(2jx) Monocytes Use Protrusive Forces to Generate Migration Paths in Viscoelastic Collagen-Based Extracellular Matrices

1. Research Interests: I am passionate about elucidating the role of physical cues in cancer progression. Cancer progression was previously thought of as solely driven by aberrant genetic mutations. But it is now understood that mutations alone do not drive disease progression. Indeed, mounting evidence has demonstrated that biophysical cues are key determinants of cancer progression. The extracellular matrix (ECM) becomes stiffer and viscoelastic as cancer progresses. These (bio)physical changes regulate the phenotype of cells found in the tumor microenvironment (TME). In addition to cancer cells, macrophages and T cells are ubiquitous in the TME. However, the impact of (bio)physical changes of the ECM on cancer cell, macrophage, or T cell phenotype is not fully understood. Precise, local, communication between macrophages and T cells potentiates effector functions. To accomplish this, macrophages and T cells have to physically migrate to find each other in the complex TME. This highlights that cell migration is essential to immunity. Furthermore, cancer cells must migrate to metastasize. These observations centralize cell migration in shaping the tumor immune landscape. Despite these findings, the precise mechanisms by which physical cues in the TME regulate cell migration, and subsequent immune response, are not fully understood.

I am eager to uncover the fundamental biophysical principles that regulate cell migration and macrophage-T cell communication once they encounter each other. More importantly, I am excited to apply these basic insights toward identification of novel therapeutic targets. Recent advances in biomaterials development, biophysical tools, and computational analyses will be leveraged to accomplish these goals. These advances now make it possible to address fascinating questions. For example, do alterations in the physical properties of the ECM play a greater role in primary tumor progression or metastasis? What role does ECM architecture play in determining the efficacy of the immune response. In the long-term, we aim to reveal how the seemingly endless complexity of the immune system can arise from a limited set of predictive physical principles. To accomplish these goals, we aim to use engineered ECM, high resolution 4D microscope imaging, multiplex flow cytometry, and image-based machine learning algorithms to elucidate biophysical principles that determine the ability of the immune system to mount an effective response against cancer.

2a. Abstract background: Circulating monocytes are recruited to tumors, where they can differentiate into macrophages that mediate tumor progression. To reach the tumor microenvironment, monocytes must first extravasate out of the vasculature and migrate through type-1 collagen rich stromal matrix. The viscoelastic stromal matrix around tumors not only stiffens relative to normal stromal matrix, but often exhibits enhanced viscous characteristics, as indicated by faster stress relaxation rate. Stress relaxation refers to a decrease in internal stresses in viscoelastic materials because of applied deformation. Despite clinically observed changes in matrix properties, the potential impact of changes in matrix stiffness or stress relaxation on monocyte migration is not understood. To address this research gap, we studied how changes in matrix stiffness and viscoelasticity impact the three-dimensional migration of monocytes through stromal-like matrices.

2b. Methods: We developed interpenetrating networks (IPNs) of type-1 collagen and alginate with independent tunability of stiffness and stress relaxation over physiologically relevant ranges. Alginate does not provide any adhesion motifs for cells to bind to and is not susceptible to degradation by mammalian proteases. The choice of collagen as the biochemical ligand is to mimic the stromal matrix where immune cells are typically found. Furthermore, the IPNs provide a confining stromal-like matrices and a three-dimensional context experienced by monocytes in vivo. Collagen fiber architecture was quantified by measuring collagen fiber length and width. Importantly, IPN stress relaxation properties are tuned independent of polymer concentration, Young’s modulus, and collagen fiber architecture. IPN stress relaxation properties were tuned by varying alginate molecular weight and tuning the amount of ionic crosslinker (Ca²⁺) added. This allows for the impact of stiffness and stress relaxation on migration to be independently demonstrated. Specifically, we can tune the characteristic stress relaxation times from ~100 seconds (fast relaxing) to 1,000 seconds (slow relaxing) while keeping the initial Young’s modulus of all the materials at ~1 kPa or ~2.5 kPa. Collagen fiber architecture was maintained by gelation of IPNs at 25°C for 2 hr, followed by transfer to 37°C incubator overnight. U937 pro-monocytic cells lines were encapsulated in the 3D alginate-collagen matrix and cell migration was assessed using confocal time-lapse microscopy.

2c. Results: We find that faster stress relaxation and higher Young’s modulus, independently enhanced the 3D migration of monocytes (Fig. 1). Increase in stress relaxation resulted in doubling of cell migration speed while increase in Young’s modulus had a lower impact (~50% increase in speed). Similar results were obtained when primary human monocytes were encapsulated in ~2.5 kPa IPNs. Migrating monocytes have an ellipsoidal or rounded wedge-like morphology, reminiscent of amoeboid migration, with accumulation of actin at the trailing edge. Surprisingly, monocytes migrate independent of matrix adhesion and Rho-mediated contractility but are dependent on actin polymerization and myosin contractility for migration. Our mechanistic studies indicate that actin polymerization at the leading edge generates protrusive forces that generate a path to migrate in the confining viscoelastic matrices. Together, our findings implicate matrix stiffness and stress relaxation as key mediators of monocyte migration and reveal an adhesion-independent mode of migration in monocytes.

2d. Implications of study: This study involved the development of stromal-like matrices with independently tunable architecture, stiffness and viscoelastic properties using a unique two-temperature gelation strategy. Furthermore, we show that monocytes cells can utilize a previously undescribed mode of migration whereby they push viscoelastic matrix to generate channels through which they can move. This finding highlights the importance of utilizing physiologically relevant materials to study cell migration. More importantly, our data raises the possibility that changes in mechanical properties of matrix regulates monocyte recruitment and trafficking. Cell recruitment is an important consideration for the development of immune-targeted therapies where preferential recruitment of certain immune cell populations is desired.

Monocytes become less migratory as they differentiate into more adhesive macrophage phenotypes. Differences in migration behavior suggests that different molecular targets will likely need to be considered depending on the whether the therapeutic goal is monocyte-depletion or macrophage-depletion. More broadly, the tunable nature of the material developed could enable mechanistic insights into the role of changes in the stromal matrix in the promotion of health and disease. Specifically, it provides a platform to study the role of viscoelasticity on migration of normal leukocytes and diseased leukocytes such as those with Leukocyte Adhesion Deficiency-1. Taken together, our data raises the possibility that ECM stiffness and viscoelasticity could determine immune cell recruitment and ultimately shape the immune response under normal and pathological conditions.

3. Teaching Interests: I am passionate about teaching because it provides me the opportunity to get students excited about science and engineering. In my view, sustained recruitment of students to intellectually engage in the pressing scientific problems of the day is essential to the long-term success of our scientific mission to improve the human condition. Over the past several years I have had the opportunity to learn from professors that were very passionate about teaching. I will be grateful to have the opportunity to have other students have similar positive experiences that I had. I am also interested in teaching because it helps students appreciate the crucial role that science plays in our everyday life which helps to facilitate broader public scientific education.

My background in chemical engineering has given me a strong grounding in general engineering and chemical engineering courses, including chemical kinetics, transport phenomena, and thermodynamics. My research portfolio that aims to utilize engineering principles to elucidate the role of (bio)physics in biological systems readily lends itself to advanced undergraduate and graduate level seminar courses or laboratory courses. These courses will give students exposure to ongoing cutting-edge research and enable to effectively use their chemical engineering education to address pressing scientific questions of the day. I am also excited to help develop a new course titled “Immuno-oncology for Engineers.” I envision this course will be offered to advanced undergraduates or graduate students. This is a new and exciting research area in the engineering community, and I strongly believe will complement the chemical engineering curriculum.