Combining gene editing with traction force microscopy to achieve a comprehensive understanding of mechanotransduction in leukocyte motility

The National Institute of General Medical Sciences awarded The Trustees of the University of Pennsylvania $438,609 on May 7, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to fund research combining gene editing with traction force microscopy to understand mechanotransduction in leukocyte motility.

The research uses CRISPR-Cas9 gene editing and traction force microscopy to conduct a directed screen of molecules implicated in cell migration, specifically upstream migration—the phenomenon in which leukocytes migrate against flow direction on surfaces presenting intercellular adhesion molecule-1 (ICAM-1). The work employs two cultured cell lines: KG1A cells as a model for hematopoietic stem and progenitor cells and HL-60 cells as a model for neutrophils, enabling comparison of how different intracellular effector molecules contribute to cell motility and establishment of universal mechanisms across cell types. Traction force microscopy measures forces exerted by cells during motility by monitoring deflection of fiduciary beads embedded within an elastic polyacrylamide gel. This project builds on more than two decades of work by the Hammer Laboratory supported largely by NIGMS.

Performance occurs in Philadelphia, Pennsylvania, with a period of performance from May 7, 2026, through February 28, 2031. The award is classified as a Project Grant.

Abstract

Leukocyte motility is critical for immunology, inflammation, and hemostasis. Immune cells exchange molecular information by direct contact, enabled by motility within secondary lymphoid organs. In inflammation, neutrophils crawl into sites of infection after adhering to blood vessel walls. Hematopoietic stem and progenitor cells (hspcs) migrate into to bone marrow after adhesion to the blood vessel wall under flow. In work largely funded by nigms, the hammer laboratory has worked to understand the chemo- mechanics of leukocyte migration for over two decades. We have used traction force microscopy (TFM), in which we measure the forces exerted by cells during motility by monitoring the defection of fiduciary beads embedded within an elastic polyacrylamide GEL. We have used this method to measure the traction forces of neutrophils and macrophages, as well as many other cell types. With the technique of TFM in hand, we are now positioned for significant breakthrough in our molecular understanding of traction stresses during leukocyte motility, owing to the development of methods to delete or alter intracellular components within a cell. Of specific interest is the fascinating phenomenon of upstream migration, in which leukocytes migrate against the direction of flow on surfaces presenting intercellular adhesion molecule-1 (icam-1). Using crispr-CAS9, we now are able do a directed screen of molecules that have been implicated in cell migration, and specifically, upstream migration, to understand precisely how these molecules contribute to the generation of traction forces in leukocytes. We will use two cultured cells lines - KG1A cells (a model hspcs) and HL-60 cells (a model neutrophil) - that allow US to compare the role of different intracellular effector molecules in cell motility to establish universal mechanisms. We will conduct a directed screen of a limited but important set of effector molecules which have been implicated in upstream migration and leukocyte motility, such as cytoskeletal regulators and RHO-gtpases. Our elucidation of traction stresses will be complemented by immuno-fluorescent staining of the spatial distribution of adhesion receptors and actin cytoskeleton to provide information about cell organization. This mira is organized in three projects. In project 1, we will perform traction mapping of KG1A cells during upstream migration. We will then screen candidate controllers of upstream migration using crispr-CAS9 and then measure how deletion affects both directional motility and traction stresses. In project 2, we will use crsipr-CAS9 to screen a family of motility modulators and study their effect on HL-60 cell chemokinesis and chemotaxis. In project 3, we will use TFM to measure the traction stresses of HL-60 cells during upstream migration and when upstream migration is reversed through crispr-CAS9 deletion. Then, we will measure the correlation between upstream migration of HL-60 cells and trans-endothelial migration on huvec monolayers.