Live-imaging studies reveal how microclots and the associated inflammatory response enhance cancer cell extravasation.

Previous clinical studies and work in mouse models have indicated that platelets and microclots might enable the recruitment of immune cells to the pre-metastatic cancer niche, leading to efficacious extravasation of cancer cells through the vessel wall. Here, we investigated the interaction between platelets, endothelial cells, inflammatory cells, and engrafted human and zebrafish cancer cells by live-imaging studies in translucent zebrafish larvae, and show how clotting (and clot resolution) act as foci and as triggers for extravasation. Fluorescent tagging in each lineage revealed their dynamic behaviour and potential roles in these events, and we tested function by genetic and drug knockdown of the contributing players. Morpholino knockdown of fibrinogen subunit α (fga) and warfarin treatment to inhibit clotting both abrogated extravasation of cancer cells. The inflammatory phenotype appeared fundamental, and we show that forcing a pro-inflammatory, tnfa-positive phenotype is inhibitory to extravasation of cancer cells.

PD-1 suppresses the maintenance of cell couples between cytotoxic T cells and target tumor cells within the tumor.

The killing of tumor cells by CD8+ T cells is suppressed by the tumor microenvironment, and increased expression of inhibitory receptors, including programmed cell death protein-1 (PD-1), is associated with tumor-mediated suppression of T cells. To find cellular defects triggered by tumor exposure and associated PD-1 signaling, we established an ex vivo imaging approach to investigate the response of antigen-specific, activated effector CD8+ tumor-infiltrating lymphocytes (TILs) after interaction with target tumor cells. Although TIL-tumor cell couples readily formed, couple stability deteriorated within minutes. This was associated with impaired F-actin clearing from the center of the cellular interface, reduced Ca2+ signaling, increased TIL locomotion, and impaired tumor cell killing. The interaction of CD8+ T lymphocytes with tumor cell spheroids in vitro induced a similar phenotype, supporting a critical role of direct T cell-tumor cell contact. Diminished engagement of PD-1 within the tumor, but not acute ex vivo blockade, partially restored cell couple maintenance and killing. PD-1 thus contributes to the suppression of TIL function by inducing a state of impaired subcellular organization.