Exploration and modulation of mechanical cues for enhanced cancer immunotherapy

The advent of immunotherapy, such as immune checkpoint blockade (ICB) and adoptive transfer of cytotoxic lymphocytes, has transformed the clinical care of cancer. However, a significant proportion of patients are resistant to immunotherapy or experience relapse following treatment. To date, most studies in the field have focused on discovering and blocking biochemical characteristics of tumors that dampen the immune response or, conversely, identifying biomolecules that enhance lymphocyte expansion and effector functions. In addition to this biochemical dimension, tumors also exhibit al-tered biomechanical properties, such as cancer-cell softening. Furthermore, the interactions between immune and cancer cells are intensely physical and influence the magnitude of the immune response. Modulating the mechanical interactions between immune cells and cancer cells, a field that we termed mechanical immunoengineering, may open new avenues in stimulating immune cells and treating can-cer.This thesis aims to understand the role of a passive mechanical cue – cancer-cell stiffness – and active forces on lymphocyte expansion and effector functions.Characterization of cancer-cell biophysics and plasma membrane cholesterol-based mechanical immune checkpoint. Recent studies suggest that cancer cell softening, a hallmark of malignancy, re-sults in lymphocyte inhibition. Our group recently identified plasma membrane (PM) cholesterol as a potential regulator of cancer-cell mechanics. Using a combination of single-cell microrheology and high-throughput deformability cytometry, I show that cellular stiffness is inversely correlated with PM cholesterol levels. Depleting PM cholesterol leads to cellular rounding and stiffening by dramatically reorganizing the cortical actin cytoskeleton, akin to cellular stiffening during mitosis.Blocking a mechanical checkpoint enhances NK cell immunological synapse formation and anti-cancer immunotherapy. Natural killer (NK) cells can mount rapid responses against cancer cells lack-ing major histocompatibility complex (MHC) class I. Thus, NK cell-based therapies may benefit pa-tients who do not respond to current T cell-targeting ICB treatments. Despite the potential of NK cells in cancer therapy, the first wave of clinical trials of checkpoint inhibitors in NK cells, either alone or in combination with existing ICB therapies, have produced mixed results. Therefore, identifying novel immune checkpoints complementary to existing ones may help overcome some of these limitations. In this chapter, I report that cancer cell softness acts as an immune checkpoint of mechanical nature on NK cells. Cancer-cell stiffening via cholesterol depletion enhances NK-cell immunological synapse formation and cytotoxicity in vitro and in vivo. Mechanical stimulation of cytotoxic lymphocytes for enhanced activation. T-cell expansion is a critical step in the manufacturing of adoptive cell transfer for cancer immunotherapy. However, ob-taining a sufficient number of T cells can be a challenge during manufacturing. Current commercial artificial antigen-presenting cells do not recapitulate the mechanical properties of dendritic cells. In particular, these passive activating surfaces lack the dynamic dimension of the T cell-antigen-presenting cell interactions. In this chapter, I develop a centrifuge-based method to apply mechanical forces on a population of T cells and show that mechanical stimulations can enhance T-cell activation.