Targeted cancer therapies, while often effective, have limited utility due to preexisting primary or acquired secondary resistance. Consequently, agents are sometimes used in combination to simultaneously affect multiple targets. MicroRNA mimics are excellent therapeutic candidates because of their ability to repress multiple oncogenic pathways at once. Here we treated the aggressive Kras;p53 non-small cell lung cancer mouse model and demonstrated efficacy with a combination of two tumor-suppressive microRNAs. Systemic nano-delivery of miR-34 and let-7 suppressed tumor growth leading to a survival advantage. This combinatorial microRNA therapeutic approach engages numerous components of tumor-cell addictive pathways and highlights the ability to deliver multiple microRNAs in a safe and effective manner to target lung tissue.

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The SRB assay has been used since its development in 1990 (Skehan et al., 1990) to inexpensively conduct various screening assays to investigate cytotoxicity in cell based studies (Vichai and Kirtikara, 2006). This method relies on the property of SRB, which binds stoichiometrically to proteins under mild acidic conditions and then can be extracted using basic conditions; thus, the amount of bound dye can be used as a proxy for cell mass, which can then be extrapolated to measure cell proliferation. The protocol can be divided into four main steps: preparation of treatment, incubation of cells with treatment of choice, cell fixation and SRB staining, and absorbance measurement. This assay is limited to manual or semiautomatic screening, and can be used in an efficient and sensitive manner to test chemotherapeutic drugs or small molecules in adherent cells. It also has applications in evaluating the effects of gene expression modulation (knockdown, gene expression upregulation), as well as to study the effects of miRNA replacement on cell proliferation (Kasinski et al., 2015).

MicroRNAs are small RNAs that negatively regulate gene expression posttranscriptionally. Because changes in microRNA expression can promote or maintain disease states, microRNA-based therapeutics are being evaluated extensively. Unfortunately, the therapeutic potential of microRNA replacement is limited by deficient delivery vehicles. In this work, microRNAs are delivered in the absence of a protective vehicle. The method relies on direct attachment of microRNAs to folate (FolamiR), which mediates delivery of the conjugated microRNA into cells that overexpress the folate receptor. We show that the tumor-suppressive FolamiR, FolamiR-34a, is quickly taken up both by triple-negative breast cancer cells in vitro and in vivo and by tumors in an autochthonous model of lung cancer and slows their progression. This method delivers microRNAs directly to tumors in vivo without the use of toxic vehicles, representing an advance in the development of nontoxic, cancer-targeted therapeutics.