Tunable three-dimensional engineered prostate cancer tissues for in vitro recapitulation of heterogeneous in vivo prostate tumor stiffness

Parallel-plate compression stiffness measurements were performed using a CellScale MicroSquisher to quantify Young’s modulus of both in vivo tumor biopsies and in vitro engineered tissues under physiological testing conditions. For in vivo measurements, excised PC-3 xenografts were sliced (~1 mm thick) and cored with a 3 mm biopsy punch to obtain disc-shaped samples from core, midpoint, and periphery regions; samples were placed in PBS and tested immediately. For EPCaTs, disc-shaped PF-based hydrogels (cast in 3 mm PDMS molds; 1 mm thickness) were tested on culture days 1, 15, and 22 to track stiffness evolution with matrix composition (0–3% excess PEGDA) and remodeling. In both cases, samples were loaded into the MicroSquisher fluid-bath chamber filled with PBS and maintained at 37°C. A 558.8 µm diameter tungsten microcantilever beam fitted with a compression platen was used to compress tissues to at least 15% deformation at a maximum compression rate of 10 µm/s for three cycles. Applied force was calculated from beam deflection, and tissue displacement was tracked by the system camera; force–displacement data were exported and converted to compressive stress–strain, with Young’s modulus computed from the slope of the linear region. These MicroSquisher-derived modulus values were central to (i) defining the in vivo xenograft stiffness range and its spatial heterogeneity, and (ii) demonstrating that EPCaTs can be tuned to reproduce the full in vivo stiffness distribution through controllable matrix formulation and culture time.