Organoids: Exploring Liver Cancer Initiation and the Possibilities of Personalized Glioblastoma Treatment


In the search for improved and high-throughput in vitro models, organoids have emerged as a promising 3D cell culture technology.1 Defined as a three-dimensional multicellular in vitro tissue construct, organoids are derived from cells that spontaneously self-organize into properly differentiated functional cell types to mimic at least some function of an organ.2 Organoid formation is driven by signaling cues in the extracellular matrix and medium, and is influenced by the particular cell types that are present.2 Compared with two-dimensional cultures, organoids incorporate more physiologically relevant cell-cell and cell-matrix interactions, and are a better reflection of the complex network found in vivo.

With significant opportunities for studies of human-specific disease mechanisms, personalized medicine, drug discovery, pharmacokinetic profiling and regenerative medicine, organoids are being pursued across a range of disciplines. Many anticipate that these cell culture models will result in more efficient translation of research into clinical success. In this article, we explore the various types of organoids under development and shine a spotlight on some of the different approaches to organoids in cancer research.

Glioblastoma organoids generated from patient tumors


Given the high drug attrition rates for cancer, there is clearly a need to better represent patients in the preclinical phase of research.3 As cancer heterogeneity is regarded as the main reason for the failure of conventional cancer therapy, the ability to more closely examine intra- and interpatient heterogeneity is crucial to understanding cancer biology and developing personalized therapies.4 Consequently, there has been an explosion of interest in creating patient-derived organoids to enable patient-specific cancer drug testing.5 In many cases, this could be achieved without the need for additional invasive procedures; tissue biopsies are routinely performed as part of cancer diagnosis, and a sample could be set aside to generate organoids.

Organoids can be derived from pluripotent stem cells (including embryonic stem cells or induced pluripotent stem cells) or neonatal or adult stem cells from healthy or diseased tissue.1,2 Cancer organoids have been generated from a range of human cancer tissues and cell lines including colon, pancreas, prostate, liver, breast, bladder and lung.6-12This year, a research group led by Hongjun Song, Professor of Neuroscience at the Perelman School of Medicine at the University of Pennsylvania, published a report in Cell detailing methods for the rapid generation of patient-derived glioblastoma organoids.13

Fresh tumor specimens were removed from 53 patient cases to produce microdissected tumor pieces that could survive, develop a spherical morphology and continuously grow in culture for at least two weeks (Figure 1). The production of glioblastoma organoids was achieved while maintaining a high level of similarity between the organoids and their parental tumors, with the expression levels of specific markers showing stability over long-term culture (48 weeks). Importantly, native cell-cell interactions were preserved by avoiding mechanical and enzymatic single-cell dissociation of the resected tumor. As Song explains, this was achieved on a clinically relevant timescale: “Normally, the treatment for glioblastoma patients starts one month after surgery. The idea is that glioblastoma organoids can be generated within two weeks and subjected to testing of different treatment strategies to come up with the best option for a personalized treatment strategy.”

Figure 1: Glioblastoma organoid generation, from fresh tumor pieces to frozen spherical organoids. Image used with permission from Jacob et al. 2020.

One concern with organoid formation and expansion is the potential variability of the serum or Matrigel that can exist across batches and sources, creating variable exogenous factors that could cause the organoid to divert. This ultimately compromises reproducibility, a major bottleneck of current organoid systems.2,13 To avoid this source of error, Song’s group used an optimized and defined medium devoid of variable factors that could contribute to the clonal selection of specific cell populations in culture.

Glioblastoma is the most prevalent primary malignant brain tumor in adults,14
and having glioblastoma organoids available for research would present significant opportunities, explains Song: “They can be used to test different drugs based on mutation profiles and to investigate mechanisms underlying tumor progression, drug sensitivity and resistance.” While the accuracy of these predictions would need to be verified, researchers hope that patient-derived organoids will be used to help inform oncologists, accelerate drug discovery, and lead to better clinical trial design.

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Live-Cell Monitoring: Optimizing Workflows for Advanced Cell Models 

As cell-based assays become technically more complex, the need to holistically capture dynamic and sometimes subtle cellular events becomes ever more important. By providing real-time imaging data of cellular events without disturbing the sample during the cell culture workflow, live-cell monitoring can support the optimization of these advanced models. Download this whitepaper to discover how live-cell monitoring can support such optimization, with a breadth of applications.

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Understanding liver cancer initiation through organoid research


Liver cancer is the fourth most lethal malignancy worldwide, where poor prognosis is largely attributed to late diagnosis of the disease.14,15 Understanding the mechanisms underlying liver cancer initiation would improve diagnostic and therapeutic strategies; however, it has been a difficult area to study due to the lack of suitable research models. Liver cancer organoids would provide a unique opportunity to mimic the early stages of cancer in vitro and could enable researchers to verify whether specific mutations are indeed capable of driving liver cancer initiation.

For this to be achieved, techniques for the culture and genetic manipulation of primary human hepatocytes need to be refined. This has mostly been pursued through the culture of liver progenitors or fetal hepatocytes, which facilitate studies of liver cancers related to stem cells.16-18 To address the need for organoids derived from functional hepatocytes, researchers across 14 universities, research institutes and hospitals in China and Japan collaborated to genetically engineer reprogrammed human hepatocytes.18 The study, published in Nature Cell Biology, details the successful generation of organoids that represented two major types of liver cancer (hepatocellular carcinoma: HCC and intra-hepatic cholangiocarcinoma: ICC), derived from directly reprogrammed human hepatocytes (hiHeps).

Lead author Lulu Sun, of the Shanghai Institute of Biochemistry and Cell Biology at the University of Chinese Academy of Sciences, provides an overview of how the liver cancer organoids were developed: “Genomic aberrations begin to occur during cancer initiation, and the normal cells gradually became malignant. We modeled this process by introducing HCC/ICC-related oncogenes into the organoids with a lentivirus. Oncogenes were selected based on their mutation frequency and previous results in animals
.” Sun notes that gradual changes in cell and organoid morphology were observed in vitro, along with changes in the expression of HCC-related markers, before the organoids were transplanted to inspect their malignancy in vivo: “We cultured these organoids in vitro for about two weeks and transplanted them into the liver lobule of immunodeficient mice. Six to eight weeks later, they formed features identical to HCCs.”

Even though numerous oncogenes have been identified through whole genome sequencing, it has been difficult to determine whether they can drive the initiation of human liver cancers. Ultrastructural analyses revealed that c-Myc, a well-known oncogene, induced HCC-initiation and a unique cellular phenotype in the hiHep organoids. In these cells, mitochondria were in unusually close contact with endoplasmic reticulum membranes. This excessive coupling between mitochondria and the endoplasmic reticulum (referred to as a MAM phenotype) was shown to facilitate HCC-initiation and when blocked, prevented the progression towards HCC, says Sun: “Not only were the expression levels of HCC-related genes in organoids reduced, but significantly reduced cancers were formed in mice.”

Resolving these alterations in mitochondrial organization represents a new potential approach to liver cancer therapies, and possibly others, Sun explains: “
Restoration of a proper MAM interface may be a useful approach in preventing c-MYC-initiated HCCs. In addition, recently, an increasing number of works captured ultrastructural alterations, including MAMs, in the course of diseases including Alzheimer’s disease and fatty liver diseases. Our results showed that the alterations between communications of organelles may also contribute to the cancer initiation process.”

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All About Organoids

Organoids are 3D cell clusters with the structural and functional features of an organ, and can be generated from induced pluripotent stem cells (iPSCs) or adult stem cells acquired from a specific patient. Consequently, organoids make it possible to study the impact of a drug on a specific disease, even a person’s own disease – they are changing the face of research and medicine as we know it. Download this eBook to discover more about organoids including their analysis and how they are effecting personalized medicine.

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Looking ahead: Organoid biobanks and efforts to improve reproducibility


Organoids have the potential to be a powerful research tool, and the possibilities have captured imaginations across disciplines. “Oligocortical spheroids” are being developed to serve as important platforms for studies of myelination in the central nervous system19 while
snake venom glands organoids could aid in the development of treatments for snake bite.20 In cancer research, organoids look set to stay, and researchers are seeking ways to maximize the impact of this growing field.

The development of organoid biobanks is regarded to be a critical step towards progressing personalized medicine and could enable greater access and collaboration across research groups. Organoid biobanks have been established for a number of cancers, and Song’s research group has added to the list with the generation of a glioblastoma organoid biobank.13 These “living biobanks” are valuable resources that provide a representative collection of well-characterized models for basic cancer research, drug screening and personalized medicine.21 Progress in the field will also need to be supported by tools and methods that can assess organoid composition, such as the use of single cell RNA-sequencing to score organoids for reproducibility, faithfulness and quality.22

References:


1.  
Foley, K. E. (2017). Organoids: A better in vitro model. Nature Methods, 14(6), 559–562. https://doi.org/10.1038/nmeth.4307

2.   Huch, M., Knoblich, J. A., Lutolf, M. P, et al. (2017). The hope and the hype of organoid research. Development, 144(6), 938–941. https://doi.org/10.1242/dev.150201

3.   Hutchinson, L., & Kirk, R. (2011). High drug attrition rates—Where are we going wrong? Nature Reviews Clinical Oncology, 8(4), 189–190. https://doi.org/10.1038/nrclinonc.2011.34

4.   Fan, H., Demirci, U., Chen, P. (2019). Emerging organoid models: Leaping forward in cancer research. Journal of Hematology & Oncology, 12(142). https://jhoonline.biomedcentral.com/articles/10.1186/s13045-019-0832-4

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5.   Drost, J., Clevers, H. (2018). Organoids in cancer research. Nature Reviews Cancer, 18(7), 407–418. https://doi.org/10.1038/s41568-018-0007-6

6.   van de Wetering, M., Francies, H. E., Francis, J. M., et al. (2015). Prospective Derivation of a Living Organoid Biobank of Colorectal Cancer Patients. Cell, 161(4), 933–945. https://doi.org/10.1016/j.cell.2015.03.053

7.   Boj, S. F., Hwang, C.-I., Baker, L. A., et al. (2015). Organoid Models of Human and Mouse Ductal Pancreatic Cancer. Cell, 160(1–2), 324–338. https://doi.org/10.1016/j.cell.2014.12.021

8.   Puca, L., Bareja, R., Prandi, D., et al. (2018). Patient derived organoids to model rare prostate cancer phenotypes. Nature Communications, 9(1), 2404. https://doi.org/10.1038/s41467-018-04495-z

9.   Broutier, L., Mastrogiovanni, G., Verstegen, M. M., et al. (2017). Human primary liver cancer–derived organoid cultures for disease modeling and drug screening. Nature Medicine, 23(12), 1424–1435. https://doi.org/10.1038/nm.4438

10. Sachs, N., de Ligt, J., Kopper, O., et al. (2018). A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity. Cell, 172(1–2), 373-386.e10. https://doi.org/10.1016/j.cell.2017.11.010

11. Lee, S. H., Hu, W., Matulay, J. T., et al. (2018). Tumor Evolution and Drug Response in Patient-Derived Organoid Models of Bladder Cancer. Cell, 173(2), 515-528.e17. https://doi.org/10.1016/j.cell.2018.03.017

12. Kim, M., Mun, H., Sung, C. O., et al. (2019). Patient-derived lung cancer organoids as in vitro cancer models for therapeutic screening. Nature Communications, 10(1), 3991. https://doi.org/10.1038/s41467-019-11867-6

13. Jacob, F., Salinas, R. D., Zhang, D. Y., et al. (2020). A Patient-Derived Glioblastoma Organoid Model and Biobank Recapitulates Inter- and Intra-tumoral Heterogeneity. Cell, 180(1), 188-204.e22. https://doi.org/10.1016/j.cell.2019.11.03

14. Ostrom, Q. T., Gittleman, H., Truitt, G., et al. (2018). CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2011–2015. Neuro-Oncology, 20(suppl_4), iv1–iv86. https://doi.org/10.1093/neuonc/noy131

15. Bruix, J., Han, K.-H., Gores, G., et al. (2015). Liver cancer: Approaching a personalized care. Journal of Hepatology, 62(1), S144–S156. https://doi.org/10.1016/j.jhep.2015.02.007

16. Hu, H., Gehart, H., Artegiani, B., et al. (2018). Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids. Cell, 175(6), 1591-1606.e19. https://doi.org/10.1016/j.cell.2018.11.013

17. Zhang, K., Zhang, L., Liu, W., et al. (2018). In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity. Cell Stem Cell, 23(6), 806-819.e4. https://doi.org/10.1016/j.stem.2018.10.018

18. Sun, L., Wang, Y., Cen, J., et al, (2019). Modelling liver cancer initiation with organoids derived from directly reprogrammed human hepatocytes. Nature Cell Biology, 21(8), 1015–1026. https://doi.org/10.1038/s41556-019-0359-5

19. Madhavan, M., Nevin, Z. S., Shick, H. E., et al. (2018). Induction of myelinating oligodendrocytes in human cortical spheroids. Nature Methods, 15(9), 700–706. https://doi.org/10.1038/s41592-018-0081-4

20. Post, Y., Puschhof, J., Beumer, J., et al. (2020). Snake Venom Gland Organoids. Cell, 180(2), 233-247.e21. https://doi.org/10.1016/j.cell.2019.11.038

21. Calandrini, C., Schutgens, F., Oka, R., et al. (2020). An organoid biobank for childhood kidney cancers that captures disease and tissue heterogeneity. Nature Communications, 11(1), 1310. https://doi.org/10.1038/s41467-020-15155-6

22. Subramanian, A., Sidhom, E.-H., Emani, M., et al. (2019). Single cell census of human kidney organoids shows reproducibility and diminished off-target cells after transplantation. Nature Communications, 10(1), 5462. https://doi.org/10.1038/s41467-019-13382-0





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