Improvements in real-time intravital imaging techniques and two-photon microscopy make it now possible to visualize the complex tumor microenvironment in vivo, over time and deeply within intact tissues. Using dynamic imaging, we addressed central aspects of tumor progression, including dynamic interactions of invading tumor cells with the tumor microenvironment and the local regulation of tumor infiltrating immune cells in eradicating tumor cells.
Immunological control of tumor progression requires the activation and expansion of tumor-specific cytotoxic T lymphocytes (CTL) followed by an efficient effector phase in the tumor lesion. To mimic in vivo dynamics of CTL effector function we established a 3D collagen matrix based assay to observe CTL effector function in real time, including active migration, sequential interactions with and serial killing of target cells. Individual CTL-target cell contacts were variable in duration (min to hours) and kinetics (stable to dynamic), comprised a median lag-phase of 90 min until apoptosis of the target cell, and were followed by CTL detachment and sequential killing of multiple target cells (up to 11/24 h). Using this model, local factors present in the tumor microenvironment were identified that interfere with or enhance the anti-tumor CTL response and serial killing. CXCL12, a chemokine with pro-migratory effects on CTL and known to be upregulated in most tumors, enhanced CTL migration, shortened interaction times with target cells and reduced the killing efficiency on a per-contact-basis which lead to near-complete abrogation of target cell killing at low CTL to target cell ratio. This effect was reversed by a CXCR4 antagonist, suggesting that enhancing serial killing by targeting the CXCR4/CXCL12 axis may improve anti-tumor immunotherapy.
Pro-migratory signals mediated by the tumor microenvironment further contribute to cancer invasion and metastasis. We investigated in vivo tumor cell invasion principles, potential guidance structures, the molecular mechanisms invasion, and the response of different tumor niches to anti-cancer therapy. Intravital fluorescence and multiphoton microscopy was used to study HT-1080 fibrosarcoma xenografts implanted into the dorsal skin-fold chamber on nude mice. After initial growth, tumors developed zones of invasive growth consisting of multicellular collective invasion strands that retained cellcell contact and the ability to proliferate while invading at velocities of up to 200 μm/day. This invasion occurred in a directed manner along blood/lymph vessels and muscle strands of the deep dermis. Therapeutic irradiation induced complete regression of the tumor main mass yet failed to eradicate perivascular invasion strands. Using knockdown and/or antibody-based treatment against β1/β3 integrins but not interference with EGFR prompted significant radiosensitization of both, tumor main mass as well as invasion strands implicating integrin-mediated adhesion and/or anti-apoptotic signaling (anoikis) in radioresistance. In conclusion, collective invasion not only supports fast angiotropic invasion but maintains an integrin-dependent survival niche.
Thus, visualizing cell kinetics in 3D in vitro and in vivo models provides novel cellular and molecular mechanisms of CTL-mediated immune defense and cancer invasion, and the role of the local tumor microenvironment therein.