Acidosis and hypoxia cooperatively impair immune cell infiltration in a microengineered tumor microenvironment

Abstract Introduction Recent advances in cancer immunotherapies, particularly chimeric antigen receptor (CAR) T and natural killer (NK) cell therapies, have shown considerable success in treating hematological malignancies, leading efforts to extend these approaches to solid tumors. The tumor microenvironment (TME), with characteristic hypoxia and acidity, is thought to hinder immune cell trafficking and impair their survival and function through immunosuppressive mechanisms. However, how the TME influences T and NK cell infiltration remains incompletely understood, and approaches to overcome its adverse effects are still limited. To address this, we developed a microfluidic in vitro platform capable of real-time monitoring of T and NK cell infiltration under controlled hypoxic and acidic conditions. Our findings reveal that hypoxia and low pH together significantly impair T and NK cell infiltration, reducing their migration by 46% and 23%, respectively. Acidic pH alone did not hinder infiltration, but in combination with hypoxia, it acutely compromised cell migration. These results offer novel quantitative insights into how environmental stressors within solid tumors hinder immune cell infiltration, laying a foundation for designing more effective immunotherapeutic strategies. Methods The microfluidic chip consists of six uniformly spaced micropillar arrays, with pillar spacing gradually decreasing from 12 μm at the inlet to 3 μm at the outlet to mimic the tumor physical barrier. The device was fabricated using standard soft lithography protocols, and the patterned PDMS layer was bonded to microscope slides via oxygen plasma treatment. Cells were suspended in growth medium at a concentration of 1 × 10⁶ cells/mL, deoxygenated with sodium metabisulfite for 1 hour, and pH-adjusted using sodium hydroxide. For hypoxia experiments, the oxygen level and pH were set to 9±2 mmHg and 6.5±0.1, respectively. Prior to cell loading, the microchannel was pre-washed for 3 minutes with either fresh or deoxygenated medium. The prepared cell suspension was then introduced into the chip using a pneumatic pump and perfused at 100 mBar for 7 minutes. This was followed by a 7-minute flush with growth medium at the same pressure to remove non-occluded cells. The number of retained cells within each of the six micropillar arrays was quantified, and an infiltration index, defined as the weighted number of cells reaching the farther arrays, was subsequently calculated. Results Our findings show that the tumor stressors substantially hinder the ability of T and NK cells to infiltrate, leading to a 46% (p = 0.005, N=6, n=480000) and 23% (p = 0.081, N=3, n=240000) decrease in their infiltration indices, respectively. Notably, an acidic environment alone did not affect immune cell infiltration. However, when combined with hypoxia, it markedly diminished their infiltration capacity. Neutralizing the acidic pH under hypoxic conditions improved the T cell infiltration index from 0.48 to 0.78 (p = 0.083, N=3, n=240000). Moreover, reoxygenation of T cells previously exposed to hypoxia fully restored their infiltration capacity, with an infiltration index of 0.90, comparable to that observed under normoxic conditions. To complement the earlier experiments, five patient-derived CAR-T cell samples were evaluated under normoxic and hypoxic conditions at pH 6.0. A considerable reduction in infiltration capacity was observed under hypoxia for CAR-T cells. The infiltration indices under normoxic and hypoxic conditions were 0.68 and 0.57, respectively (p = 0.0472, N=5, n=400000). All statistical comparisons were performed using the Mann–Whitney U test. N and n represent the number of samples and individual cells, respectively. Conclusions The results demonstrate that a hypoxic and acidic microenvironment together, significantly alters cell behavior, reducing infiltration efficiency through the microphysiological micropillar arrays. The pronounced decrease in the number of cells reaching the final, narrowly spaced regions, along with the lowered infiltration index suggests that oxygen deficiency and acidosis amplify the physical barrier properties of the tumor microenvironment. These findings highlight the individual contributions of hypoxia and pH in disrupting cellular immunotherapy trafficking and indicate that strategies aimed at oxygenation within solid tumors may substantially restore immune cell infiltration.