Indenting multicellular spheroids with various tip geometries.

Spheroids are of great interest in the study of cancer as they can partially mimic the tumour microenvironment, thus allowing to investigate several aspects of cell – microenvironment interactions in healthy and diseased conditions, including those pertaining to mechanobiology. Atomic Force Microscopy (AFM) is a versatile tool for studying biological samples and their mechanobiological properties. In AFM, the tip shape and dimensions determine the contact geometry between the tip and the sample and the length scales at which the mechanical properties are probed. Given the complex multiscale structure of spheroids, the choice of tip geometry and size would allow, in principle, to dissect the mechanical response of the overall system into the contributions of the constituents, from the single cell level to the cellular aggregate. In this work, we studied the mechanical properties of spheroids derived from four cell lines (A549, NHLF, HT-29, and CCD-18Co cells). Our studies revealed that using different contact geometries in the fitting procedure results in significantly different Young’s modulus values, highlighting the multiscale response of these complex cellular systems and the importance of a precise experimental design and choice of the AFM probe for the nanomechanical measurements. We observed that the location of F-actin filaments is correlated with the rigidity of the spheroids.