Dynamic pillar-perfusion platform for screening enzyme-induced self-assembling peptide therapeutics in 3D breast cancer spheroids.

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.