Shelter in place: Live CLL cells inside the bone marrow fibroblasts and its implication in residual disease persistence Open Access

• Live cell-in-cell is driven by BTK but not BCL2 inhibitors and it represents a novel mechanism underlying MRD persistence.

• CXCL12-CXCR4 axis plays a key mechanistic role in live CIC and targeting CXCR4 may offer a new therapeutic approach to minimize MRD.

Bruton tyrosine kinase inhibitors (BTKi) have been successful in treating B-cell malignancies including chronic lymphocytic leukemia (CLL). However, a deep response to BTK inhibition is uncommon. It is unknown what mechanism results in minima residual disease (MRD) persistence. Cell-in-cell (CIC) describes the microscopic observations of embodiment of an intact cell by another whole cell. CIC has been sporadically described by pathologists in human fixed tissues for over a century. However, its biological, pathological and clinical significance remain obscure. In the current study, we investigated human primary CLL samples using a clinically faithful ex vivo model system that accurately recapitulates the lymphoma tumor microenvironment (TME). We observed that CLL cells were actively internalized by BMF and remained alive and mobile for days. We hypothesized that live CIC may represent a new mechanism for tumor cells to evade therapies and survive as residual disease. Indeed, CIC events were identified directly in the bone marrow of patients being treated with BTK inhibitors. Using confocal microscopy, we demonstrated that the ex vivo exposure to BTKi drove CLL cells into BMF. We further showed that CIC inside the BMF were indeed protected from drug-induced apoptosis compared to cells that stayed outside. Mechanistically, we identified that CXCR4 receptor was required for CIC as CXCR4 antagonists and CRISPR-mediated genetic depletion completely abrogated CIC. Altogether, human direct evidence and ex vivo data implicate CIC in disease persistence. Targeting tumor-stroma interaction via CXCR4 inhibition could constitute a new therapeutic approach to minimize MRD and future relapses.