4-Dimensional Intravital Microscopy: A New Model for Studies of Leukocyte Recruitment and Migration in Hepatocellular Cancer in Mice

Introduction Although it is accepted that the immune system plays a role in the prognosis of hepatocellular carcinoma (HCC), the exact mechanisms of leukocyte recruitment into HCC are poorly understood. Progress in the study of this aspect has been hindered by technical limitations. Materials and Methods In the present study, we describe the use of 4D intravital microscopy which represents an advantageous technology for the investigation of the microvascular system and leukocyte migration in HCC. To establish 4D intravital microscopy, we used a HCC tumor model in transgenic mice expressing enhanced green fluorescent protein in specific leukocyte subpopulations and combined digital time-lapse recording, laser scanning confocal microscopy, and 3D reconstruction. Using this technology, we studied the intra- and extravascular leukocyte adhesion and migration in HCC in vivo at the single-cell level. Results We showed that although vessel density in HCC was lower than in normal liver, tumor tissue was moderately infiltrated with leukocytes of lymphoid and myeloid origin. Most tumor-infiltrating leukocytes migrated in a random manner frequently changing direction of migration in the tumor tissue. The migration velocity of myeloid and lymphoid leukocytes in HCC tissue was not different. Discussion These results demonstrated that 4D intravital microscopy has potential to be a powerful tool in the study of mechanisms of leukocyte recruitment and intratumoral migration in HCC.