Biomedical Researcher University of Louisville Prospect, Kentucky, United States
Introduction:: Cancer progression is highlighted by the emergence of aberrant mechanical features in the tumor microenvironment that present pathological signals to drive tumor malignancy. Alterations to extrinsic signals such as tumor ECM stiffness and composition have been well described; however, the development of solid stresses within the tumor and its effect on tumor evolution is poorly understood. Recent reports have suggested that intra-tumoral stress guides cancer cell escape from tumors, highlighting an emergent mechanobiological driver of cancer progression; however, these aspects are difficult to investigate with standard in vitro tools and requires the development of advanced biophysical tools. To address this, we have developed a microfluidic platform that generates deformable alginate microbeads that are able to quantify compressive stresses generated within a growing glioblastoma (GBM) tumorsphere.
Materials and Methods:: Microfluidic devices were designed using AutoCAD 2023 and include two semicircular inlets and one circular outlet with diameters of 2.5mm and 4.0mm, respectively. Sylgard 184 elastomer and curing agent were mixed in a 10:1 ratio, degassed, and poured onto SU-8 photoresist molds. 1% wt medium viscosity alginate fluorescein (Creative Pegworks) served as the dispersed phase and soy bean oil (Thermo-Fisher) served as the continuous phase. Dispersed phase flow rate was maintained at 50µL/hr and continuous phase flow rates varied from 150-450µL/hr. Microbeads were collected in TBS with 1% CaCl2. Particle analysis was performed using ImageJ (FIJI). Hanging drop method was used to encourage encapsulation of the microbeads by GBM tumorspheres. Time-lapse fluorescent microscopy was used to longitudinally track tumor development and bead position. Atomic force microscopy (AFM) was used to calculate bead stiffness. Finite element based-models were used to approximate bead deformation and subsequently calculate compressive stress.
Results, Conclusions, and Discussions:: To optimize force probe analyses, beads production must be high throughput, monodisperse, and uniform in mechanical properties. Through video assessments, alginate beads were generated at a rate of 5 beads/second enabling ~20,000 beads per h (Figure 1B). To determine bead size characteristics, we utilized ImageJ particle analysis of the collected and determined an average diameter of 5.6µm with a standard deviation of 3.0µm and average circularity of 0.82 (Figure 1 C-D). We then evaluated microgel mechanics via AFM force mapping of 512 force curves and determined a median Young’s modulus of 2.56kPa (Figure 1E). In preliminary experiments, we have successfully integrated force probes into GBM tumorspheres embedded in 3D hyaluronic acid hydrogels, showing proof or principle (Figure 1F). Here, we present the development of a force probe platform capable of quantifying compressive stress in a 3D context. We have demonstrated an approach that generates monodisperse and mechanically uniform force probes in a high throughput manner. Using this system, we are able to begin interrogating the magnitude and dynamics of intra-tumoral stress accumulation in tumors. Additionally, this platform opens the door for new inquiries of the impact of compressive stress in other contexts, including developmental biology, stem cell biology, and fibrotic disease.
