2. In situ analysis of tumor-infiltrating T lymphocytes
Our observations that some vaccinated melanoma patients could display a clear tumor regression with a very low or sometimes undetectable anti-vaccine T cell response led us to analyse the other anti-tumor T cells present in these patients, namely the T lymphocytes that recognized tumor-specific antigens different from those of the vaccines. Detailed analyses carried out in two vaccinated patients clearly indicated that anti-tumor T lymphocytes were already present prior to vaccination, both in blood and in some tumors. It is obvious that there is a seemingly pacific coexistence between tumor cells and tumor-specific T lymphocytes that occurs in many cancer patients. The reasons for this coexistence may well be the key towards improving the clinical efficacy of cancer vaccines. Our approach is to gain information about human tumor-infiltrating or tumor-associated T cells through an in situ genetic analysis.
We work on tumour material obtained through a tight collaboration with the clinical teams in charge of the vaccination of melanoma patients. The most important information will probably be obtained from the minority of patients who will display tumor regression, and whom we cannot identify beforehand. Tumour samples are processed simultaneously for histological analysis including immunochemical detection of immune cells, for complete gene profiling on a fragment of the tumor, and for laser microdissection on frozen material. A small piece is systematically put into culture to derive a melanoma cell line.
We use laser microdissection to isolate small numbers (1-50) of tumour cells or T lymphocytes infiltrating the tumour. The advantage of this methodology over the gene profiling of the complete tumour, which we carry out also, is to localize a given biological process within the tumour. It is obvious that ought to the infiltration of the tumour by various types of stromal cells, T cells are in different microenvironments depending on their location within the tumour. The difficulty of the approach is to maintain RNA integrity throughout the process: we measured that the RNA recovery in microdissection is usually around 1%, which is too low for the analysis of 1-50 cells. Over the last year we worked out every aspect of these experiments, and improved the recovery by a factor of 10-20, depending on whether or not an histological staining is required. We can now measure with RT-qPCR the expression of 6-8 genes on a microdissected fragment of about 50 cells. We are currently setting up a global cDNA amplification, in order to carry out complete gene profiling (Affymetrix microarrays) on fractions of the samples. We have adapted and are comparing two methods of amplification that can theoretically be used on single cells. We verified that they could indeed be used on single micromanipulated cells of human T lymphocyte clones.
To know more... (pdf chapter of the last de Duve Institute report)
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