Engineering probiotic biohydrogen micro-factories to initiate reductive stress for boosting tumor vulnerability

Disruption of redox homeostasis profoundly affects cellular metabolism and activities. While oxidative stress is extensively studied in cancer therapies, research on reductive stress remains in its infancy. Molecular hydrogen (H₂), a well-known antioxidant, holds significant potential to induce reductive stress due to its strong antioxidative properties, making it a promising candidate for cancer therapy. However, it remains a major challenge to develop a sustainable H₂ delivery system in vivo. Herein, we designed a micro-factory by engineering a gel-based microcapsule that encapsulates Enterobacter aerogenes, a.k.a. probiotic biohydrogen microcapsules (PBMCs), enabling the sustained H₂ generation within tumor microenvironment. Notably, PBMCs effectively suppressed the proliferation of eight tumor cell lines as well as drug-resistant cancer cells. The prolonged H₂ release from PBMCs induced reductive stress, as evidenced by a significant increase in the GSH/GSSG ratio in 4T1 cells. Moreover, PBMCs displayed significant antitumor effects in breast, melanoma and liver cancer models. The inhibition of PI3K-AKT pathway and the activation of MAPK pathway were identified as key mechanisms responsible for inducing tumor cell cycle arrest and apoptosis. The PBMCs also exhibited synergistic effects in combination with chemotherapeutics, resulting in robust inhibitions of preinvasive carcinoma growth and commonly associated pulmonary metastasis. Overall, our study introduces an innovative strategy to manipulate reductive stress in the tumor microenvironment through in situ H₂ generation, thereby enhancing tumor vulnerability.