Poster P17 - Mechanical fibrous cues drive apoptotic responses

Introduction: Close to one billion cells die every second in our bodies, which is crucial to maintaining homeostasis. Any imbalance can lead to severe disorders such as cancer and autoimmune diseases. Programmed cell death, called apoptosis, follows canonical intrinsic or extrinsic pathways. In both, cells round up and, for a brief period, are held in position by filamentous actin retraction fibers (RFs). Traditionally, when studied in flat 2D, differences have been observed between the apoptotic pathways. How these pathways differ in loose connective tissue environments comprising a sparse network of fibers is poorly documented. In our present study, using quantitative confocal microscopy, we develop new knowledge in apoptotic outcomes of cells attached to in vivo mimicking single or multiple fibers. We demonstrate that RF patterning determines cell mechanical stability. Generally, fiber networks have delayed apoptosis in both pathways, and we discover that for cells with low mechanical stability, the caspase duration flips between the two pathways. We also present force-caspase relationships highlighting the role of cellular contractility in apoptosis. Overall, we establish a new contextually relevant paradigm for studying critical cell behavior.

Materials and

Methods: Our experimental setup used the non-electrospinning spinneret-based tunable engineered parameters platform (STEP) technique. We created aligned 200 nm fibers with interfiber spacing ranging from 10 and 20 μm to constrain cells to be attached to two and single fibers, respectively. We used human colon cancer cells that expressed a caspase FRET sensor (Fig 1. A). To examine MOMP, we added the TMRM dye, and to confirm cell death, we used the SYTOX dye. We also used a plasma membrane dye to visualize RF patterning. We induced both extrinsic and intrinsic apoptosis pathways using TRAIL+ Cycloheximide or Raptinal, respectively. We then used confocal fluorescent time-lapse imaging to examine apoptosis parameters and nanonet force microscopy (NFM) to measure cell forces.

Results, Conclusions, and Discussions: Cells attached to one fiber had a spindle shape, and cells attached to two fibers had an elongated shape compared to a stellate shape for cells on flat 2D (Fig 1. B). Once the cells are rounded up after apoptosis induction, they are mechanically held in place by RFs. Cells on one fiber had the least number of RFs, while cells on flat had an isotropic distribution of RFs. We measured the cell body displacement and found that cells attached to single fibers had the largest displacements (Fig 1. C), indicating they were least mechanically stable. We found that regardless of fiber geometry, MOMP occurred faster on fibers. Caspase duration, on the other hand, was the longest on 2D in the extrinsic pathway but shortest in the intrinsic pathway compared with fiber networks (Fig 1.D). We discovered that cells on one fiber, signifying the least mechanical stability, had the shortest extrinsic caspase duration, which flipped to the longest during intrinsic conditions. Using NFM, we develop caspase-MOMP-force master curves showing a force drop that coincided with MOMP and increased caspase activity (Fig 1. E). Overall, for the first time, we demonstrate that the intrinsic and extrinsic apoptotic pathways respond to mechanical stability differently, and apoptotic responses in ECM-mimicking fibrous environments differ from those observed in traditional 2D.

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