Engineered Mycobacterium smegmatis-based intrapleural immunotherapy remodels the pleural immune microenvironment for malignant pleural effusion treatment

Background Malignant pleural effusion (MPE) is a common and devastating complication of advanced malignancies, particularly lung cancer, with limited therapeutic options and poor prognosis. Immune checkpoint inhibitors and cytokine therapies have shown promise in solid tumors but are constrained by dose-dependent toxicity and limited efficacy in the pleural cavity. Engineered Mycobacterium smegmatis ( M. smegmatis ) co-expressing a PD-L1 inhibitor and interleukin-15 immunocytokine (Ms-PI15) represents a novel in situ vaccine platform capable of sustained local drug delivery with favorable biosafety profiles. Here, we investigated the therapeutic efficacy and underlying mechanisms of intrapleural Ms-PI15 administration in a murine MPE model. Methods A syngeneic MPE mouse model was established by intrapleural inoculation of luciferase-expressing Lewis lung carcinoma (LLC-Luc) cells into C57BL/6 mice. Mice were treated with intrapleural injections of PBS, Ms-vector (empty vector control), or Ms-PI15. Tumor burden was monitored by bioluminescence imaging. Pharmacokinetics and biodistribution of Ms-PI15 were evaluated using DIR fluorescence imaging and bacterial colony enumeration. Flow cytometry and immunohistochemistry were employed to characterize immune cell populations in the tumor microenvironment, spleen, and thoracic draining lymph nodes (TDLNs). Biosafety was assessed by histopathological examination and serum biochemistry. Results Intrapleural administration of Ms-PI15 significantly suppressed MPE development and pleural tumor growth compared with PBS and Ms-vector controls. DIR fluorescence imaging and CFU enumeration demonstrated that Ms-PI15 accumulated predominantly in the pleural tumor microenvironment and TDLNs, peaking at 72 h post-administration, with minimal dissemination to distant organs. Mechanistically, Ms-PI15 remodeled the immune microenvironment by promoting maturation of CD80 + CD86 + myeloid dendritic cells (mDCs), driving M1 polarization of tumor-associated macrophages (increased CD86 + , decreased CD206 + ), and significantly enhancing infiltration of CD8 T cells and NK1.1 + NK cells in the pleural tumor, spleen, and TDLNs. No significant histopathological abnormalities or serum biochemical alterations were observed in major organs following Ms-PI15 treatment, confirming its favorable biosafety profile. Conclusion Intrapleural administration of the engineered M. smegmatis Ms-PI15 effectively inhibits MPE progression by remodeling the pleural immune microenvironment toward an antitumor phenotype, while maintaining excellent biosafety. These findings provide a preclinical foundation for the development of intracavitary bacterial immunotherapy as a novel treatment strategy for malignant pleural effusion.