In the mid-2000s researchers found that they could take stem cells outside the body and coax them to assemble into three-dimensional masses—tiny, rudimentary organs or tumors. It’s one of the most significant recent breakthroughs in science. With the right microenvironment, these cell cultures, called organoids, can show similar characteristics to their organ or tumor counterparts. This allows researchers to perform experiments that would be impossible in living models or in standard, two-dimensional cell cultures. Organoids are now complementing and advancing the approach to basic biological research and drug development.
Over the past decade, researchers have been furiously working to create and refine organoid technology, creating living tissues modeling those of the kidney, liver, gut, prostate, lung—even the brain—and more, as well as many kinds of tumors. One extraordinarily important class of organ was missing from the list until recently: immune organs like the lymph node or spleen.
According to Ankur Singh, Mechanical and Aerospace Engineering/Biomedical Engineering, the absence of immune organoids has been due to the organs’ incredible complexity, including their ability to respond uniquely to each disease. That has not stopped Singh. His lab is engineering the first immune organoid platform, a tool for solving the many mysteries of how the immune system works to fight infection and disease. In addition, Singh models how cells in the immune tissues become cancerous, with an eye toward finding new targets and therapies to stop deadly lymphomas.
A First of its Kind, Infection-Fighting Organoid
The immune organs are complex, and their processes are dynamic. Therefore, Singh has focused on one subanatomical structure of the lymph node—an exceptionally important one that continuously protects us from infection. The zone he’s interested in is made up of mostly B cells, the cells responsible for producing antibodies. When the body is under attack by a pathogen, the B cells cluster and transform into germinal centers. These centers activate and mutate B cells to generate disease-specific antibodies to fight the invading pathogen.
Singh’s group has been able to create organoids that represent these germinal centers, complete with B cells and supporting cells. The lab starts by taking naive B cells from mouse models or humans, cells that haven’t been programmed to respond to infections. These cells are immersed, with supporting cells, in a tunable hydrogel that Singh’s group developed. They can specially tailor the gel to mimic the natural environment of the immune tissues, so that the B cells begin to assemble and behave like they would in the body. The result is that, for the first time, Singh’s organoid can induce true germinal centers that can activate B cells outside of the body.
“It’s the first of its kind to be functional,” Singh says. “Although it has been possible to activate B cells ex vivo, the resulting cells don’t behave like germinal center B cells. Our organoids provide a multidimensional platform, with bona fide germinal center B cells that have been validated in number of ways. And with the hydrogel matrix, you can control which signals, how much you present them, what combination you create, where and for how long.”
Singh’s group has already shown how the organoid platform out-performs two-dimensional models. They are now working to hone the technology so that the organoids can produce antibodies specific to various diseases. This would greatly enhance the ability to study how the immune system succeeds or fails in fighting different infections as well as the development of vaccines.
“This technology has opened a gateway to unexplored areas of infection, immunity, and cancer,” Singh says. “I can imagine the immune organoid will be useful for fundamental immunology discoveries, for discovering immunotherapeutics against infectious agents, other immune diseases, and even the immunogenic or cancerous effects of chemicals (or hydrocarbons) in the environment. Also, what drugs and small molecule chemicals could mount some sort of immunity? All of those things could be studied in this organ system.”
A Lymphoma Designer System for Mimicking Individual Tumor Environments
Singh is also deeply interested in understanding how the same immune cells that protect us can become cancerous. A strong effort in his lab focuses on lymphoid malignancies, particularly lymphomas, which are highly prevalent, deadly, and often difficult to treat. The lymphomas Singh studies—non-Hodgkin lymphomas, tumors of the B and T cells—will take the lives of 20,000 people in 2017 alone. Prior to Singh’s work, no synthetic, designer tumor model for this lymphoma existed outside the body.
In 2013, Singh began working on this problem with Weill Cornell Medicine colleagues Ari M. Melnick, Hematology and Medical Oncology/Pharmacology, and Leandro Cerchietti, Hematology and Medical Oncology. “We discovered that lymphomas varied in their requirement of survival signals, based on which subclass they belonged to. Even within the same tumor subclass, two patient samples can be different,” Singh says. “So we engineered a designer system that was able to address that tumor heterogeneity.”
Using tumor cells, Singh can engineer a tumor-hydrogel organoid with synthetic biomaterials. The components of the hydrogel mimic those of the tumor microenvironment from individual patients and can selectively engage with surface proteins on tumor cells. Singh and Melnick, with support from the National Institutes of Health and the Department of Defense, are also looking into the effectiveness of a new class of inhibitor for these subclasses of tumors, testing the potential therapeutic on various engineered tumor organoids.
Singh’s group is currently in the process of moving the organoids from dishes to chips—small platforms that allow researchers to integrate and control relevant factors, such as fluidics from blood vessels or chemical gradients. This allows researchers to better mimic the organ’s natural environment. The chips are being designed to connect to each other. Theoretically, scientists could eventually simulate a body-like system made of organs-on-chips.
“Even in its current form as a subanatomical part—the antibody-forming germinal center—our organoid can be connected to an existing organ-on-a-chip model,” Singh says. “Then people can do developmental or therapeutic evaluation or immunogenic studies.”
Singh’s lab has received funding from the National Cancer Institute to develop a lymphoma-on-chip technology, called LETSSGo (Lymphoma-on-Chip Engineered Technology for Single-Organoid Sequencing and Genomics). This technology would help determine the causes of drug resistance in one of the most devastating subtypes of lymphoma. It will allow tumors to grow in the tissues while researchers collect integrated genomic analyses. “There are many questions that can be asked,” Singh says. “We’re looking for what factors affect resistance, one step at a time.”
Singh says one of the most rewarding things about his work is knowing that his lab’s technology can be used by others. “From the beginning, I wanted to understand this big black box of immunology and germinal centers. I took a deeper look into a structure I wanted to create and the functionality I wanted to achieve,” Singh says. “Now there are people across the states and abroad who are interested in using our technology because this is the first functional system. We have a lot more to prove, but we’re approaching a lot of breakthroughs, too.”
“That’s the power of the whole system. It’s leading to new discoveries and better validation of the therapeutic targets.”
Fast-Tracking Drug Development
Singh can create 200 tumor organoids in a day, and he’s working on a new technology that would increase it to 500 per minute, using automated approaches. With so many copies, researchers can test multiple treatments, alone or in combination, and conduct genetic tests almost simultaneously and with much more accuracy than in two-dimensional models. The organoids may also outpace animal models—where human tumors are engrafted in immunocompromised mouse models—removing an essential factor in the body’s natural response to the tumor.
Currently, there’s less than a 10 percent success rate for cancer therapeutics in clinical trials, and it can take upward of a decade and cost as much as $2 billion for a new drug to be approved for patients. With Singh’s organoid technologies, finding successful targets and therapeutics could be vastly accelerated. “That’s the power of the whole system,” Singh says. “It’s leading to new discoveries and better validation of the therapeutic targets.”
Singh’s organoids are a key part of Cornell’s PATh program (Progressive Assessment of Therapeutics), directed by Kristy L. Richards, Biomedical Sciences, Hematology and Medical Oncology at Weill Cornell Medicine, which aims to accelerate drug development for lymphomas and to model a better protocol for drug development in general.
“Cornell is a phenomenal place with potential for strong program-building,” Singh says. “In addition to incredibly intelligent students, we have a very unique programmatic strength, with the Veterinary School, the facilities we have, and then Weill Cornell Medicine. There are very few places in the country where I could have these kinds of collaborations.”