high-dose ionizing radiation leads to depletion of Lgr5+ CBCs, and thereby severely reduces the stem cell pool and disrupts the abilities of intestinal epithelium to repair and self-renew. Active proliferating Lgr5-expressing CBCs are more radiosensitive in comparison to Bmi1+ QISCs
Several studies have reported that induced Notch signaling following irradiation plays an essential role in tissue regeneration in response to irradiation damage and contributes to radiotherapy resistance in cancer cells.
Unlike the reported additive function of Notch1 and Notch2 in normal ISC homeostasis, proper functioning and signaling events of both receptors are required for post-radiation recovery.
Using the organoid-forming assay, irradiation-induced Notch activation through up-regulation of Notch receptors, but not by activation of the Wnt/β-catenin pathway in Paneth cells, has been shown, leading to dedifferentiation of Paneth cells into stem-like cells to replenish villus epithelial cells.
Organoids containing proliferative markers, such as Lgr5 or Ki-67, can be established from Notch-inactive secretory progenitors or Paneth cells through Notch re-initiation after radiation treatment.
Moreover, irradiation drives activation of a range of Notch target genes along with elevated proliferation-promoting transcription factors in Paneth cells, which normally lack stemness to form organoids alone, to obtain multipotency and dedifferentiate. Yes-associated protein as well as Stat3 were also observed in irradiated Paneth cells compared with nonirradiated controls. These data imply the existence of Notch pathway reprogramming after radiation injury to be involved in a possible radioprotective signaling network that connects cell survival, proliferation, and DNA-repair mechanisms for epithelial regeneration. By contrast, colonic Atoh1-expressing secretory progenitors, but not Notch1-active absorptive progenitors, can produce Lgr5+ stem cells and organoid formation on DSS treatment.
It remains unknown whether this process is through Notch reactivation or if other Notch receptor-expressing cells could also regain stem cell properties.
Chemotherapeutic agents, such as 5-fluorouracil, camptothecin-11 (irinotecan), and doxorubicin, are commonly used in treating colorectal cancer (CRC). Similar to radiotherapy, such drugs and their metabolites can cause toxicities to actively proliferating cells, thereby damaging the intestinal stem cell niche and producing acute gastrointestinal adverse effects. The loss of CBCs that resulted from chemotherapeutic drugs would stimulate remaining stem cells by a rapid Notch and Wnt activation for regeneration purposes, evidenced by an acute expansion of Wnt- and Notch-related gene expression.
Notch and Wnt activities were proposed as injury signals in a study where a small molecule, the p53 up-regulated modulator of apoptosis inhibitor, was evaluated to reduce stem cell exhaustion in colon caused by the chemotherapeutic drug camptothecin.
In vitro survival assay was performed on mouse and human primary colon organoids to test the protective responses of p53 up-regulated modulator of apoptosis inhibitor against camptothecin-induced injury and Notch/Wnt expansion. This study and others also revealed a difference between mouse and human organoids as well as between organoids and mice crypts in the sensitivity of Notch/Wnt targets to p53 up-regulated modulator of apoptosis inhibitor and camptothecin.
In a doxorubicin-induced injury model, Notch activation enabled dedifferentiation of some Paneth cells into multipotent stem cells in the mouse small intestine.
The study combined lineage-tracing method with organoid-forming assay and successfully obtained clonogenic small intestine organoids from those dedifferentiated Paneth cells on Notch activation.
Collectively, those studies highlighted the translational potential and contribution of intestinal organoids in modeling regeneration process powered by Notch signaling on DNA-damaging agent treatment.
In contrast to radiotherapy and chemotherapy, DSS disrupts intestinal barrier function by elevating the general colonic mucosal permeability, rather than specifically targeting active stem cells, resulting in a persistent infiltration of inflammatory cells, translocation of microbial pathogens, and failure to restore mucosal barrier integrity. DSS-induced acute and chronic intestinal erosions are commonly used models to study repair mechanisms in inflammatory bowel diseases, which includes Crohn disease and ulcerative colitis. Multiple studies have uncovered molecular mechanisms in the epithelial regeneration process on mucosal injury using organoid-based technologies. It has been discovered that a transient yet robust activation of yes-associated protein synergizing with Olfm4, a Notch direct target gene in stem cells, is crucial for intestinal healing from DSS-induced injury.
Organoid staining and imaging reveal that Notch induction requires glycoprotein 130, a coreceptor for IL-6. Through the glycoprotein 130–Src family kinase–yes-associated protein axis, Notch1, Notch3, and Dll3 expression levels are up-regulated to support proliferation.
Together, these data show that organoid morphologic characteristics can be indicators of which phase the cells are in during intestinal regeneration. In summary, these findings not only provide insights into the Notch signaling network in response to mucosal erosion, irradiation, or chemotherapy, but also support the idea that organoids can be great tools to modulate Notch-dependent regeneration in various intestinal injury models. However, caution should be taken when applying organoid models to study dedifferentiation and regeneration because of the differences between intestinal organoids and in vivo epithelial homeostasis or when interpreting fetal organoids and re-emergence of fetal gene signatures during regeneration in injury models.
Applying Organoid Systems to Study Notch Signaling in Intestinal Cancer
Notch signaling also plays an important role in tumorigenesis. Although Notch activation was identified as associated with both tumor-promoting and tumor-suppressing outcomes, depending on either different type of cancer or different stage of cancer, it is well established that overexpression of Notch pathway components advances intestinal cancer progression. CRC patients have higher expressions of NOTCH receptors, Jagged ligands, and Notch target genes compared with healthy individuals.
In the assessment of Notch for the maintenance of adenoma proliferation, pan-Notch inhibition using dibenzazepine induces differentiation of intestinal adenoma cells into goblet cells.
More important, Wnt activation in mice via loss of function in adenomatous polyposis coli (APC+/–) mice only led to adenoma formation in the small intestines. However, conditional Notch activation in the APC mutant (Notch/APC) mice significantly induced development of intestinal adenomas and dysplastic lesions in the colon,
confirming that Notch is necessary to trigger intestinal tumorigenesis synergistically with Wnt signaling.
High interpatient heterogeneity and intratumor heterogeneity are key features of CRC and represent the biggest obstacles for therapy development. Commonly used cancer models cannot fully recapitulate the original tumors from patients, causing many novel therapeutic agents to fail in clinical trials. For instance, anti-tumor drug screening is heavily dependent on genetically engineered animals. Although animal models are essential tools for cancer research, they hardly reflect the complex genetic or histologic heterogeneity found in human tissues. The classic two-dimensional cell culture derived from patients is also frequently used for in vitro experiments. However, experimental cancer cell lines continue to evolve and display distinct molecular features in the in vitro culture environment. In addition, only a small subset of cancer cells possesses stem cell properties that allow them to expand over multiple generations. Therefore, the original genetic diversity can be lost after several passages. Transplantation of fresh patient-derived tumor xenografts into immunodeficient mice can successfully maintain primary tumors ex vivo, but the process requires extensive resources and is extremely time-consuming.
To better understand molecular mechanisms involved in gut tumorigenesis and metastasis and to uncover novel therapeutic targets, investigators use organoid culture-based techniques to help analyze mice and patient tumor samples as they can faithfully recapitulate the genetic, histologic, and morphologic heterogeneity of parental tumors.
Sole activation of Notch signaling in the mouse gut was found to be insufficient to induce tumorigenesis, whereas spontaneous intestinal adenomas develop in a tumor suppressor gene p53-depleted background.
demonstrated that activation of Notch combined with the loss of p53 (NICD/p53−/−) in mice triggers epithelial-to-mesenchymal transition in the gut, and develops highly metastatic tumors. Organoids from NICD/p53−/− mice adopted tumor organoid properties, including a sphere-like structure, growth factor independency, hyperproliferation, and lack of secretory cell differentiation. However, organoids from single mutant of either NICD or p53−/− mice cannot grow independently of the growth factors (R-spondin, EGF, and Noggin). These mutant organoids and patient-derived CRC organoids both showed persistent elevation of yes-associated protein, histone methyltransferase MLL1, as well as the Notch target gene, HEY1.
Therefore, a combination of activated Notch and depleted p53 function may be required to lock cells into a regenerative state required for tumorigenesis. Thus, these data provided insights in modeling colon cancer using organoids with transgenes that alter Notch signaling.
CRC is now the second leading cause of cancer-related deaths in the United States.
Given the fact that a poor prognosis of CRC patients is associated with disease spreading to other organs, such as the liver, brain, and lungs, the establishment and study of reliable metastatic disease models are of great clinical importance. Splenic organoid transplantation of mouse intestinal tumors is an efficient method to generate liver metastases and to evaluate colonizing capacity, overcoming the weakness in traditional genetically engineered mice that often fail to develop highly penetrant metastases.
Transcriptome profiling of CRC tumor tissues and organoids shows that a Notch1 gene signature is specifically associated with the transcriptome-based consensus molecular subtype 4.
This subtype of CRC is characterized by induced epithelial-to-mesenchymal transition, elevated transforming growth factor-β signaling, stroma invasion, and the worst prognosis. In this study, conditional knockout of p53 in mice with constitutive Notch1 and Kras activation meant that mice developed highly malignant intestinal adenocarcinoma that had the ability to spread to distant organs. By injecting Kras activation organoids into recipient mouse spleens, elevated neutrophil infiltration was observed in liver metastases, indicating Notch1 activation drives transforming growth factor-β–dependent neutrophil recruitment in rewiring the tumor microenvironment to drive cancer progression.
A more recent study reported that Notch3 blockade via use of a Notch3-antagonistic antibody significantly inhibited tumor progression in a carcinogen AOM-induced transgenic murine model, which is based on concomitant deletion of p53 and activation of AKT signaling (Trp53–/–AktE17K).
This model also resembles consensus molecular subtype 4. However, this study showed that up-regulation of Notch3 but not Notch1 is associated with cancer progression and the consensus molecular subtype 4 subtype using mouse tumor organoids or The Cancer Genome Atlas data. Moreover, they confirmed enhanced lung and liver metastases in vivo using Notch3-overexpressed organoids in the orthotopic transplantation model. Another study reported that Notch3 up-regulation featured aggressive CRC xenografts in immunocompromised mice, and silencing Notch3 can impair proliferation of CRC cells.
So far, the function of NOTCH3 in the intestinal epithelium has not been widely studied; therefore, whether NOTCH3 could represent a new therapeutic target to treat consensus molecular subtype 4 patients, without interfering with Notch regulation in intestinal stem cell homeostasis, still needs further investigation. Together, those studies showed a translational potential of tumor organoid transplantation in studying CRC metastasis, through either splenic or orthotopic transplantation.
Potential Applications
Organoid culture has emerged as an effective and reliable model for drug screening. Data collected from organoid biobanks derived from tumor samples and adjacent healthy tissues are consistent with results from large-scale sequencing analyses of CRC,
proving that patient-derived CRC organoids can accurately represent corresponding patient tumors' genetic alterations. Similar observations in metastatic gastrointestinal cancers were reported by Vlachogiannis et al.
They showed that genomic, transcriptional, and morphologic profiling of patient-derived CRC organoids largely overlaps with those from patient biopsies.
for the first time, utilized patient-derived CRC organoids to evaluate antitumor effects of botanical oligomeric proanthocyanidins, which exhibited a cancer stem-like cell targeting property, using both in vitro and in vivo systems. The same group identified anti-tumor mechanisms of oligomeric proanthocyanidins by down-regulating expression of stem-like genes, including NOTCH1, Jagged 1, and the cleaved or active form of NOTCH1. However, testing oligomeric proanthocyanidins on healthy tissue organoids was not included in this study. Given the fact that Notch signaling is also conserved during normal ISC homeostasis, the toxicity of oligomeric proanthocyanidins on patients' intestinal epithelium remains unclear. Therefore, patient-derived healthy organoids should always be included in organoid-based drug screening protocols to develop selectively targeted drugs and predict treatment outcomes. Furthermore, great benefits are foreseen in applying organoid-based drug discovery to research in the therapeutic modulation of Notch signaling. Unlike pan-Notch inhibitors, which display on-target dose-limiting toxicity, fine-tuned suppression of specific components involved in Notch signaling has shown great potential in anticancer therapies with less toxicity to healthy tissues.
In addition to the potential applications mentioned above (Figure 2), organoids have been shown useful in co-culture platforms to study the role of Notch signaling in epithelium–immune cell cross talk. For example, recombinant IL-22 enhanced ISC regeneration.
Such growth-inducing properties were found in human and mouse organoids in an IL-22–dependent manner. However, IL-22 treatment did not increase Wnt or Notch pathway gene expression.
Adhesive interactions between mononuclear phagocytes and intestinal epithelium perturb normal epithelial differentiation and serve as a therapeutic target in inflammatory bowel disease.
wherein co-cultures of mononuclear phagocytes and organoids showed direct E-cadherin–mediated Notch activation. This resulted in cystic change of organoids and goblet cell depletion, which may contribute to inflammatory bowel disease–related colitis induced by impairment of transforming growth factor-β signaling in mononuclear phagocytes.
Adhesive interactions between mononuclear phagocytes and intestinal epithelium perturb normal epithelial differentiation and serve as a therapeutic target in inflammatory bowel disease.
Collectively, these studies illustrate that the organoid system has the potential as a successful platform to study direct interactions between immune cells and the intestinal epithelium (Figure 2). However, the in vivo relevance of the co-culture platform composed of intestinal organoids with single types of immune cells needs further studies in the future.
Figure 2Organoid-based approaches and applications for studies of Notch signaling in the intestinal epithelium. (Figure generated with BioRender.com, Cleveland, OH.) ISC, intestinal stem cell.
In summary, using organoid culture as a tool to investigate Notch signaling in the intestine has vast academic and therapeutic potential, in terms of exploring signaling pathway network, establishing disease models, and developing personalized drugs and therapeutics. On the other hand, some limitations still exist. A major limit is that the intestinal organoid is more reflective of a regenerative state than the normal homeostasis of the epithelium. To date, the stem cell niche factors for human organoid culture medium have not been defined, although Fujii et al
have refined organoid culture condition by switching p38 inhibitor to a combination of insulin-like growth factor 1 and fibroblast growth factor 2 to better model healthy and diseased human intestinal epithelium. Further optimization is still required to reflect more closely in vivo homeostasis for mouse and human guts. The current organoid embedding matrix mainly relies on Matrigel, which is the reconstituted basement membrane secreted by mouse tumor cells. Improving the current Matrigel extracellular matrix makeup or developing alternative synthetics may not only reduce costs of developing organoid systems but can also be tailored to experimental needs (ie, to explore extracellular matrix–regulated Notch signaling in ISC homeostasis, regeneration, and tumorigenesis). It has been acknowledged that epithelial Notch signaling interplays with other nonepithelial components that are currently absent in the epithelial organoid structure, which includes blood vessels, mesenchymal cells, neural cells, microbiota, and immune cells. It is well known that Notch plays an essential role in neovascularization and regulation of immune cells in the context of tumor growth and metastasis.
The pluripotent stem cell–derived organoid-based technique made it possible to grow a surrounding and supportive mesenchyme and even functional human small intestine tissue in mice by such organoid xenografts.
Future advances in the co-culture system and pluripotent stem cell–derived organoids have the potential to complement the absence of nonepithelial cell types. The idea of combining a gene editing approach, such as CRISPR/Cas9, with organoid transplants
to modulate Notch-induced restoration of organ functions holds promise for development of engineered regenerative medicine. However, this so-called mini-gut transplantation is still in an early stage of development. Finally, although tumor organoids are considered genomically stable and more closely reflect tumor heterogeneity in vivo compared with cancer cell lines, optimization for growing and long-term maintaining patient organoid heterogeneity is required. Nevertheless, despite these limitations, the capability of organoids in recapitulating molecular and physiological manifestations complements existing two-dimensional culture systems and animal models. The use of intestinal organoids has significantly contributed to further define the Notch signaling pathway and validate its impact on the epithelium by means of controlling homeostasis, regeneration, and carcinogenesis. The various applications of organoids also hold great promise that have potential to lead to discoveries of novel therapeutic targets for treating intestinal disorders.
References
Sato T.
Stange D.E.
Ferrante M.
Vries R.G.J.
Van Es J.H.
Van Den Brink S.
Van Houdt W.J.
Pronk A.
Van Gorp J.
Siersema P.D.
Clevers H.
Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.
Loss of intestinal crypt progenitor cells owing to inactivation of both Notch1 and Notch2 is accompanied by derepression of CDK inhibitors p27Kip1 and p57Kip2.
Adhesive interactions between mononuclear phagocytes and intestinal epithelium perturb normal epithelial differentiation and serve as a therapeutic target in inflammatory bowel disease.
Supported in part by National Cancer Institute research funding CA222064 (L.Z.), NIH research funding HL103827 (L.Z.), and the Department of Pathology, Case Western Reserve University , faculty startup fund (L.Z.).
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