Softer substrates mechanical primes sustained and metabolically fit CD8+ T cells for anti-tumor activity

Adoptive T cell therapy for solid tumors is limited by poor persistence of CD8+ T cells, a dysfunction that is often programmed during ex vivo expansion. Here, we show that, when biochemical inputs are held constant,substrate mechanics alone can direct durable anti-tumor function in primary human CD8+ T cells. Using polyacrylamide (PA) hydrogels of defined stiffness (soft [~]1 kPa; stiff [~]55 kPa) in both flat and bead formats, we first establish that contact geometry dominates early activation, whereas substrate stiffness governs the 14-day expansion trajectory. Across the rapid expansion protocol, flat PA substrates sustain proliferation, limit PD-1/LAG-3 acquisition, and preserve a balanced effector-regulatory cytokine profile. In contrast, Dynabeads-expanded cells exhibit net cell loss and a more pronounced decline in cytokine output over time. To define the underlying programs, RNA-seq identifies a 125-gene biomimetic core shared by both PA conditions but absent from Dynabeads, encompassing proliferation, OXPHOS, mechanobiology, and a stem-like precursor (Tpex) signature. Consistent with these transcriptional differences, metabolic profiling shows that flat soft PA best preserves dual glycolytic and mitochondrial capacity at day 14, indicating enhanced bioenergetic flexibility. Functionally, PA-primed CD8+ T cells display superior cytotoxicity against MDA-MB-231 and MCF-7 breast cancer cells in both 2D and collagen-based 3D co-cultures, with this advantage maintained under matrix constraints that mimic solid tumor microenvironments. Together, these findings establish substrate mechanics as a tunable and functionally decisive design parameter for engineering durable, solid-tumor-effective CD8+ T cell products in preclinical in vitro models of solid tumors.