Intrapleural nano-immunotherapy promotes innate and adaptive immune responses to enhance anti-PD-L1 therapy for malignant pleural effusion

Malignant pleural effusion (MPE) is indicative of terminal malignancy with uniformly fatal prognosis. Often, two distinct compartments of tumor microenvironment, the effusion and disseminated pleural tumors, co-exist in the pleural cavity, presenting a major challenge for therapeutic interventions and drug delivery. Clinical evidence suggests that MPE comprises abundant tumor associated myeloid cells with the tumor-promoting phenotype, impairing antitumor immunity. Here, we developed liposomal cyclic dinucleotide (LNP-CDN) for targeted activation of STING signaling in macrophages and dendritic cells and showed that, upon intrapleural administration, they induce drastic changes in the transcriptional landscape in MPE, mitigating the immune cold MPE in both the effusion and pleural tumors. Moreover, combination immunotherapy with blockade of PD-L1 potently reduced MPE volume and inhibited tumor growth not only in pleural cavity but also in lung parenchyma, conferring significantly prolonged survival of MPE-bearing mice. Furthermore, the LNP-CDN-induced immunological effects were also observed with clinical MPE samples, suggesting the potential of intrapleural LNP-CDN for clinical MPE immunotherapy. Overall design: The LLC MPE mice (n = 3/treatment) were treated with intrapleural PBS, LNP-CDN (1 µg), anti-PD-L1 Ab (10F.9G2; 30 µg; BioXCell) or LNP-CDN plus anti-PD-L1 Ab. 48 h later, pleural fluid cells were collected and pooled from n = 3 MPE mice under each treatment. Biological replicates were then processed at the single-cell suspension stage with an equivalent number of cells from each replicate. Four thousand cells from each of the mixed samples for each condition were loaded onto the 10x Genomics Chromium System. Library preparation was performed using 10x Genomics Chromium Single cell 3' reagent kit V3 according to the manufacturer's instructions by Cancer Genomics Shared Resource (CGSR) of the Wake Forest Baptist Comprehensive Cancer Center and passed quality control. Libraries were sequenced using a NovaSeq 6000 (one library per lane) at the CGSR.