The dilemma of cancer biomechanics assessment: Living soft matter from a rheological perspective

Biomechanical properties of cancer models, both in vitro (cellular) and ex vivo (tissue-based), are increasingly recognized as valuable biomarkers, offering critical insights for distinguishing pathological states from healthy counterparts, particularly in cancer progression. However, mechanobiology of living matter exhibits substantial variability, being sensitive to a range of factors that can be grouped into biological characteristics, substrate properties (e.g., surface type, ECM composition, topography, functionalization), and methodological parameters, including technique, experimental settings (e.g., indentation depth, loading rate, applied force, temperature), and tip properties (e.g., shape and size). Each of these variables can shift measured elasticity, typically reported as Young's modulus, by orders of magnitude, complicating the interpretation of mechano-markers. A systematic analysis of these discrepancies is largely lacking. This review critically evaluates published data, rescaling and comparing results to provide a comprehensive view of mechanobiology across 2D and 3D models, whether cellular or tissue-based. Notably, a consistent trend emerges: biomechanical properties fluctuate markedly with applied stress due to stress-induced stiffening or softening, making probe-sample contact area, influenced by probe geometry or indentation depth, a key determinant of measurements. Moreover, most studies focus solely on elastic measurements, often neglecting viscoelasticity, which is essential to capture the time-dependent behaviours of living tissues.