(PHILADELPHIA) – Using advanced new microscopy techniques in concert with sophisticated transgenic technologies, scientists at The Wistar Institute have for the first time created three-dimensional, time-lapse movies showing immune cells targeting cancer cells in live tumor tissues. In recorded experiments, immune cells called T cells can be seen actively migrating though tissues, making direct contact with tumor cells, and killing them.
Insights from this new view of the body's on-board defenses against cancer may open the way for improved immunotherapies to treat the disease.
With a series of movies made under different experimental conditions, the researchers resolved important questions about the mechanisms by which T cells act against cancer. Their findings, published online November 20, will appear in the November 27 print edition of The Journal of Experimental Medicine.
"We've taken the first real-time look at the final phase of the immune system's response to cancer cells," says Wolfgang Weninger, M.D., an assistant professor in the Immunology Program at Wistar and senior author on the new study. "This has enabled us to delineate the rules of T cell migration and engagement directly within the intricate microenvironment of tumors."
The scientists used a leading-edge instrument called a two-photon microscope, able to peer inside living tissues. The microscope tracked and recorded the movements in three dimensions over time of T cells in a transgenic mouse developed by Weninger and Ulrich von Andrian at Harvard Medical School in which the cells fluoresce green. In addition, for this study, tumor cells in the mice were engineered to fluoresce blue.
In one group of the mice, a vaccine developed by Wistar professor and study co-author Hildegund C.J. Ertl, M.D., was used to activate the T cells that recognize a molecule on the surface of the tumor cells. Such molecules are referred to by immunologists as antigens. In a second group, no such vaccine was given.
Movies captured with the two-photon microscope then recorded the unfolding scene in the so-called tumor microenvironment. How would the green T cells behave in the two groups of mice?
"In the animals that received the vaccination, we saw two waves of activity after the T cells entered the tumor microenvironment," Weninger says. "Early on, the T cells didn't actively migrate through the tissue. This, we found, was because they were interacting directly with the tumor cells in place. In several instances, we were actually able to see the tumor cells dying – the first time that has ever been observed in real-time in living animals. Then, once the tumor cells had been destroyed, we saw a dramatic change in the behavior of the activated T cells. They began to migrate actively, searching for any other tumor cells that might remain in the area."
In contrast, in the mice that did not receive the vaccination, the T cells were much sparser and, importantly, distinctly inactive in their migration. Consequently, tumor cell death was very rare under these conditions.
The researchers then designed a second set of experiments to complement the first. In the first set of experiments, the T cells were varied in two groups of mice while the target tumor cells were uniform. In the second set, the T cells were uniform while the tumor cells varied in two groups of mice according to whether or not they presented a specific antigen to which the T cells would respond.
T cells were removed from mice without tumors, activated in the test tube, and then reintroduced into mice carrying tumors that either did or did not express the antigen. This procedure, referred to as adoptive transfer, is an immunotherapy strategy against cancer being tested in a number of human clinical trials. In some of those trials, a patient's own T cells are removed, tested for their ability to recognize the patient's cancer cells, activated and expanded greatly in numbers in the laboratory, and then returned to the patient.
The hope in these trials is that these enhanced T cell populations will specifically target and destroy the patient's cancer. To date, despite a few remarkable successes, these trials have proven frustratingly uneven. Greater insights into the mechanisms of interaction between T cells and tumor cells could provide vital new information to advance these efforts.
"Through adoptive transfer," Weninger explains, "we were able to compare two situations, one in which the T cells recognize something on the tumor and one in which they don't. When the T cells recognized the antigen, they interacted directly with the tumor cells. After tumor cell destruction, they became actively migratory, hunting for more tumor cells. In the absence of antigen, the T cells did not interact with tumor cells, and could not sustain an active migratory behavior within tumors."
"There are several significant conclusions from these experiments," Weninger says. "First, it is now possible to visualize the behavior of the individual cellular components of the tumor microenvironment in real-time. Second, we have demonstrated that T cells physically interact with tumor cells, which had not been shown before. Finally, it's the presence of antigen that determines how T cells migrate and interact with the tumor cells.
"These experiments set the basis for unraveling the molecular requirements for T cell migration and T cell-tumor cell interactions. We should then be able to use results from this research to further improve immunotherapeutic strategies against cancer in patients."
The lead author on The Journal of Experimental Medicine study is Paulus Mrass, M.D. In addition to senior author Weninger, the other Wistar-based co-authors are Lai Guan Ng, Sachin Daxini, Marcio Lasaro, Amaya Iparraguirre, Lois L. Cavanagh, Ph.D, and Ertl. The remaining co-authors are Hajime Takano and Philip G. Haydon at the University of Pennsylvania and Ulrich H. von Andrian at the CBR Institute for Biomedical Research, Harvard Medical School.
Support for the research came from the National Institutes of Health, the W.W. Smith Charitable Trust, the Max Kade Foundation, and the Cancer Research Institute. Haydon has equity interest in the company Prairie Technologies Corporation, which builds and markets the two-photon microscope used in this project.
The Wistar Institute is an international leader in biomedical research, with special expertise in cancer research and vaccine development. Founded in 1892 as the first independent nonprofit biomedical research institute in the country, Wistar has long held the prestigious Cancer Center designation from the National Cancer Institute. Discoveries at Wistar have led to the creation of the rubella vaccine that eradicated the disease in the U.S., rabies vaccines used worldwide, and a new rotavirus vaccine approved in 2006. Wistar scientists have also identified many cancer genes and developed monoclonal antibodies and other important research tools. Today, Wistar is home to eminent melanoma researchers and pioneering scientists working on experimental vaccines against flu, HIV, and other diseases. The Institute works actively to transfer its inventions to the commercial sector to ensure that research advances move from the laboratory to the clinic as quickly as possible. The Wistar Institute: Today's Discoveries – Tomorrow's Cures. On the web at www.wistar.org.
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