Lyophilized bacteria-infected tumor cells for targeted immunotherapy of lung metastases and associated fibrosis.

Lung metastasis remains a major clinical challenge, often associated with poor prognosis due to its highly immunosuppressive microenvironment and fibrosis-induced complications. Current treatment strategies, including chemotherapy, radiotherapy, and immunotherapy, have shown limited efficacy in addressing lung metastases, and less attention has been given to their associated fibrosis. Here, we develop a 'cell-in-cell' delivery platform (i.e., lyophilized bacteria-infected tumor cells (LyoBT)) to simultaneously target lung metastasis and their associated fibrosis. This approach leverages the intrinsic lung tropism of tumor cells and the immunostimulatory properties of both tumor cells and bacteria, while mitigating tumorigenic and pathogenic risks through lyophilization. Notably, bacterial infection led to phenotypic changes in tumor cells. Specifically, characterization of LyoBT revealed upregulated expression of CD47, CD44, and E-cadherin, further enhancing lung targeting. Furthermore, increased calreticulin (CRT) exposure in LyoBT coupled with bacterial immune-stimulatory properties, promoted anti-tumor immunity. In a melanoma lung metastasis model, LyoBT demonstrated efficient accumulation in the lungs, leading to robust anti-tumor immune activation and significant inhibition of tumor progression. Notably, LyoBT also reduced fibrosis-associated immune cell infiltration and cytokine release, alleviating lung metastasis-induced fibrosis. Furthermore, LyoBT served as a drug delivery platform for immune checkpoint inhibitors (aPD-L1), with LyoBT@aPD-L1 demonstrating enhanced therapeutic efficacy. Our findings highlight the potential of LyoBT as a dual-functional strategy to combat both lung metastases and their associated fibrosis, offering a promising new avenue for bacterial-based cancer immunotherapy.