Gurpreet Kaur Bamrah, Shoolini University
Cancer ranks as a leading cause of death in the world, with 19.6 million new cases and over 10 million casualties in 2020 alone. With such a high fatality rate, it is necessary to continue advanced research and development of novel cancer treatments and therapies to achieve a better survivability rate and to cope with the growing number of patients.
While there is a considerable amount of success in already existing treatments like surgery, chemotherapy, and radiotherapy, it is not enough to be relied on. There is a substantial hindrance of inherent resistance observed within the cancer cells. Intra-tumor and inter-tumor heterogeneity increase diagnosis difficulties since not all the cells within the tumor tissue are responsible for disease progression. Different regions of the same tumor show varied drug responses, growth rate, immunogenicity, and plasticity. Therefore, it is important to develop a pre-clinical model of tissue clustered cells derived either from the patient’s tumor tissue or stem cell tissue, that mimics the micro-environment of tumor characteristics and genetic diversity in vitro. This is where the development of organoids takes place.
An Introduction to Organoids
Organoids are tiny organ-like structures that are harvested from the patient (Patient-derived Organoid or PDO) or induced pluripotent stem cells (iPSC). These are three-dimensional, self-replicating, and self-organizing structures that model the in vivo constitution of the organs or tumor-derived organs to improve understanding of cancer cell resistance. Organoids are used to develop novel cancer treatments by improving the targeting of specific cells in tumors, deciphering the response of treatment, and help predict accurate and complete drug responses.
Since organoids are obtained from tumor biopsies, patient-specific response to cancer can be predicted, thus guiding the treatment towards the personalized medicine sector, and improving patient outcomes. The multi-fold range of human tumor cells in molecular heterogeneity and plasticity brings in new challenges for precision medicine. This can be managed by testing drug responses on such patient-derived preclinical models. It also gives a detailed understanding of how an organ works, thus providing new insights into human development and disease.
Organoid Culturing Process
Organoids are miniature forms of organs that are derived from stem cells or cancer stem cells. Culturing of different organoids depends on the origin of the tumor tissue. This starts with a resection biopsy of the tumor, followed by enzymatic or mechanical digestion of the tissue. The tissue is cultured into a basement membrane (BME) like extracellular matrix hydrogel e.g., Matrigel or Culturex BME., these can also be cultured to a floating primary culture followed by seeding in BME or culturing at an air-water interface using transwell inserts. Organoids are then passed every 1-2 weeks in a media that vary according to a tissue of origin. For instance, the addition of nicotinamide is used to maintain the colon-organoid culture, R-spondin, epidermal growth factor (EGF) is used for long-term maintenance of small intestine organoid. Due to such variable criteria including biopsy site, biopsy size, organoid expansion rate, and cell number requirement, the analysis pattern is different for each type of cancer organoid.
Till now, human tumor organoids extracted from the colon, pancreas, prostate, breast, gastric, lung, kidney, oropharyngeal, bladder, and ovarian tumor tissues have been cultured and used in the field of cancer research and personalized medicine.
Organoids can also be grown from adult stem cells (ASCs) and embryonic stem cells (ESCs) that are capable of self-organizing into 3D structures and resemble tissue of origin (for ASCs) or direct to differentiation (for ESCs). These mini-organs mirrors the genetic and phenotypic facet of tumor epithelium including the intra-tumor heterogeneity. Ergo, it maintains the mutation pattern of the tumor during long-term culture without any genetic alterations.
Different Cells Deriving Organoids
An organoid refers to a group of epithelial cells that are removed from an animal’s body along with its stromal composition. Most of the organoids formed are thus epithelial in origin (carcinomas). Modeling of other cancer types including sarcomas and leukemia is a work that is foreseen in the future. These are cultured from a niche of cells that gives rise to a particular organ organoid and further modeled for drug testing. Organoids derived from pluripotent stem cells (PSCs) are precursors of different organs due to their nature.
Brain organoids from PSCs can reconstruct the tissue build on parts of the brain. First cultured were cerebral organoids that are a congregation of diverse cells representing various brain parts. Optic organoids and retina organoids are extractions from ESCs and iPSCs respectively. These are used in transplantation into mice or primates, retinal degeneration modeling, and further clinical applications. Cultured region-specific organoids include the forebrain, cerebellum, cortex, hippocampus, midbrain, and hypothalamus.
Organoids of the foregut from PSCs comprise of thyroid, lung, liver, pancreas, and stomach. These are generated through stepwise differentiation that mimics the in vivo process.
Organoids of the small intestine and colon from PSCs are achieved through manipulations of growth factors to construct intestine tissues. This is used in transplantation assay for damaged colonic epithelium using a mouse model.
ASCs derivatives of organoids are responsible for tissue maintenance and injury repair. A small intestine organoid is constructed using a cocktail of factors, promoters, and niche cells like Lgr5+ stem cells (leucine-rich repeat-containing G-protein coupled receptor 5). Organoids of the liver and pancreas built from a single Lgr5+ stem cell are used in injury repair, as the proliferation of the cells increases under injured conditions. These have also been used in the transplantation of mouse pancreatic cells to rescue liver failure.
Other ASCs derived organoids include genetically stable prostate organoids from luminal and basal cells. Tissue-specific organoids include the mammary gland, fallopian tube epithelium, taste bud, gastric, and airway organoids.
Using Organoids in Cancer Research and Treatment
The Organoid culture has high accessibility and visibility, making it more useful in studying tumor development. Starter culture requires very few cells that can propagate fast into an organoid under proper culture conditions, which makes the whole process seamlessly efficient and technological. This novel 3D cultural organization that recapitulates the in vivo environment of the tumor, is modeled to study disease progression, embryonic development, anatomic patterns of the body, organ morphogenesis, signaling pathways, drug screening, and application of precision personalized medicine.
The patient-derived cancer organoids maintain the heterogeneity and genetic features of original cancer tissue, this improves analysis and prediction of treatment which is personalized to a patient by enhancing drug response and minimizing side effects. It used to be a laborious process to formulate an in vitro model for malignant prostate cancer because of difficulty in propagating cancer cells. With the organoid culture system, it is now easy to recapitulate frequent mutations in prostate cancer, this enables us to carry out various genetic and pharmacological studies on prostate cancer cells.
The mortality rate of ductal cancer was extremely high due to late diagnosis. A standard organoid model is expected to study tumorigenesis of this fatal disease. Organoid banks of pancreatic cancer, which is rapidly generated from a benign or malignant tumor., colorectal cancer organoids from different clinical stages, and adjacent cancer cells have been generated to study disease progression and genetic and drug testing as it reveals DNA, RNA alterations in colon cancer due to gene sequencing.
Human pathological models such as organoids have been used to test high-throughput drugs for individual patients. Human-derived organoid for cystic fibrosis has been successfully made to correct gene mutation and is used in clinical trials. Application of such models in analyzing gene-drug association and guidance through specific drug prescription precise to a patient. Future beholds use of these organoids to test drug toxicity, drug metabolism, and toxic reactions, ergo, for regenerative medicine and signal to model.
Organoids are also used in tissue engineering apart from cancer research. CRISPR-Cas 9 technology is used to study gene mutations in organoids. Transplantation of fully operative organoids back into a patient to quicken the process of tissue repair is one such use.
Biobanks of patient-derived and healthy organoids are cryopreserved, their genetic characteristics, DNA makeup, and tumor genetics are made accessible worldwide for advanced research and development. Biobanks will help niche treatment screening in a personalized manner. Maintaining such depositories will not only contribute to the knowledge of cancer research, but it will also lead to excelling innovations and development and eventually contribute to cancer eradication.
Conclusion
Organoids are ‘mini-organs extrapolated from both cancerous tissue and normal cell tissues, that have heightened our understanding of disease development, drug discovery, tissue engineering, and personalized medicine. In vivo heterogeneity of tumor tissue is recapitulated in vitro conditions although duplicating the tumor microenvironment is a bit of a challenge, e.g., localizing different gradients of hypoxic condition, lack of immune cells, blood vessels, and neurons. These challenges can be overcome by co-culturing lymphocytes, macrophages, the input of endothelial cells, and the addition of stromal components that facilitate tumor progression, disease succession, and evolution, analyzing drug-cell association and consequently contributing to drug discovery and development.
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References:
- Xia, X., Li, F., He, J., Aji, R., & Gao, D. (2019). Organoid technology in cancer precision medicine. Cancer letters, 457, 20-27.https://doi.org/10.1016/j.canlet.2019.04.09
- Nagle, P. W., Plukker, J. T. M., Muijs, C. T., van Luijk, P., & Coppes, R. P. (2018, December). Patient-derived tumor organoids for prediction of cancer treatment response. In Seminars in cancer biology (Vol. 53, pp. 258-264). Academic Press. https://doi.org/10.1016/j.semcancer.2018.06.005
- Bleijs, M., van de Wetering, M., Clevers, H., & Drost, J. (2019). Xenograft and organoid model systems in cancer research. The EMBO journal, 38(15), e101654.https://doi.org/10.15252/embj.2019101654
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