Saba Ghassemi

Cells test substrate rigidity by local contractions on submicrometer pillars

Cell growth and differentiation are critically dependent upon matrix rigidity, yet many aspects of the cellular rigidity-sensing mechanism are not understood. Here, we analyze matrix forces after initial cell–matrix contact, when early rigidity-sensing events occur, using a series of elastomeric pillar arrays with dimensions extending to the submicron scale (2, 1, and 0.5 μm in diameter covering a range of stiffnesses). We observe that the cellular response is fundamentally different on micron-scale and submicron pillars. On 2-μm diameter pillars, adhesions form at the pillar periphery, forces are directed toward the center of the cell, and a constant maximum force is applied independent of stiffness. On 0.5-μm diameter pillars, adhesions form on the pillar tops, and local contractions between neighboring pillars are observed with a maximum displacement of ∼60 nm, independent of stiffness. Because mutants in rigidity sensing show no detectable displacement on 0.5-μm diameter pillars, there is a correlation between local contractions to 60 nm and rigidity sensing. Localization of myosin between submicron pillars demonstrates that submicron scale myosin filaments can cause these local contractions. Finally, submicron pillars can capture many details of cellular force generation that are missed on larger pillars and more closely mimic continuous surfaces.

Force generated by actomyosin contraction builds bridges between adhesive contacts

Extracellular matrices in vivo are heterogeneous structures containing gaps that cells bridge with an actomyosin network. To understand the basis of bridging, we plated cells on surfaces patterned with fibronectin (FN)‐coated stripes separated by non‐adhesive regions. Bridges developed large tensions where concave cell edges were anchored to FN by adhesion sites. Actomyosin complexes assembled near those sites (both actin and myosin filaments) and moved towards the centre of the non‐adhesive regions in a treadmilling network. Inhibition of myosin‐II (MII) or Rho‐kinase collapsed bridges, whereas extension continued over adhesive areas. Inhibition of actin polymerization (latrunculin‐A, jasplakinolide) also collapsed the actomyosin network. We suggest that MII has distinct functions at different bridge regions: (1) at the concave edges of bridges, MIIA force stimulates actin filament assembly at adhesions and (2) in the body of bridges, myosin cross‐links actin filaments and stimulates actomyosin network healing when breaks occur. Both activities ensure turnover of actin networks needed to maintain stable bridges from one adhesive region to another.

Decoding information in cell shape.

Shape is an indicator of cell health. But how is the information in shape decoded? We hypothesize that decoding occurs by modulation of signaling through changes in plasma membrane curvature. Using analytical approaches and numerical simulations, we studied how elongation of cell shape affects plasma membrane signaling. Mathematical analyses reveal transient accumulation of activated receptors at regions of higher curvature with increasing cell eccentricity. This distribution of activated receptors is periodic, following the Mathieu function, and it arises from local imbalance between reaction and diffusion of soluble ligands and receptors in the plane of the membrane. Numerical simulations show that transient microdomains of activated receptors amplify signals to downstream protein kinases. For growth factor receptor pathways, increasing cell eccentricity elevates the levels of activated cytoplasmic Src and nuclear MAPK1,2. These predictions were experimentally validated by changing cellular eccentricity, showing that shape is a locus of retrievable information storage in cells.

CAR T cell immunotherapy for human cancer

Adoptive T cell transfer (ACT) is a new area of transfusion medicine involving the infusion of lymphocytes to mediate antitumor, antiviral, or anti-inflammatory effects. The field has rapidly advanced from a promising form of immuno-oncology in preclinical models to the recent commercial approvals of chimeric antigen receptor (CAR) T cells to treat leukemia and lymphoma. This Review describes opportunities and challenges for entering mainstream oncology that presently face the CAR T field, with a focus on the challenges that have emerged over the past several years.

Fabrication of elastomer pillar arrays with modulated stiffness for cellular force measurements

The mechanical properties of a cells environment can alter behavior such as migration and spreading, and control the differentiation path of stem cells. Here we describe a technique for fabricating substrates whose rigidity can be controlled locally without altering the contact area for cell spreading. The substrates consist of elastomeric pillar arrays in which the top surface is uniform but the pillar height is changed across a sharp step. Preliminary results demonstrate the effects on cell migration and morphology at the step boundary.

α-Actinin links extracellular matrix rigidity-sensing contractile units with periodic cell-edge retractions

During cell migration, the cell edge undergoes periodic protrusion–retraction cycles. Quantitative analyses of the forces at the cell edge that drive these cycles are provided. We show that α-actinin links local contractile units and the global actin flow forces at the cell edge and present a novel model based on these results.

Gold-Tipped Elastomeric Pillars for Cellular Mechanotransduction.

We describe a technique for the fabrication of arrays of elastomeric pillars whose top surfaces are treated with selective chemical functionalization to promote cellular adhesion in cellular force transduction experiments. The technique involves the creation of a rigid mold consisting of arrays of circular holes into which a thin layer of Au is deposited while the top surface of the mold and the sidewalls of the holes are protected by a sacrificial layer of Cr. When an elastomer is formed in the mold, the Au adheres to the tops of the molded pillars. This can then be selectively functionalized with a protein that induces cell adhesion, while the rest of the surface is treated with a repellent substance. An additional benefit is that the tops of the pillars can be fluorescently labeled for improved accuracy in force transduction measurements.

The CPT1a inhibitor, etomoxir induces severe oxidative stress at commonly used concentrations

Etomoxir (ETO) is a widely used small-molecule inhibitor of fatty acid oxidation (FAO) through its irreversible inhibitory effects on the carnitine palmitoyl-transferase 1a (CPT1a). We used this compound to evaluate the role of fatty acid oxidation in rapidly proliferating T cells following costimulation through the CD28 receptor. We show that ETO has a moderate effect on T cell proliferation with no observable effect on memory differentiation, but a marked effect on oxidative metabolism. We show that this oxidative metabolism is primarily dependent upon glutamine rather than FAO. Using an shRNA approach to reduce CPT1a in T cells, we further demonstrate that the inhibition of oxidative metabolism in T cells by ETO is independent of its effects on FAO at concentrations exceeding 5 μM. Concentrations of ETO above 5 μM induce acute production of ROS with associated evidence of severe oxidative stress in proliferating T cells. In aggregate, these data indicate that ETO lacks specificity for CTP1a above 5 μM, and caution should be used when employing this compound for studies in cells due to its non-specific effects on oxidative metabolism and cellular redox.

203. Shortened T Cell Culture with IL-7 and IL-15 Provides the Most Potent Chimeric Antigen Receptor (CAR)-Modified T Cells for Adoptive Immunotherapy

Adoptive T cell immunotherapy involves the isolation, ex vivo expansion and reinfusion of patient T cells. The efficacy of adoptive immunotherapy is dependent on the ability of T cells to engraft, expand and persist upon adoptive transfer. In this therapy, T cells are cultured ex vivo using natural or artificial antigen presenting cells that deliver signal 1 (TCR/CD3) and signal 2 (e.g. CD28 co-stimulation) along with exogenously added cytokines. IL-2 is the most commonly used cytokine for ex vivo T cell culture; however, there is renewed interest in IL-7 and IL-15 due to their ability to enhance the survival and proliferation of stem cell memory (Tscm) and central memory (Tcm) T cells. We show that primary human T cells freshly isolated from peripheral blood are heterogeneous with substantial numbers of Tscm and Tcm cells in addition to effector differentiated T cells. During ex vivo culture, these cells progressively differentiate into a population of T cells with a predominantly CD45RO+, CD27-, CCR7- effector differentiated phenotype. Exogenous IL-7 and IL-15 delay this transition in T cell phenotype and preserve a greater proportion of Tscm and Tcm cells in the final ex vivo culture product. We hypothesize that limited ex vivo culture of T cells in the presence of IL-7 and IL-15 rather than IL-2 will enhance engraftment and persistence of T cells in vivo contributing to enhanced efficacy in adoptive transfer. We show that T cells can be harvested and viably frozen from ex vivo cultures as early as day 3 following activation. Early activated T cells expressing a chimeric antigen receptor targeting CD19 (CART-19) show potent yet specific cytotoxicity and cytokine production in vitro. We investigated the therapeutic potential of cells harvested at day 3 versus later time points using a Nalm-6 leukemic cell xenograft mouse model. We demonstrate that day 3 CART-19 cells show potent anti-leukemic activity compared to day 5 or day 9 cells. Comparing CART19 cells cultured in either IL-2 or IL-7/15, we show that mice treated at a 10-fold lower dose with day 3 cells cultured in IL-7/15 exhibit the greatest anti-leukemic efficacy compared with day 9 cells where the latter fail to control leukemia. In summary, we show that limiting T cell culture ex vivo to the minimum required for lentiviral transduction in the presence of IL-7 and IL-15 provides the most efficacious T cells for adoptive T cell immunotherapy.

Reducing Ex Vivo Culture Improves the Antileukemic Activity of Chimeric Antigen Receptor (CAR) T Cells

The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Limiting ex vivo culture time of CD19-specific CAR T cells during manufacturing yielded improved persistence and effector function in vivo. The success of chimeric antigen receptor (CAR)–mediated immunotherapy in acute lymphoblastic leukemia (ALL) highlights the potential of T-cell therapies with directed cytotoxicity against specific tumor antigens. The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Most protocols for T-cell engineering routinely expand T cells ex vivo for 9 to 14 days. Because the potential for engraftment and persistence is related to the state of T-cell differentiation, we hypothesized that reducing the duration of ex vivo culture would limit differentiation and enhance the efficacy of CAR T-cell therapy. We demonstrated that T cells with a CAR-targeting CD19 (CART19) exhibited less differentiation and enhanced effector function in vitro when harvested from cultures at earlier (day 3 or 5) compared with later (day 9) timepoints. We then compared the therapeutic potential of early versus late harvested CART19 in a murine xenograft model of ALL and showed that the antileukemic activity inversely correlated with ex vivo culture time: day 3 harvested cells showed robust tumor control despite using a 6-fold lower dose of CART19, whereas day 9 cells failed to control leukemia at limited cell doses. We also demonstrated the feasibility of an abbreviated culture in a large-scale current good manufacturing practice–compliant process. Limiting the interval between T-cell isolation and CAR treatment is critical for patients with rapidly progressing disease. Generating CAR T cells in less time also improves potency, which is central to the effectiveness of these therapies. Cancer Immunol Res; 6(9); 1100–9. ©2018 AACR.