Our immune system finds it difficult to eliminate tumours effectively. Deciphering the strategies it implements may increase the immune system's effect on tumour cells and thus improve the clinical perspectives for anticancer immune therapy. At the Institut Curie, INSERM and CNRS researchers have used two-photon microscopy to demonstrate, for the first time in vivo and real-time, how T lymphocytes infiltrate a solid tumour in order to fight it.
These "defenders" methodically encircle the enemy positions and "patrol" until they meet a tumour cell, which they have previously learnt to recognise. They then halt to eliminate it, before resuming their rounds. The rapidity of the advance achieved by T lymphocytes is indicative of either the absence of an adversary, or defeat of the immune system in the battlefield.
This scenario was published in The Journal of Experimental Medicine.
How is a tumour destroyed by T lymphocytes? This scenario has recently been visualised by researchers at the Institut Curie. The original images obtained and assembled in twelve video sequences are the result of close collaboration between a specialist in two-photon microscopy, Luc Fetler, an INSERM scientist in the CNRS/Institut Curie "Physical Chemistry Curie" Unit1, and immunologists, notably Alexandre Boissonnas, in the INSERM "Immunity and Cancer" Unit at Institut Curie.
Our body's defences against an infection or tumour are based on a string of actors, some of them generalists, the others highly specialised. Cytotoxic T lymphocytes belong to the second category. To achieve their task, their cell surface carries a membrane receptor which is complementary to the antigen in the pathological cells to be eliminated. Alerted by the presence of this antigen, the T lymphocytes are activated. Having identified an infectious or tumour cell, they bind to it and target it with a fatal load of enzymes.
When T lymphocytes infiltrate a tumour...
Before this work by Alexandre Boissonnas and Luc Fetler, no-one had ever observed at the cellular scale what happens when activated T cells arrive in a solid tumour. The novel experimental model developed by these Institut Curie scientists reveals the strategy adopted by these cells to destroy the tumour.
Recognition of the tumour antigen determines T lymphocyte behaviour
To arrive at their conclusion, the researchers used an animal model to observe the route followed by T lymphocytes in tumours endowed with a particular antigen, ovalbumin (OVA) and in tumours which served as controls and were devoid of this antigen. When the tumours reached 500 to 1000 mm3, eight to ten days after the injection of tumour cells with or without the antigen, the researchers then injected the mice with a large number of OVA antigen-specific T cells.
What happened after the day of transfer? As expected, only the tumour with the OVA antigen disappeared, after about a week. In the interval, the two-photon microscope (see box) made it possible to examine the scene in situ over the first 150 micrometres of the tumour. Each image provided a photograph of the different cell populations, blood vessels and collagen fibres present. And using a series of successive images, it was possible to reconstitute the trajectory of a T lymphocyte.
Using this method, the researchers thus examined the different protagonists - T lymphocytes and tumour cells - during two distinct periods of tumour development. In the antigen-devoid tumour, T cells "patrolled" at a consistently high rate (approximately 10 micrometres per minute), at all stages of development. However, T lymphocyte behaviour varied in antigen-containing tumour; when the tumour stopped growing because of the lymphocyte injection three or four days previously, the defenders patrolled slowly (4 micrometres per minute) and halted frequently. Their mean rate reached a plateau at 4 micrometres per minute. Then, during later stages when the tumour was regressing, most T lymphocytes resumed their rapid mobility.
To summarise therefore, the T lymphocyte trajectories were confined to regions containing high levels of live tumour cells, while they were broader and fluid in regions littered with dead tumour cells. The Institut Curie scientists concluded that presence of the antigen halted the T lymphocytes which were then occupied by recognising and killing their adversaries.
Furthermore, by analysing the distribution of T lymphocytes throughout the respective tumours, the scientists noted that these defenders were always present at the periphery, but that the presence of the antigen was essential to in-depth penetration, leading to effective tumour elimination.
These results were validated in two types of experimental tumour generated from two lines of cancer cells.
It is now up to the clinicians to verify whether the in-depth infiltration of T lymphocytes could constitute a criterion for a good prognosis.
A clearer understanding of how the immune system functions is essential to optimise one of the most promising options for future cancer treatment: immune therapy.
For many years now, the Institut Curie has been participating actively in the development of innovative strategies in this field. Two clinical studies are currently under way at the Institute: one in patients with choroidal melanoma and the other in women with cervical cancer.
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The two-photon microscope, plunging into living tissues
For several years, the Institut Curie has been placing particular emphasis on imaging techniques to decipher the intimate mechanisms of life. Two-photon microscopy (M2P) has been helping the work of Institut Curie biologists for ten years. Its ability to illuminate an site situated at a depth of up to 0.5 mm can avoid a time-consuming succession of sections and samples within the thickness of a fixed tissue. The principle of M2P is based on the excitation of molecules which are intrinsically fluorescent in cells, or fused with a protein such as GFP or CFP (green- or cyan-fluorescent protein). The infrared laser used to excite these molecules emits ultra-short impulses (100 femtosecondes3, 80 million times a second) which are so intense that the two photons can be absorbed simultaneously by a molecule. This absorption of photon pairs only occurs at the focal point, thus enabling the generation of clear, penetrating images and the direct and real-time filming of cell movements in a living tissue.
T lymphocyte tactics in tumours
The scientists injected mice with tumour cells expressing a green fluorescent molecule: some cells were endowed with an antigen, ovalbumin, while the others (which served as controls) were devoid of this antigen. When the tumours reached 500 to 1000 mm3, eight to ten days later, the scientists injected the mice with a large number of antigen-specific T lymphocytes. On these two-photon microscopy photographs we can see, left, that the tumour expresses the antigen and the tumour cells (in green) are few in number as they have been destroyed by T lymphocytes. On the right, the tumour is devoid of the antigen and the tumour cells have not been destroyed. The blue labelling corresponds to collagen fibres and the red labelling to blood vessels.
1 UMR 168 CNRS/Institut Curie "Physical Chemistry Curie", headed by Jean-François Joanny 2 INSERM Unit 653Institut Curie "Immunity and Cancer", headed by Sebastian Amigorena
© A. Boissonnas, L. Fetler/Institut Curie References « In vivo imaging of cytotoxicT cell infiltration and elimination of a solid tumor » Alexandre Boissonnas1,3, Luc Fetler2,3, Ingrid S. Zeelenberg1, Stephanie Hugues1 and Sebastian Amigorena1
1 Unité Inserm 653, Immunité et Cancer, Pavillon Pasteur, Institut Curie, 26 rue d'Ulm, F-75245 Paris cedex 05.
2 CNRS UMR 168, Laboratoire de Physico-Chimie Curie, Institut Curie, 26 rue d'Ulm, F-75245 Paris cedex 05.
3 Auteurs en contribution équivalente
The Journal of Experimental Medicine, February 19, 2007
Institut Curie Catherine Goupillon/Céline Giustranti Tel. 01 44 32 40 63/64 Fax 01 44 32 41 67 firstname.lastname@example.org
3 femto (f): préfix which means 10 -15 , or a millionth of a billionth of the unit cited.