Bioengineering & Translational Medicine · Published 2026-04-09 · DOI 10.1002/btm2.70135
Abstract Enzyme‐induced self‐assembling peptides (EISAPs) are a promising class of enzyme‐activated anticancer therapeutics, yet their translational screening is limited by the lack of 3D tumor models that effectively capture drug penetration, self‐assembly dynamics, and treatment response. To address this need, we developed a pillar–perfusion 3D breast cancer spheroid platform to screen a six‐peptide panel—P1 (Fmoc‐FF‐pTyr), P2 (Fmoc‐FF‐pThr), P3 (RGD‐FF‐pTyr), P4 (NBD‐FF‐pTyr), P5 (Nap‐FF‐pTyr), and P6 (Nap‐FF‐pThr)—under static and dynamic flow. Hydrogel optimization identified a 2% gelatin/1% alginate matrix enabling >90% spheroid transfer efficiency and stable non‐invasive morphology, while Matrigel‐based embedding generated invasive spheroids mimicking metastatic behavior. Across the peptide panel, P1 and P5 produced the strongest cytotoxic effects, with dynamic perfusion further enhancing activity (P1 viability ∼55% at 100 μM). Co‐treatment with P1 + 5 μM Doxorubicin resulted in enhanced viability loss and complete inhibition of invasion. Fluorescence imaging of NBD‐FF‐pTyr confirmed progressive intratumoral penetration and core accumulation over 5 days. RT‐qPCR analysis demonstrated peptide‐ and subtype‐specific transcriptional effects, with P1 in MCF‐7 spheroids significantly downregulating BCL2, BRCA2, and TP53, while P5 in MDA‐MB‐231 spheroids produced the strongest repression of survival and DNA‐repair pathways. These findings establish a dynamic, high‐throughput 3D platform for comparative EISAP screening and demonstrate the therapeutic potential of enzyme‐responsive peptides in complex tumor microenvironments.
Abstract from DOAJ. Public domain (CC0 1.0).
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