A novel dual-release scaffold for fluorescent labels improves cyclic immunofluorescence

Abstract

Cyclic immunofluorescence is a powerful method to generate high-content imaging datasets for investigating cell biology and developing therapies. This method relies on fluorescent labels that determine the quality of immunofluorescence and the maximum number of staining cycles that can be performed. Here we present a novel fluorescent labelling strategy, based on antibodies conjugated to a scaffold containing two distinct sites for enzymatic cleavage of fluorophores. The scaffold is composed of a dextran decorated with short ssDNA that upon hybridization with complementary dye-modified oligos result in fluorescent molecules. The developed fluorescent labels exhibit specific staining and remarkable brightness in flow cytometry and fluorescence microscopy. We showed that the combination of DNase-mediated degradation of DNA and dextranse-mediated degradation of the dextran as two complementary enzymatic release mechanisms in one molecule, improves signal erasure from labelled epitopes. We envision that such dual-release labels with high brightness and efficient and specific erasure will advance multiplexed cyclic immunofluorescence approaches and thereby will contribute to gaining new insights in cell biology.

Fig. 1. Conjugate design with proposed mechanisms of release. Conjugates are comprised of antibodies and oligonucleotides bound to dextran. Complementary oligos are modified with Cy5 fluorophore for direct primary staining. First mechanism (top): cleavage of dsDNA by DNase I. Second mechanism (bottom): cleavage of carbohydrate backbone by dextranase. Dual-release (middle) shows the combination of both enzymes acting simultaneously.

Fig. 2. Stain and release flow cytometric analysis of SUP-T1 cells labelled with anti-CD4 conjugates (1 nM each). (A) Schematic representation of aCD4-Dex-dsDNA-Cy5 (top), aCD4-dsDNA-Cy5 (middle), and aCD4-Cy5 (bottom), the respective dot plots showing intensity after 10 min of staining and dual-release respectively. CD4+ signal is on the y-axis and forward scatter on the x-axis. aCD4-Dex-dsDNA-Cy5 shows approximately a 3-fold increase in staining intensity compared to the controls, while the dual-release in the presence of dextranase and DNase I leads to decrease of the fluorescence intensity for both oligo-based constructs. (B) Quantification of fluorescent signal at given time points and release pathway of flow cytometry experiment. Data is shown in MFI ± SD of the analyzed cell pool. (C) Quantification of release efficiency at given time points of release. Release efficiency is depicted in percent of signal reduction after release step.

Fig. 3. Flow cytometric analysis of SUP-T1 cells labeled with 5 nM aCD4-Dex-oligo hybridized with either aBiotin-PE or aBiotin-APC highlighted in red or blue, respectively. In both cases approximately a 3-fold increase in staining intensity compared to the controls was observed. Signal is given in mean fluorescence intensity (MFI) ± SD and release efficiency is depicted in percent of signal reduction after release step.

Fig. 4. Confocal laser-scanning microscopy images of fixed AsPC-1 cells stained with aEGFR-Dex-dsDNA-dye conjugates and signal quantification of initial fluorescence and during the course of release. (A) First row: release of aEGFR-Dex-dsDNA-Cy5 mediated by dextranase only; second row: release of aEGFR-Dex-dsDNA-Cy5 mediated by DNase only; third row: dual-release of aEGFR-Dex-dsDNA-Cy5 with both enzymes; fourth row: release of aEGFR-Dex-dsDNA-Cy5 conjugates while counterstained with non-cleavable aEpCAM-PE; fifth row: control with both non-cleavable anti-EGFR and anti-EpCAM conjugates. The columns indicate membrane staining after certain time points of release with t = 0 min being the initial staining before release. Cy5 signal is depicted in red, PE in green, and Vio515 in violet. Scale bar is 50 μm and is a representation for all images. LUTs for release with aEGFR-Dex-dsDNA-Cy5 are shown on the first panels and were kept constant for each release. (B) Signal quantification of (A), which showed faster and more efficient fluorophore cleavage when using both release reagents. Signal decrease of controls was also observed, however, it was accounted to photobleaching. (C) Quantification of dual-release mode of aBiotin-Vio515 labeled probes with aEpCAM-PE counterstaining. The releasable label was cleaved from the antibody while PE signal remains on the membrane. The data represent the mean average ± SE (aEGFR-Dex-dsDNA-Cy5 + Dextranase: n = 43; aEGFR-Dex-dsDNA-Cy5 + DNase I: n = 56; aEGFR-Dex-dsDNA-Cy5 dual release: n = 67; aEGFR-Dex-dsDNA-aBiotin-Vio515 + aEpCAM-PE dual release: n = 41; aEGFR-Cy5 + aEpCAM-PE dual release: n = 87). All signals are background-subtracted.

Fig. 5. Cyclic immunofluorescence of three targets on fixed AsPC-1 cells demonstrated in confocal microscopy with oligo-based conjugates. (A) Selective target staining located on the membrane observed in the appropriate channels. The first round of image acquisition was started with aEGFR-Dex-dsDNA-aBiotin-Vio515, followed by aEpCAM-Dex-dsDNA-Cy5, and ended with aCD66c-PE which is not releasable. The workflow of this experiment is indicated in the upper part of the figure. Duration of every staining and every release step was 10 min. Images were acquired with all three lasers to show successful release with the same ROI indicated within the white squares. Plates were taken out of the microscope for wash and re-staining, thus, slight shifts in ROI are visible. LUTs for each channel are shown on the right and were kept constant for each channel. Scale bar is 50 μm and is a representation for all images. (B) Quantification of (A) showing full release after 10 min. The data represent the mean average ± SE (each release with n = 47).