As a pilot proof of principle study, we defined certain criteria for our project which were most likely to translate into an in-vivo tumor setting. Given the timeframe of the project, we restricted the scope of our experiments to generating and characterising Affi-OMVs and THP-OMVs, and testing their uptake.
To demonstrate that Duonco is functional as a complete system, the following criteria should be met:
We isolated OMVs from bacterial cultures via the protocol specified on the experiments page. OMVs can be detected indirectly by probing for the presence of outer membrane proteins. The most common protein used for this purpose is Outer Membrane Protein A (OmpA). OmpA is a constitutively expressed 37 kDa outer membrane protein present in E.coli[1]. We confirmed the presence of OmpA in the OMV fraction isolated from E.coliΔtolR and E.coliΔnlp1 (Fig 1)
Fig 1: Anti-OmpA Western blot of OMV fractions from E.coliΔtolR and E.coliΔnlp1. Equal volumes were loaded for the OMV samples. Bands were detected at the expected size of 37 kDa, indicating the presence of OmpA and, by extension, OMVs. Cell pellets of both strains were also analysed. The presence of excess samples in these lanes hindered the proper resolution of proteins during electrophoresis, causing a hazy and diffused appearance.
The presence of OMVs was subsequently confirmed by transmission electron microscopy (Fig 2).
Fig 2: (A, B) Cryogenic electron microscope images of E.coliΔtolR OMVs suspended in PBS. Spherical structures with lipid bilayers are distinctly visible, confirming the presence of OMVs.
During the isolation of OMVs, cell debris may co-sediment, causing inflated protein concentration values, as well as introducing unwanted bacterial cell components. Isolated OMVs were found to be free of viable cells by plating on LB agar. SDS PAGE analysis revealed that OMVs were highly enriched for proteins ~35 kDa in size, which we hypothesise are members of the outer membrane protein (OmP) family (Fig 3). Very faint bands were noted for other molecular weights, indicating the general absence of cell debris. Overall, the OMV isolation protocol performed well and primarily yielded OMVs, with minimal contamination.
Fig 3: SDS PAGE of OMVs and cell pellets their corresponding source strains. Excess sample for bacterial pellets caused the appearance of a smear. OMV samples had intense bands ~35 kDa. This SDS PAGE gel is a duplicate of the samples analysed by Western blot (Fig 1).
Affi-OMVs containing ClyA-V5-Affi and THP-OMVs containing ClyA-3XFLAG-THP were isolated. We analysed the OMV fraction by Western blotting and found evidence that proteins correctly localised to the OMV fraction.
Fig 4: (A) Anti-FLAG Western blot of bacterial cells (before and after induction) and THP-OMV fraction. A band at 38 kDa corresponding to ClyA-3XFLAG-THP is visible in all lanes, suggesting the production and localization of ClyA-3XFLAG-THP to the OMV fraction. A lower weight band (~27 kDa) is visible in cells following induction (B) Anti-V5 Western blot of bacterial cells (before and after induction) and Affi-OMV fraction. A band at 43 kDa corresponding to ClyA-V5-Affi is visible in all lanes, suggesting the production and localization of ClyA-V5-Affi to the OMV fraction. Additional bands are also noted. Equal volumes were loaded for the induced and uninduced cell samples for both blots. Expression is also noted in the uninduced samples, possibly due to the leaky behaviour of the lac operon (Fig 17, results).
We measured cell viability following treatments of different concentrations of THP-OMVs expressing ClyA-3XFLAG-THP, as compared to a control treatment of PBS. SK-BR-3, a HER2+ breast cancer cell line, was utilised for this assay. The mean percentage viability is reported as the viability of SK-BR-3 cells treated with THP-OMVs normalised to the mean viability following PBS treatment (Fig 4). As the ClyA domain of both ClyA-3XFLAG-THP and ClyA-V5-Affi is effectively the same, we hypothesise that we would obtain similar results for Affi-OMVs expressing ClyA-V5-Affi on the surface.
Fig 5: Cytotoxicity of THP-OMVs as measured using the MTT assay with SK-BR-3 cells. The mean percentage viability is reported as the viability of THP-OMV treated SK-BR-3 cells normalised to the mean viability following PBS treatment. Viability is reported as mean ± standard error of mean of three biological replicates. Concentrations of THP-OMVs indicated are working concentrations.
We aimed to test the selective internalisation of Affi-OMVs and THP-OMVs into a cell line overexpressing both target markers, HER2 and CX3CR1, compared to a negative control cell line with low expression of the markers. The test cell line was SK-BR-3, and the negative control was HEK 293T [2]. They were chosen due to relative differences in the expression of HER2 and CX3CR1 [3][4].
We designed different treatments to study internalisation (Table 1, Fig 6). Internalisation was detected with immunocytochemistry (ICC) and Western blot. We have adapted portions of this experiment from [5].
| Treatment | SK-BR-3 (1) | SK-BR-3 (2) | SK-BR-3 (3) | SK-BR-3 (4) | HEK 293T(5) |
|---|---|---|---|---|---|
| Type of OMVs used for treatment | Native OMVs | Affi-OMVs | THP-OMVs | Affi-OMVs +THP-OMVs | Affi-OMVs +THP-OMVs |
| OMV quantity | 5 μg | 5 μg | 5 μg | 5 μg + 5 μg | 5 μg + 5 μg |
| Protein probed | OmpA | V5 tag | 3XFLAG tag | V5, 3XFLAG | V5, 3XFLAG |
Fig 6: Workflow to study internalisation.
ICC can enable visualisation of internalisation following Affi-OMV and THP-OMV treatment. We performed ICC on SK-BR-3 cells treated with native OMVs using two different fixatives, methanol and PFA, in order to decide which fixative was most compatible with this cell line. Through our standardisation experiments, we found that methanol fixation yielded better results. Total protein content in the OMVs was used as a proxy measure for OMV quantity. We incubated SK-BR-3 and HEK 293T cells with treatments specified in Table 1 for 4-5h at 37°C. After washing, fixation and staining, we visualised the localization of OMVs by confocal microscopy. We also lysed cells after incubation and analysed them via Western blot. (Fig 7, 8, 9, 10, 11, 12).
Fig 7: Fig 6: Native OMVs were incubated for 4 hours with SK-BR-3 cells. OMVs (green) are visualised by rabbit anti-OmpA antibodies and anti-rabbit Alexa Fluor 488. Signal was detected from the extracellular region.
Fig 8: Western blot against V5 tag. Affi-OMVs were incubated for 4 hours with SK-BR-3 cells and HEK 293T. We observed a band at the expected size for ClyA-V5-Affi in all three lanes. Greater signal is detected from SK-BR-3 than HEK 293T. We approximately loaded equal sample of lysed cells between SK-BR-3 and HEK 293T. Affi-OMVs serves as a positive control for signal and protein size. These results are suggestive of selective internalisation.
Fig 9: Affi-OMVs were incubated for 4 hours with SK-BR-3 cells. Affi-OMVs (green) expressing ClyA-V5-Affi are visualised by rabbit anti-V5 antibodies and anti-rabbit Alexa Fluor 488. Signal was detected at the periphery of cells, indicating cell binding and possibly internalisation.
Fig 10: THP-OMVs were incubated for 4 hours with SK-BR-3 cells. THP-OMVs (green) expressing ClyA-3XFLAG-THP are visualised by anti-FLAG antibodies and anti-mouse Alexa Fluor 488. Signal is detected from the cytoplasm of the cell, indicating internalisation.
We also performed a Western blot against 3X FLAG of lysed SK-BR-3 and HEK 293T cells that were incubated with THP-OMVs, however the blot was inconclusive. We believe that there was not enough protein for detection, we will repeat the blot by loading more cell lysate in the future.
Fig 11: Affi-OMVs and THP-OMVs were co-incubated for 4 hours with SK-BR-3 cells. Affi-OMVs (red) expressing ClyA-V5-Affi are visualised by anti-V5 antibodies and anti-rabbit Alexa Fluor 633. THP-OMVs (green) expressing ClyA-3XFLAG-THP are visualised by anti-FLAG antibodies and anti-mouse Alexa Fluor 488. Signal from both OMVs was detected from the cytoplasm, suggesting internalisation.
Fig 12: Affi-OMVs and THP-OMVs were co-incubated for 4 hours with HEK 293T cells. Affi-OMVs (red) expressing ClyA-V5-Affi are visualised by anti-V5 antibodies and anti-rabbit Alexa Fluor 633. THP-OMVs (green) expressing ClyA-3XFLAG-THP are visualised by anti-FLAG antibodies and anti-mouse Alexa Fluor 488. Signal was detected from the extracellular media.
We performed a blinded image analysis of the internalisation studies. From this preliminary data we arrived at the following conclusions:
To summarise, we have developed a functional, flexible, and adaptable dual OMV system, and provided a preliminary proof of principle. More concrete experiments are required as mentioned above. Conceptually, once established in cell culture systems, similar analyses pipelines can be extended to systems recapitulating in-vivo tumours in higher detail like 3-D cell cultures and patient derived organoids. Ligand-receptor combinations may be optimised for different cancers and patients using high-throughput approaches. One can thus imagine a future when real-time protein expression data from in-vivo tumours is used to inform the construction of specific OMVs to maximise therapeutic output.