How Organoids Are Fueling Infectious Disease Research and Personalized Medicine

There are two fundamental questions that persist across all subfields of biology: “What are the cues that govern the formation and stability of the differentiated state, and can we recapitulate morphogenesis in culture?”¹ These questions are important because having the ability to recapitulate tissue function and dissect disease progression in vitro opens many doors for drug discovery and allows researchers to explore questions that can’t be answered in vivo.

Alongside efforts to recapitulate tissue function, there has been an increasing appreciation for the importance of 3D architecture in functional differentiation.¹ While 2D cell cultures have been useful in many ways, they differ from in vivo conditions significantly, thereby making it difficult to translate findings to the clinic. Similarly, animal studies have been critical to enabling progress across healthcare sectors; however, species differences in absorption, distribution, metabolism, excretion and toxicity (ADMET) limit predictivity. Together with the notoriously high cost and failure rate of drug development, there is an urgent need for more predictive research models across all fields.

Here, we discuss organoids in the context of personalized medicine and infectious disease and explore how they provide a new window into disease and fuel the discovery of new therapeutics.

 
Organoids used to pursue a more personalized approach to medicine

To address the issue of treatments working well for some patients, but not for others, there has been a move towards pursuing a more personalized approach to medicine – particularly for cancer research. Subsequently, approaches to the development of cancer therapeutics have included drugs targeted at specific mutations; for instance, EGFR and PIK3CA-targeted drugs.^4,5 Unfortunately, however, these targeted drugs are not always effective in treating the tumor with the specific mutation, highlighting a knowledge gap that exists in understanding tumor responses.⁶

Patient-derived organoids (PDOs) have the potential to make personalized medicine significantly more successful, by serving as predictors for individualized tumor treatment responses. Partly, this is because of their convenient development timeline; personalized organoids can be grown within weeks from the time of biopsy, providing a way forward for precision medicine.⁷ Ovarian cancer PDOs, for example, have shown promise as important tools for clinical decision making; 36 whole-genome characterized PDOs from 23 patients were studied, and organoid responses to chemotherapeutics were compared with those of the patients. Organoids were subject to a range of treatments, including drugs that would not be expected to be effective for the tumor type (based on whole-genome sequencing data).⁸ Importantly, clinical response scores and PDO drug responses to a combination treatment were correlated, and a high responsiveness to at least one tested drug was identified. PDO subtypes can also be genetically manipulated, allowing researchers to probe the effect of individual genes and entire gene cassettes on other gene expression patterns, functions and interactions, as well as drug effects across a greater variety of disease states and genetic environments.^9,10

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Tumor heterogeneity remains one of the biggest challenges for cancer treatment. As a result of this heterogeneity, PDOs derived from different locations within the same tumor show differential responses. ⁸ With this in mind, one might wonder how to go about defining an “acceptable” level of predictivity. For Else Driehuis, researcher at the Clevers group at Hubrecht Institute and clinical molecular biologist in pathology in training at the University Medical Center Utrecht, the answer is straightforward: “I believe a reasonable measure is quite simple actually: it should perform better than what we have now. Simply, we can show that for a particular patient subset, for a particular drug, we can do better than the current selection criteria. That criteria may simply be a tumor type and stage in the clinic. Let’s say all patients with disease X in stage Y get radiotherapy as the standard of care. 40% of them will benefit, the others don’t. If, based on organoid screens we can get that number up, then implementing the use of organoids should be worth it.”

For Driehuis and her colleagues, this goal has driven the development of a wide range of organoids, including pancreatic cancer organoids^11-13 and oral mucosal organoids as potential platforms for personalized cancer therapy.¹⁴ The drive to implement organoids in healthcare is also supplemented with practical considerations: “However, we should weigh the benefits against potential downsides such as costs and treatment delays, etc – they are important aspects to consider,” says Driehuis. Other groups have suggested that the issue of heterogeneity could also be mitigated somewhat in clinical settings by developing multiple organoids from different locations within the tumor. ⁸ In this way, cancer subtypes can be analyzed side-by-side for a comparison of drug response.

“In another example,” adds Driehuis, “the treatment of patients with metastasized colorectal cancer with EGFR-targeting antibodies is nowadays only allowed if no mutations are identified in downstream genes such as KRAS. Still, amongst that group of patients that get the drug, we see quite variable response rates. If we can improve that with organoid screens, should we not ask ourselves if that should be the standard instead of genetic screening?”

The implementation of organoids will require usability, explains Driehuis: “If organoid screens are to be implemented in the clinic, they need to work in a routine diagnostic setting. If we can get those challenges manageable though, patients that will not benefit can then perhaps receive another treatment, or at the very least not be harmed by the side-effects of these treatments.”

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Organoids serving as a window to viral mechanisms

“For infectious diseases, that’s a much narrower field. There are really only a few infectious diseases that have been modeled in organoids,” says Jürgen Knoblich, scientific director at the Institute of Molecular Biotechnology, Austrian Academy of Sciences. “Most is still in a research-phase. But organoids are playing a major role now in the SARS-CoV-2 epidemic.” Indeed, organoids are being used to help unravel the mechanisms of SARS-CoV-2 infection. In May 2020 – only months after the pandemic began – SARS-CoV-2 was shown to infect human kidney organoids, resulting in infectious viral progeny.¹⁵ Their findings lay a foundation for exploring multi-organ dysfunction in patients with COVID-19. Organoids are also conducive to high-throughput drug screening for SARS-CoV-2, as shown through the use of lung organoid models derived from human pluripotent stem cells.¹⁶ Organoids can also be used to compare the susceptibility of different species to a virus, which could be useful for assessing the infectivity of emerging flu viruses in humans.¹⁷

As discussed in an aptly named review article “COVID-19: organoids go viral”, organoids are becoming increasingly popular for infectious disease studies. ¹⁶ “The general reason for that,” explains Knoblich, “is because many pathogens are very host-specific, meaning that the traditional drug development pipeline, which often involves animal models, does not work so well – or only works if you involve primates. And of course, nobody wants to do that.” Knoblich’s own career in organoid work has revolved around developing cerebral organoids for modeling microcephaly, which has had major implications for modeling the Zika virus.¹⁸ Organoids were used to show that the Zika virus can cause microcephaly, which helped the World Health Organization class the situation as a global pandemic.

More recently, Knoblich and colleagues extended these findings, whereby they uncovered key differences in encephaly-inducing mechanisms caused by the Zika virus, versus those of the herpes simplex virus.¹⁸ “We found that although, medically, both viruses have the same effect – they cause microcephaly – the actual cellular mechanism is fundamentally different. Zika viruses invade the cell and causes them to die. Herpes simplex viruses also invades the cells, but changes their shape. And so, if you wanted to treat this, you would have to use fundamentally different approaches.”

One key finding was that both viruses fail to potently induce an important pathway: the type I interferon system. “Normally, a cell will detect the virus, and start the interferon response. It will start to reduce its own production of proteins, so that the virus cannot hijack that, and it will start to degrade everything in the cell that it doesn't like, and it will stop dividing, and go into defense mode.” This process, which relies on intricate and rapid machinery, was observed in real time in the organoids. However, the fetal brain organoids were different, says Knoblich: “Very few cells showed this response, and it was delayed. The brain of a human fetus is not sufficiently protected against viruses. We reasoned that the final outcome of the innate immune response is the production of interferons, and so we applied interferons to the organoids.” Organoid defects could be rescued with the application of interferons, and interestingly, different subtypes of interferons were required (interferon-ß for Zika virus, interferon-α2 for the herpes simplex virus).¹⁸ These findings reveal important differences between the viruses that have not been observed in 2D cultures.

Working towards implementing organoids in healthcare

Organoids are expected to play a major role in the future of healthcare; they have the potential to improve personalized medicine and serve as key platforms for critical studies of infectious disease and many other disciplines. For organoids to fulfil their potential across a range of research areas, however, there is a need for greater standardization, explains Driehuis: “We need to make screening quick, reproducible, easy and feasible for implementation in healthcare. That’s the challenge ahead I believe, and we aren’t there yet. It’s quite a big challenge actually, because first of all, we’re dealing with living cells – so the variability introduced by different technicians and conditions is likely to have more of an effect than in DNA library preparation performed prior to DNA sequencing, for example.”

Other challenges are specific to studies of pathogenic viruses, such as required safety levels complicating the use of robotics in high-throughput drug screens.¹⁹ Also, not all infectious diseases are suited to organoid studies, explains Knoblich: “We studied another virus that’s not in the paper, a cytomegalovirus which is actually a much bigger health problem than the Zika virus. With the cytomegalovirus, we could recapitulate the pathology, but not the microcephaly. It just takes too long; it takes months and months for the virus to spread in the organoid. Experimentally, it’s just a nightmare, as it’s logistically very difficult. So that’s why we settled on the herpes simplex virus; it’s very aggressive and within a few days, the whole organoid is destroyed.”

Looking ahead, the application of genetic engineering is likely to open many doors; for instance, by enabling manipulations of specific signaling pathways in cancer, and the identification of infected cells through the use of fluorescent reporters of different genes. ^19,20 Improving the faithful representation of certain tissues remains a priority across the board, such as finding ways to develop more complex co-cultures that include immune cells. PDOs, on the other hand, have their own challenges, says Driehuis: “If you take a piece of tumor, it doesn’t only contain tumor cells, but also immune cells, connective tissue and perhaps even tumor-adjacent healthy tissue. We need to figure out a way to look for the effect on the tumor cells only. I believe potentially imaging and artificial intelligence-driven algorithms can help us out there.”

References

1. Simian M, Bissell MJ. Organoids: A historical perspective of thinking in three dimensions. J Cell Biol. 2016;216(1):31-40. doi: 10.1083/jcb.201610056

2. Lancaster MA, Knoblich JA. Organogenesis in a dish: Modeling development and disease using organoid technologies. Science. 2014;345(6194):1247125-1247125. doi: 10.1126/science.1247125

3. Shamir ER, Ewald AJ. Three-dimensional organotypic culture: experimental models of mammalian biology and disease. Nat Rev Mol Cell Biol. 2014;15(10):647-664. doi: 10.1038/nrm3873

4. Sukrithan V, Deng L, Barbaro A, Cheng H. Emerging drugs for EGFR-mutated non-small cell lung cancer. Expert Opin Emerg Dr. 2018;24(1):5-16. doi: 10.1080/14728214.2018.1558203

5. DiGrande S. FDA Approves first breast cancer drug to target PIK3CA mutation plus companion diagnostic test. AJMC. https://www.ajmc.com/view/fda-approves-first-breast-cancer-drug-to-target-pik3ca-mutation-plus-companion-diagnostic-test. Published May 29, 2019. Accessed August 26, 2021.

6. Aboulkheyr Es H, Montazeri L, Aref AR, Vosough M, Baharvand H. Personalized cancer medicine: an organoid approach. Trends Biotechnol. 2018;36(4):358-371. doi: 10.1016/j.tibtech.2017.12.005

7. Clevers H. Modeling development and disease with organoids. Cell. 2016;165(7):1586-1597. doi: 10.1016/j.cell.2016.05.082

8. Witte CJ de, Valle-Inclan JE, Hami N, et al. Patient-derived ovarian cancer organoids mimic clinical response and exhibit heterogeneous inter- and intrapatient drug responses. Cell Rep. 2020;31(11). doi: 10.1016/j.celrep.2020.107762

9. Monteil V, Kwon H, Prado P, et al. Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2. Cell. 2020;141(4). doi: 10.1016/j.cell.2020.04.004

10. Driehuis E, Clevers H. CRISPR/Cas 9 genome editing and its applications in organoids. Am J Physiol-Gastrointest  Liver Physiol. 2017;312(3):G257-G265. doi: 10.1152/ajpgi.00410.2016

11. Driehuis E, van Hoeck A, Moore K, et al. Pancreatic cancer organoids recapitulate disease and allow personalized drug screening. PNAS. 2019;116(52):26580-26590. doi: 10.1073/pnas.1911273116

12. Driehuis E, Kretzschmar K, Clevers H. Establishment of patient-derived cancer organoids for drug-screening applications. Nat Protoc. 2020;15(10):3380-3409. doi: 10.1038/s41596-020-0379-4

13. Tiriac H, Belleau P, Engle DD, et al. Organoid profiling identifies common responders to chemotherapy in pancreatic cancer. Cancer Discov. 2018;8(9):1112-1129. doi: 10.1158/2159-8290.CD-18-0349

14. Driehuis E, Kolders S, Spelier S, et al. Oral mucosal organoids as a potential platform for personalized cancer therapy. Cancer Discov. 2019;9(7):852-871. doi: 10.1158/2159-8290.CD-18-1522

15. Han Y, Duan X, Yang L, et al. Identification of SARS-CoV-2 inhibitors using lung and colonic organoids. Nature. 2021;589(7841):270-275. doi: 10.1038/s41586-020-2901-9

16. Clevers H. COVID-19: Organoids go viral. Nat Rev Mol Cell Biol. 2020;21(7):355-356. doi: 10.1038/s41580-020-0258-4

17. Lancaster MA, Renner M, Martin C-A, et al. Cerebral organoids model human brain development and microcephaly. Nature. 2013;501(7467):373-379. doi: 10.1038/nature12517

18. Krenn V, Bosone C, Burkard TR, et al. Organoid modeling of Zika and herpes simplex virus 1 infections reveals virus-specific responses leading to microcephaly. Cell Stem Cell. 2021;28(8):1362-1379.e7. doi: 10.1016/j.stem.2021.03.004

19. Geurts MH, van der Vaart J, Beumer J, Clevers H. The organoid platform: promises and challenges as tools in the fight against COVID-19. Stem Cell Rep. 2021;16(3):412-418. doi: 10.1016/j.stemcr.2020.11.009

20. Hofer M, Lutolf MP. Engineering organoids. Nat Rev Mater. 2021;6. doi: 10.1038/s41578-021-00279-y