Biomarkers of the immune response in primary colorectal cancer

Aim: The object of this project is to characterise lymphocytes in colorectal cancer (CRC), to identify subpopulations that reflect immune activation or suppression. Our aim is to validate the methods used so that they may be used in future early vaccine trials to assess the effect of such trials on the immune microenvironment of this disease. Methods: We recruited 34 patients undergoing surgery for CRC. Lymphocytes were harvested from blood (PBL) and tumour (TIL). Flow cytometry and immunohistochemistry were used to detect subpopulations within TIL, by identifying 18 markers for T cell homing and two of immune suppression (CD25 and Foxp3). To assess function, cells were stimulated and the supernatant was tested in cytokine ELISA. Results: Using flow cytometry, the average number of CD4+CD25+ TIL was 9.98% (range 2–31) compared to 5.6% (0–27.5) for PBL (p = 0.1972). Immunohistochemical staining for Foxp3 was positive in samples obtained from 14 patients. Expression of homing markers differed between PBL and TIL, with the proportion expressing CXCR6 higher (p,0.0001)). Using flow cytometry to calibrate TIL input numbers enabled more accurate measurement of functional status. TIL released both interferon-c and IL-10, associated with positive and negative antitumour responses respectively, but at levels lower than PBL. Conclusions: We have confirmed the presence of immunosuppressive cells in CRC, however, there is not an increase in expression when compared to PBL. The percentage expression of the chemokine receptor CXCR6 is higher in TIL when compared to PBL. Subpopulations within TIL expressing particular cell surface markers may contribute differently to the immune microenvironment, and work is currently on going to isolate these cells and determine their functional properties. The methods we have developed to assess the immune response at the tumour site can be used to analyse the effect of future vaccine trials.