In-vitro immune-modulation of triple-negative breast cancer through targeting miR-30a-5p/MALAT1 axis using nano-PDT...

Triple negative breast cancer (TNBC) is an immunogenic tumor; however, its tumor immune microenvironment (TIME) is densely packed with immune suppressive cytokines and immune checkpoints. The immune-suppressive features of TNBC TIME represent a considerable obstacle to any immunotherapeutic approach. The objective of this study was to develop a multimodal in-vitro strategy to manipulate the TNBC TIME and enhance patients' outcomes by employing carefully tailored hybrid chitosan-lipid Nanoparticles (CLNPs), metformin and chlorin e6 (Ce-6)-mediated PDT, alone or combined. Special focus is directed towards evaluation of the role of the selected treatment agents on the non-coding RNAs (ncRNAs) involved in tuning the immuno-oncogenic profile of TNBC, for instance, the miR-30a-5p/MALAT1 network.

This study enrolled 30 BC patients. CLNPs and ce-6-loaded CLNPs with different physicochemical features were synthesized and optimized using ionotropic gelation. The intracellular concentration and effects on MDA-MB-231 cellular viability were investigated. UHPLC was used to quantify ce-6. MDA-MB-231 cells were transfected with miR-30a-5p oligonucleotides and MALAT1 siRNAs using lipofection to investigate the interaction between MIF, PD-L1, TNF-α, IL-10, and the miR-30a-5p/MALAT1 ceRNA network. qRT-PCR was used to evaluate IL-10, TNF-α, and MIF expression levels, whereas flow cytometry was used for PD-L1.

Immunophenotyping of BC biopsies revealed significantly elevated levels of immunosuppressive markers, including IL-10, TNF-α, PD-L1, and MIF in BC biopsies compared to its normal counterparts. Upon patient stratification, it was shown that MIF and IL-10 are upregulated in TNBC patients compared to non-TNBC patients. Nonetheless, immune suppressive biomarkers expression investigated in the current study was generally correlated with signs of poor prognosis. CLNPs with mean particle size ranging from 50-150 nm were obtained. CLNPs exhibited different patterns of intracellular uptake, cytotoxicity and modulation of the immunosuppressive markers based on their physicochemical properties and composition. In particular, CLNP4 in-vitro effectively reduced IL-10, TNF-α, MIF, and PD-L1. Loading of Ce-6 into CLNP4 (Ce6-CLNPs) improved the in-vitro cytotoxic effects via PDT. In addition, PDT with Ce6-CLNP4 enhanced the expression of tumor-suppressive miR-30a-5p and decreased oncogenic lncRNA MALAT1 expression in MDA-MB-231 cells, suggesting a potential for modulating the TNBC immuno-oncogenic profile.

This study demonstrated that CLNPs and Ce-6-mediated PDT can modulate several key immunosuppressive factors and the miR-30a-5p/MALAT1 axis in TNBC cells. These findings provide a rationale for further in-vivo investigation of this multimodal therapeutic strategy.