Mitochondrial dsRNA from B-ALL cells stimulates mesenchymal stromal cells to become cancer associated fibroblasts.

Cancer associated fibroblasts (CAF) arising from bone marrow-derived mesenchymal stromal cells (MSC) are prominent in B-precursor acute lymphoblastic leukaemia (B-ALL). We have previously shown that CAF formation is triggered by exposure to reactive oxygen species-inducing chemotherapy and that CAF support chemoresistance by donating mitochondria to the cancer cells, through tunnelling nanotubes. In the present study, we show that exposure of MSC to ALL cell lines, patient-derived xenografts and primary cells or their conditioned media can also trigger CAF formation. Using bulk RNA sequencing in cell lines, we show that the MSC to CAF transition is accompanied by a robust interferon pathway response and we have validated this finding in primary cells. Using confocal microscopy and flow cytometry, we identify the take-up of leukaemia cell-derived mitochondrial dsRNA by MSC as a proximate trigger for the MSC to CAF transition. We show that inhibition of dsRNA formation in ALL cells by treatment with low-dose ethidium or the mitochondrial transcription inhibitor IMT1 or degradation of dsRNA in conditioned media by 100°C exposure ablates the ability of the ALL conditioned media to stimulate MSC to CAF transition. Our data reveal a novel and previously undescribed mechanism by which cancer cells induce a CAF phenotype in stromal cells, showing how B-ALL cells can directly induce the previously described niche-mediated protection within the bone marrow.

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.