Design and construction of exosomes carrying miRNAs capable of suppressing the synthesis of HER2. The objective of the first series of experiments was to design miRNAs targeting HER2. To this end we have constructed 7 miRNAs from which to select a miRNA that was the most effective in blocking the synthesis of HER2 in a HER2 positive cancer cell line and in a cell line transfected with a plasmid encoding HER2. The miRNAs were cloned in a miRNA expression vector named pcDNA6.2-GW/EmGFP-miR-neg downstream of an open reading frame encoding EGFP as described in Materials and Methods.
In the initial screening, SK-OV-3, a HER2-high expressing cell line and HEp-2, a HER2 negative cell line transfected with a plasmid encoding HER2 were transfected with the plasmids encoding the miRNAs. Of the 7 miRNAs tested the miRNA No.1 and No.4 were most effective in suppressing the accumulation of HER2. On the basis of these results, miRNAs No.1 and No.4 were selected for further studies and designated as miR-HER2-1 and miR-HER2-4 respectively.
Next, we modified the two miRNAs by the addition of sequences embodying exosome-packaging-associated motifs (EXO-motifs). The efficacy of suppressing HER2 by miR-HER2-E1 and miR-HER2-E4 was then retested in SK-OV-3 cells and in HEp-2 cells expressing HER2. On the basis of the results shown in Fig. 1a and b, we selected miR-HER2-E1 for further studies. The sequences of miR-HER2-1, miR-HER2-4, miR-HER2-E1 and miR-HER2-E4 are listed in Materials and Methods.
Production and characterization of exosomes containing miR-HER2-E1. In all experiments described in this report we used exosomes produced in HEK-293 cells. The properties of these exosomes were investigated as follows. Briefly:
HEK-293 cells were remain untreated (293) or transfected with 10 µg of non-target (NT) or miR-HER2-E1 plasmids. After 52 h the extracellular medium was collected and the exosomes were purified as described in Material and Methods. The purified exosomes were tested for size distribution and for the presence of proteins associated with exosomes. Typically, exosomes contain Alix, CD9, Annexin V, Flotillin-1, and TSG101.
To test for protein content, purified exosomes were lysed by RIPA lysis buffer. Next, 15 µg of solubilized exosome protein subjected to electrophoresis in denaturing gels, and reacted with antibodies to above proteins (Fig. 2a). As expected the exosome proteins were present in purified exosomes derived from HEK-293, NT or miR-HER2-E1 transfected cells.
Exosomes purified from HEK-293 cells transfected with miR-HER2-E1 were analyzed by Izon’s qNano technology as described in Materials and Methods. The results (Fig. 2b) show that the size distributions of exosomes generated by the cell lines transfected with NT or miR-HER2-E1 were nearly identical.
Figure 2c shows the expression of mature miR-HER2-E1 by qPCR analysis. As expected the exosomes were purified from parental HEK-293 cells and the NT-transfected cells did not contain detectable amounts of miR-HER2-E1.
Exosome-delivered miR-HER2-E1 downregulated the accumulation of HER2 and reduced the viability of cells expressing high levels of HER2. In this series of experiments, we examined whether miR-HER2-E1 produced in HEK-293 cells and delivered via exosomes effectively blocked the accumulation of HER2. We report 2 series of experiments.
In the first replicate cultures each containing 2.5 × 105 SK-OV-3 cells were exposed for 72 h to (i) 0.1 µg, 5 µg, or 20 µg of purified exosomes carrying miR-HER2-E1, or (ii) 20 µg of purified exosomes produced in NT transfected HEK-293 cells, or (iii) remain untreated (Con). The HER2 cell lysates were subjected to electrophoresis in denaturing gels and reacted with antibodies to HER2. Figure 3a shows that the accumulation of HER2 decreased in SK-OV-3 exposed to exosomes containing miR-HER2-E1 in a dose dependent manner.
The second series of experiments was designed to determine whether the miR-HER2-E1 expressing exosomes were also able to suppress the accumulation of exogenous HER2. HEp-2 cells were transfected with a plasmid encoding HER2 for 36 h, then exposed for 36 h to 20 µg of purified exosomes produced in miR-HER2-E1 or NT transfected HEK-293 cells. Figure 3b shows that the expression of exogenous HER2 decreased in cells exposed to exosomes containing miR-HER2-E1.
HER2 positive cancer cells respond to treatment with anti-HER2 antibody suggesting that HER2 is an essential cell surface protein. To test the hypothesis that the exosome-delivered miR-HER2-E1 kill HER2 dependent cells by blocking the replenishment of the protein in HER2-positive SK-OV-3, HCT116 cancer cells and HER2-negative MDA-MB-231, HEp-2 cells were each seeded in 96-well plates and exposed to 1 µg of exosomes purified from HEK-293 cells transfected with miR-HER2-E1. After 72 h of incubation, the relative cell viability was determined by the CCK8 assay. The obtained values were normalized with respect to the values of mock-treated control group as described in Materials and Methods. The results (Fig. 4) show the following:
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SK-OV-3 cells exposed to exosome carrying miR-HER2-E1 exhibited 60% cell viability as compared to the viability of SK-OV-3 cells treated with NT-packaged exosomes;
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HCT116 cells exposed to exosomes carrying HER2-E1 exhibited 40% cell viability as compared to 90% cell viability of HCT116 cells treated with NT-packaged exosomes;
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HEp-2 and MDA-MB-231, the two HER2-negative cells exhibited 90–100% cell viability on exposure to exosomes carrying miR-HER2-E1.
These results suggest the following: (i) miR-HER2-E1 kills HER2 dependent cells by blocking the replenishment of the protein. (ii) miR-HER2-E1 had no effect on the viability of cells that were not dependent on HER2 for viability.
Anti-tumor efficacy of miR-HER2-E1 delivered by administration of exosomes in vivo. In this series of experiments Balb/c derived nude mice were injected subcutaneously into flanks with 5 × 106 SK-OV-3 cells (HER2 positive), HCT116 (HER2 positive) or MDA-MB-231 (HER2 negative) cells. Once the tumors averaged 90 mm3 in size they were injected intratumorally with 50 µl containing 10 µg of exosomes purified from miR-HER2-E1, or NT transfected HEK-293 cell 6 times in total on days 1, 4, 7, 10, 13, 16 respectively. The size of tumors was measured on days 1, 4, 7, 10, 13, 16, 19 and 22. The results (Fig. 5a, b and c) show that the exosomes carrying miR-HER2-E1 caused a reduction in the volume of SK-OV-3 tumors (Fig. 5a) or in HCT116 tumors (Fig. 5b), but not in tumors induced by MDA-MB-231 tumor cells (Fig. 5c). These results are consistent with the results obtained in cell culture studies.
Enhancement of anti-tumor activity by targeting exosome to HER2 positive cells. The experiments described earlier in the text showed that exosomes carrying miR-HER2-E1 entered into both HER2 positive and HER2 negative cells ultimately killed only HER2 positive cells. The objectives of the studies described below were to enhance the entry of the exosomes into HER2 positive cells. To achieve this objective, we constructed two cell lines designed to produce novel exosomes. The 293-miR-XS HER2 exosome carried on their surface a peptide which enabled exosome to adhere to the HER2 on the surface of HER2 positive cells. The 293-miR-XS exosome lacked the HER2 adhesion peptide. Both exosomes packed miR-HER2-E1. Details of the construction and properties of the two cell lines are described in Materials and Methods.
Two series of control experiments are relevant here. First, verified the presence of miR-HER2-E1 in exosomes produced in both cell lines. As illustrated in Fig. 6a, the exosomes produced by both cell lines carried miR-HER2-E1 as determined by analyses of cell pellets and purified exosomes.
Next, the exosomes produced by the two cell lines were purified and tested by ELISA to verify that they differed in their ability to adhere to HER2 protein. In brief, exosomes purified from 293-miR-XS and 293-miR-XS-HER2 stable cell lines were coated onto the 96-well ELISA plates. After removal of the coating solution and blocking nonspecific binding by bovine serum albumin (BSA), the coated exosomes were incubated with HER2 protein, and then exposed to HRP-conjugated rabbit anti-HER2 antibody. The plates were then rinsed again, followed by exposure to tetramethyl benzidine substrate (TMB) for color development. The reaction was stopped by the addition of a stop solution. Plates were read on a BioTek microplate reader at a wave length of 450 nm. As shown in Fig. 6b, purified exosomes produced by 293-miR-XS-HER2 preferentially bound HER2 protein.
Anti-tumor efficacy of exosomes carrying miR-HER2-E1 and adhering to HER2. In this series of experiments nude mice derived from Balb/c were injected subcutaneously into flanks with 5 × 106 SK-OV-3 cells. Tumors averaging 80 mm3 were injected intravenously on days 1, 4, 7, 10, 13, 16, 19 and 22 with 3 µg/animal (Fig. 7a) or 30 µg/animal (Fig. 7b) of exosomes purified from 293-miR-XS-HER2, 293-miR-XS or parental HEK-293 cells. The sizes of tumors were measured on days 1, 4, 7, 10, 13, 16, 19, 22, 25 and 28.
The results of these experiments are shown in Fig. 7a and b. The results show that exosomes purified from 293-miR-XS-HER2 cells were significantly more effective in reducing the growth of HER2-positive tumors as compared to exosomes purified from HEK-293 or 293-miR-XS cells. The results also show that the shrinkage of tumors injected with 3 µg or 30 µg of 293-miR-XS-HER2 exosomes /animal was virtually identical suggesting that the 3 µg dose was close to or exceeded the amounts required to kill the susceptible cells.