Immunhistochemische Subtypisierung der lokalen lymphozytären Zellinfiltration und Expression von ICAM-1 nach instillativer Behandlung mit stimulativen CpG-ODN am orthotopen, murinen Harnblasenkarzinommodell

The intravesical application of the tuberculosis vaccine Bacille Calmette Guérin (BCG) has been used for the last 40 years and to date is the most effective adjuvant treatment for non-muscle-invasive bladder cancer (NMIBC). However, the detailed mechanism of action remains to be determined. Exploring the active ingredients of BCG, unmethylated cytosin- and guanin-rich sequences were identified. This subsequently gave rise to the development of synthetic CpG-oligodeoxynucleotides (CpG-ODN) with a nuclease-resistant, phosphorothioate-modified backbone, inhering strong immunostimulatory properties. CpG-ODN aim at toll-like-receptor 9 (TLR9), a pattern-recognition-receptor. By different signalling cascades, TLR9 activation increases the transcription and expression of cytokines and factors, which conclusively leads to a Th1-dominated, cell-mediated immune response. Antineoplastic potential of some CpG-ODN has been verified for a variety of solid tumours in animal models, including the MB49-model used in this study. In a first series, in 36 C57/Bl6 mice, a MB49-transitional cell carcinoma was implanted via transurethral instillation. Twelve of these received a control treatment with PBS, the other 24 were repeatedly instilled with CpG-ODN following different schedules. On day 13, the animals were sacrificed, and the bladders were harvested for immunohistochemical subtyping of the lymphocytic infiltration by staining CD3, CD4, CD8a and CD19. Hereby an augmented T-cell-infiltration (CD3) of the tumour bearing tissue was observed, whereas a further subdifferentiation between T-helper-cells (CD4) and cytotoxic T-cells was not achievable on the applied semiquantitative scale. Staining of CD 19 showed a decreasing density of B-cells over the course of therapy, possibly indicating a suppression of the humoral Th2-response in favour of a Th1-polarization. In a second series, 12 mice were divided in 3 groups. The first group was treated with CpG-ODN instillation without preceding tumour transfer, the second underwent tumour implantation without further treatment, while the last cohort received both, tumour and treatment. The animals were sacrificed on day 7 and bladders were resected and processed to finally immunohistochemically evaluate the presence of the surface protein Intercellular Adhesion Molecule 1 (ICAM-1), already known to be enhanced after BCG-Treatment in a murine model. After exposition to CpG-ODN a massive increase of the expression of ICAM-1 was observed not only by infiltrating immunocytes, but also by the cancer cells, suggesting a crucial role in the mode of action. The complex of ICAM-1 with its receptor Lymphocyte Function-Associated Antigen 1 (LFA-1) might exceed pure adhesion properties by representing a key aspect in the establishment and maintenance of Th1-type immune response. The crucial role of ICAM-1 in transmigration of immune cells and in the establishment of the cytotoxic immunological synapse suggests CpG-ODN as promising adjuvant for modern checkpoint inhibitors. Further research will certainly need to illuminate the features and functions of ICAM-1 in CpG-ODN treatment to prospectively allow transferring this promising approach into the human system.