Real-time immune activity within tumours

Scientists have for the first time created three-dimensional, time-lapse movies showing immune cells targeting cancer cells in live tumour tissues.

Researchers at The Wistar Institute achieved the breakthrough using advanced new microscopy techniques in concert with sophisticated transgenic technologies.

In recorded experiments, immune cells, called T cells, can be seen actively migrating though tissues, making direct contact with tumour cells and killing them.

Insights from this new view of the body's on-board defences 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.

"We've taken the first real-time look at the final phase of the immune system's response to cancer cells," said senior author Wolfgang Weninger, an assistant professor in the Immunology Program at Wistar.

"This has enabled us to delineate the rules of T cell migration and engagement directly within the intricate microenvironment of tumours."

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, tumour cells in the mice were engineered to fluoresce blue.

Recording immune cells in action

In one group of the mice, a vaccine developed by Wistar professor and study co-author Hildegund Ertl was used to activate the T cells that recognise a molecule on the surface of the tumour 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 tumour microenvironment.

"In the animals that received the vaccination, we saw two waves of activity after the T cells entered the tumour microenvironment," Weninger explained.

"Early on, the T cells didn't actively migrate through the tissue.

"This, we found, was because they were interacting directly with the tumour cells in place.

"In several instances, we were actually able to see the tumour cells dying – the first time that has ever been observed in real-time in living animals."

He added: "Then, once the tumour cells had been destroyed, we saw a dramatic change in the behaviour of the activated T cells: they began to migrate actively, searching for any other tumour 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, tumour cell death was very rare under these conditions.

Second set of experiments

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 tumour cells were uniform.

In the second set, the T cells were uniform while the tumour 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 tumours, activated in the test tube, and then reintroduced into mice carrying tumours 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 recognise 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 tumour cells could provide vital new information to advance these efforts.

Finding out how T cells work

"Through adoptive transfer, we were able to compare two situations, one in which the T cells recognise something on the tumour and one in which they don't," Weninger said.

"When the T cells recognised the antigen, they interacted directly with the tumour cells.

"After tumour cell destruction, they became actively migratory, hunting for more tumour cells.

"In the absence of antigen, the T cells did not interact with tumour cells, and could not sustain an active migratory behaviour within tumours."

Weninger concluded that it is now possible to visualise the behaviour of the individual cellular components of the tumour microenvironment in real-time.

He added that his team have demonstrated that T cells physically interact with tumour cells for the first time, and also that it is the presence of antigen that determines how T cells migrate and interact with the tumour cells.

"These experiments set the basis for unravelling the molecular requirements for T cell migration and T cell-tumour cell interactions," said Weninger.

"We should then be able to use results from this research to further improve immunotherapeutic strategies against cancer in patients."

Their findings, published online on 20 November, will appear in the 27 November print edition of The Journal of Experimental Medicine.

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