Giving organoids room to grow

technology feature Giving organoids room to grow These 3D structures derived from human cells can be an improvement over simple cell lines, but organoids can still lack important physiological cues for development. Finding the right in vivo environment can take things a step further. Michael Eisenstein P ancreatic cancer is a stealthy foe, typically only revealing itself when effective treatment options are limited—or nonexistent. Roughly 90% of patients receive their diagnosis at advanced stages of disease, when surgery is no longer an option. David Tuveson’s team at the Cold Spring Harbor Laboratory hopes to flush this disease out of hiding with sophisticated models that can essentially re-enact the earliest stages of onset and progression for actual patients’ tumors: ‘organoids,’ transplanted in vivo. While cell lines are often the fastest and most economical tool for exploring biological questions, they can also be a poor surrogate for complex living organisms. These cells are separated from their physiological context, grown on flat artificial surfaces, and often contain extensive genetic abnormalities that allow them to grow continuously in such conditions. For cancer research in particular, such cultures bear little resemblance to the diverse and complex cellular ecosystems found within real tumors. Organoids are 3D tissues formed from patient-derived cells, which are cultivated in a manner that promotes assembly into multicellular structures that mirror key features of their origin tissue. Under the right conditions, researchers can generate organoids that replicate components of the liver, kidney, intestine, and brain—as well as virtually any tumor. “You can get an organoid that looks a lot like a human cancer does under the microscope,” says Jatin Roper, who uses these models to study colorectal cancer at Duke University. Although powerful on their own, organoids grown in a dish can only develop so far without guidance from the symphony of physiological cues produced in the body. To unlock this potential, researchers have been transplanting organoids into rodent hosts. In vivo, the organoids can flourish and offer an unprecedented view of diverse biological processes. “We can recapitulate the early stages of pancreatic cancer, before it invades and spreads,” says Tuveson. “That’s valuable because we don’t really see humans with early pancreatic cancer.” Room to grow. Transplanting organoids into the right environment can help them reach even greater research potential. Credit: Chris Winsor / Moment / Getty The findings from these models could enable earlier diagnoses or reveal new treatment options. Many other investigators are now turning to similar transplantation systems to study organ development and disease pathology, and even laying preclinical groundwork for organoid-based regenerative medicine. However, for these implants to flourish, researchers must also ensure that the organoids are finding the supportive conditions they need to feel at home within their rodent recipient. No place like home Left to their own devices in vitro, organoids can quickly self-assemble into relatively complex structures. For example, Takanori Takebe’s team at Cincinnati Children’s Hospital routinely converts human pluripotent stem cells into ‘liver buds’— highly organized assemblies of multiple cell types that resemble the developing embryonic liver. But as they grow, these organoids are threatened by starvation due to lack of a working circulatory system. Lab Animal | VOL 48 | JULY 2019 | 193–195 | www.nature.com/laban “Maintenance is really challenging in vitro,” says Takebe, “After 24 days of culture, they’re dying off.” Other organoid types can be more resilient, but nevertheless fail to reach their full functional potential outside the context of a living body—for example, tumor organoids comprised primarily of cancer cells may lack essential features that contribute to growth and response to therapy. “The traditional understanding of cancer was that it is a genetic disease driven by signaling pathways,” says Roper. “But we’re realizing more and more that many other cell types in the body are also responsible for cancer progression.” Indeed, there is now considerable research into how the ‘tumor microenvironment’ formed by a patient’s vasculature, immune system, and other surrounding cells influences pathology. Transplantation models allow researchers to essentially plug immature organoids into living systems, thereby allowing them to develop further and even integrate with their host. “The complexity we observe 193 technology feature is remarkable,” says Jason Spence of the University of Michigan, who works extensively with intestinal organoids. “Organoids remodel from a very simple fetal-like structure to a complex tissue with crypts and villi that form after transplantation.” However, researchers must overcome a number of technical challenges to ensure the stable integration, or ‘engraftment’, of these implants. As with any real-estate deal, location is key. Early in vivo efforts tended to entail implantation beneath the surface of the skin, a site that is easy to access and monitor. Such subcutaneous implants may survive and grow more robustly than they would in vitro, but they are still vulnerable to arrested development due to a lack of tissue-specific growth and developmental cues. Orthotopic transplantation, in which the organoid is placed into its native tissue environment or a near equivalent, tends to yield better, more physiological results, explains Toshiro Sato, a researcher at Keio University who was among the early pioneers of the organoid field. “But orthotopic transplantation is less accessible than subcutaneous transplantation, and thus technically more difficult.” As a postdoc in Ömer Yilmaz’s lab at MIT, Roper helped devise a strategy that uses colonoscopy to guide the injection of colorectal organoids at specific sites within the mouse intestine1, a technique that requires specialized equipment and expertise. But the results are worth the effort. “We can model early tumors called adenomas as well as the metastatic process, from the initiation of the tumor through invasion of the muscle layer and spreading to the liver,” Roper says. Other organoids pose an even more daunting challenge. For example, Hongyan Zou and colleagues at the Icahn School of Medicine at Mount Sinai recently grappled with the implantation of large organoids modeling the human dorsal forebrain in neonatal mice2. This required a delicate surgical procedure to ensure that there was room to implant the organoid within the skull without seriously damaging the surrounding tissue. But Zou’s efforts ultimately proved successful—“the engraftment rate was close to 100%,” she says, noting that they subsequently observed efficient infiltration of mouse blood vessels into the newly transplanted organoids. That being said, some organoids can still flourish at non-orthotopic si (...truncated)