Hydrogel microtumor arrays of patient melanoma recapitulate phenotypes and drug sensitivity

Patient-derived tumor xenograft (PDTX) models are considered the gold standard for preserving tumor heterogeneity and recapitulating patient drug responses, yet their use is limited by high cost, long timelines, and variable engraftment success. Here, we present the application of a soft lithography patterned polyacrylamide microtumor array platform designed to mimic biophysical aspects of the melanoma tumor microenvironment, including physiological stiffness, extracellular matrix (ECM) composition, and spatial confinement, which together recapitulate in vivo drug sensitivity for the evaluation of therapeutics. The microtumor array platform is comprised of approximately 100 distinct features per well, with a diameter of approximately 350 μm per feature, each imposing spatial confinement with peripheral geometric cues to confined cells. Using two BRAF^V600E mutant melanoma cell lines, Mela14 (treatment-resistant) and Mela16 (treatment-naïve), we investigated whether microtumor arrays could restore cancer stem cell (CSC) characteristics, using ABCB5 and CD271 marker expression, and recapitulate PDTX drug response phenotypes to the BRAF/MEK inhibitor combination dabrafenib/trametinib (DT). Immunofluorescence analysis revealed that CSC marker expression was diminished on conventional tissue culture plastic (TCP) but restored to levels comparable to primary and PDTX tissue upon five days of spiral-patterned hydrogel confinement. Drug response assays demonstrated that microtumor array primed Mela14 cells retained DT resistance, whereas Mela16 cells remained responsive, paralleling PDTX outcomes. Global proteomics indicated that spiral confinement upregulated pathways in Mela14 associated with drug resistance, metastasis, cell repair and survival, while spiral patterned Mela16 showed upregulated pathways associated with cellular homeostasis and increased proteome plasticity. These findings suggest that polyacrylamide microtumor arrays can reproduce key features of the in vivo melanoma microenvironment, which may enable rapid, reproducible, and clinically relevant drug sensitivity testing. Therefore, this platform offers potential as a complementary preclinical model for personalized medicine and therapeutic discovery in cancer.