Extensional Flow Microfluidics Reveal Size-Dependent Core-Shell Mechanical Heterogeneity in Pancreatic Tumor Spheroids

Understanding the mechanical behavior of cancer cell aggregates is important for investigating metastatic potential and developing effective therapeutics. Tumor cell clusters are major contributors to metastasis and can undergo extreme deformation to migrate through capillaries smaller than the cluster size, suggesting complex multicellular mechanics. Despite their clinical significance, the mechanics of multicellular spheroids remain underexplored. In this study, the mechanical behavior of spheroids formed from the pancreatic cancer cell line PANC-1 was investigated using a cross-slot microfluidic device. Suspended spheroids from two size ranges were stretched in non-contact extensional flow (small spheroids mostly 40 < D0 < 75 μm, large spheroids mostly 90 < D0 < 145 μm). Digital image correlation analyses of intra-spheroid strains revealed strong deformation differences for small and large spheroids while analyses based on 2D projected contours of the spheroid shape did not show trends. Smaller spheroids deformed relatively homogeneously and exhibited significant overall stretching. In contrast, larger spheroids displayed heterogeneous deformation characterized by a stiff central core and a deformable outer shell. To investigate the origin of these mechanical differences, cellular morphology and the expression of stiffness-associated proteins, including vimentin and E-cadherin, were assessed from confocal microscopy. Larger spheroids display significantly lower nuclear sphericity than small spheroids, indicating more cellular packing with increasing spheroid size. Additionally, vimentin expression was concentrated in the outer shell of large spheroids, whereas small spheroids exhibit vimentin expression throughout the aggregate. Thus individual cell morphology and cytoskeletal networks seemed to contribute to the core-shell biomechanical behavior of the large spheroids and lack thereof for small spheroids. Taken together, this study suggests that as spheroids grow, they transition from an irregular and disorganized cluster at low cell numbers to a coordinated and hierarchical structure at sufficient cell numbers that corresponds to diameters ≳ 100 μm.