USMB-shMincle: a virus-free gene therapy for blocking M1/M2 polarization of tumor-associated macrophages

Abstract

Mincle is essential for tumor-associated macrophage (TAM)-driven cancer progression and represents a potential immunotherapeutic target for cancer. Nevertheless, the lack of a specific inhibitor has largely limited its clinical translation. Here, we successfully developed a gene therapeutic strategy for silencing Mincle in a virus-free and tumor-specific manner by combining RNA interference technology with an ultrasound-microbubble-mediated gene transfer system (USMB). We identified a small hairpin RNA (shRNA) sequence shMincle that can silence not only Mincle expression but also the protumoral effector production in mouse bone marrow- and human THP-1-derived macrophages in the cancer setting in vitro . By using our well-established USMB system (USMB-shMincle), the shMincle-expressing plasmids were delivered in a tissue-specific manner into xenografts of human lung carcinoma A549 and melanoma A375 in vivo . Encouragingly, we found that USMB-shMincle effectively inhibited the protumoral phenotypes of TAMs as well as the progression of both A549 and A375 xenografts in a dose-dependent manner in mice without significant side effects. Mechanistically, we identified that USMB-shMincle markedly enhanced the anticancer M1 phenotype of TAMs in the A549 and A375 xenografts by blocking the protumoral Mincle/Syk/nuclear factor κB (NF-κB) signaling axis. Thus, USMB-shMincle may represent a clinically translatable novel and safe gene therapeutic approach for cancer treatment.

Smad3 Promotes Cancer‐Associated Fibroblasts Generation via Macrophage–Myofibroblast Transition

“…TRI reagent (Molecular Research Center), reverse transcription system (Promega), and SYBR Green Supermix (Bio‐Rad) were used for RNA extraction, complementary DNA (cDNA) synthesis, and real‐time polymerase chain reaction (PCR), respectively, according to manufacturer's protocol. [ 53 , 54 ] α ‐SMA, VEGF, CD31, F4/80, LysM, and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) primers used in this study are listed in Table 3 . Gene expression levels normalized with GAPDH of three experiments were expressed as mean± standard error of the mean (s.e.m.).…”

Strategic Decoy Peptides Interfere with MSI1/AGO2 Interaction to Elicit Tumor Suppression Effects

“…The underlying mechanism by which Cilengitide effectively reduced GBM progression and metastasis was the preferential interruption of EGFRvIII/integrin β3 complex formation [44][45][46]. Interestingly, another recent study of ours that revealed that MSI1 promoted GBM tumorigenesis via upregulation of macrophage inhibitory factor 1 (MIF1) and M2 polarization [47] may provide another implication for the potential application of virus-free gene therapy based on blocking M1/M2 polarization of tumor-associated macrophages (TAMs) [48].…”

“…The 27 peptides were then incubated with His-tagged recombinant AGO2 and the immunoblotting results revealed that peptides 11 and 26 from C-terminal MSI1 appeared to be two of the strongest binding peptides to AGO2 among the array (Figure 2C). To investigate whether the identified peptides could indeed bind with AGO2 in cells, we utilized a 13 amino acid cell-penetrating peptide (CPP) from HIV-1 TAT (48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60) and conjugated to our candidate peptides, namely Pep11 and Pep#26, to facilitate their cellular uptake (Figure 3A) [30]. As shown in our co-immunoprecipitation competition experiments, control peptide (CP) could not interfere with the binding complex formation between MSI1 and AGO2 (Figures 3B and S1).…”

“…Peptide transfection was carried out with Proteojuice (Millipore 71281, Merck Co., Darmstadt, Germany) following the manufacturer's instruction. For the in vivo compatible cell-penetrating peptide (CPP)-modified peptides, we tested two different CPPs at the C-terminal ends, including TAT (48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60) from HIV [55][56][57].…”

LncRNA-Dependent Mechanisms of Transforming Growth Factor-β: From Tissue Fibrosis to Cancer Progression

“…Therefore, post-delivery monitoring and optimizing effective concentration of lncRNAs therapeutics are critical for translational application. Novel noninvasive ultrasound microbubble-assisted (USMB) delivery largely reduced the concentrations of lncRNA-targeting agents in nontargeted tissue [166], contributing to the safety and effectiveness in preclinical studies [73,80,167]. Thus, USMB represents a realistic approach to translating lncRNA-targeted therapeutics with added value in postdelivery monitoring and assessment with its imaging function.…”