A photocleavable peptide-tagged mass probe for chemical mapping of epidermal growth factor receptor 2 (HER2) in human cancer cells

Abstract

Human epidermal growth factor receptor 2 (HER2) testing has great value for cancer diagnosis, prognosis and treatment selection. However, the clinical utility of HER2 is frequently tempered by the uncertainty regarding the accuracy of the methods currently available to assess HER2. The development of novel methods for accurate HER2 testing is in great demand. Considering the visualization features of in situ imaging and the quantitative capability of mass spectrometry, integration of the two components into a molecular mapping approach has attracted increasing interest. In this work, we reported an integrated chemical mapping approach using a photocleavable peptide-tagged mass probe for HER2 detection. The probe consists of four functional domains, including the recognition unit of an aptamer to catch HER2, a fluorescent dye moiety (FITC) for fluorescence imaging, a reporter peptide for mass spectrometric quantification, and a photocleavable linker for peptide release. After characterization of this novel probe (e.g., conjugation efficiency, binding affinity and specificity, and photolysis release efficiency), the probe binding and photolysis release conditions were optimized. Then, fluorescence images were collected, and the released reporter peptide after photolysis was quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A limit of quantification (LOQ) of 25 pM was obtained, which very well meets the requirements for clinical laboratory testing. Finally, the developed assay was applied for HER2 testing in four breast cancer cell lines and 42 pairs of human breast primary tumors and adjacent normal tissue samples. Overall, this integrated approach based on a photocleavable peptide-tagged mass probe can provide chemical mapping including both quantitative and visual information of HER2 reliably and consistently, and may pave the way for clinical applications in a more accurate manner.

Fig. 1. Schematic representation of the photocleavable peptide-tagged mass probe for chemical mapping of HER2. The probe consists of four functional domains (i.e., the aptamer HB5 capable of targeting the HER2 protein, a fluorophore for fluorescence imaging, a photocleavable linker to release the reporter peptide and a reporter peptide as the target of mass spectrometry detection). The probe was prepared by a Michael addition reaction of the maleimide labeled at the C-terminus of AVLGVDPFR-PL-K(FITC)FK-Mal with a disulfide at the 5′ end of the aptamer HB5. Then, the newly synthesized probe was incubated with cells and tissue samples. Afterward, we performed in situ fluorescence imaging based on the FITC fluorophore. After that, photolysis was initiated to rapidly release the reporter peptide, and the reporter peptide was ultimately quantified by LC-MS/MS. In this way, the distribution and levels of HER2 could be acquired sequentially.

Fig. 2. Characterization of the photocleavable peptide-tagged mass probe HB5-FITC-PL-Peptide. (A) The structure of the HB5-FITC-PL-Peptide probe and a schematic representation of probe synthesis by the Michael addition reaction. (B) HPLC chromatograms before and after conjugation of the aptamer HB5 and AVLGVDPFR-PL-K(FITC)FK at the wavelengths of DNA (260 nm) and peptide (220 nm). The HPLC conditions are described in the ESI. As is shown, almost all HB5 can be exhausted in the presence of excess peptides. The peptide does not coelute under these HPLC conditions and affect the purity of the product (i.e., HB5-FITC-PL-Peptide). (C) HPLC chromatograms of the probe before and after photolysis at the wavelength of DNA (260 nm) and the corresponding LC-MS/MS chromatogram. (D) Specificity evaluation of HB5-FITC-PL-Peptide by flow cytometry. HB5-FITC-PL-Peptide has relatively strong binding to HER2-positive cells (BT474 and SK-BR-3) and negligible binding to HER2-negative cells (MDA-MB-231 and MCF-7), using random DNA as a control. (E) Optimization of the incubation time and photolysis time. Cells were incubated with HB5-FITC-PL-Peptide for 0.25 h to 3.5 h. The amount of bound probes reached a maximum at approximately 2 h. Cells were incubated with the probe for 20 min to 120 min. The peptide release reached a plateau at ∼100 min.

Fig. 3. Fluorescence images of cells treated with the probe HB5-FITC-PL-Peptide, including the HER2-positive breast cancer cells BT474 and SK-BR-3 and HER2-negative breast cancer cells MDA-MB-231 and MCF-7. First, enzyme-free cell dissociation buffer was used in cell collection to prevent damage to surface proteins. Before incubation with the probe, the cells were blocked by treatment with binding buffer. The cells were first stained with DAPI. After washing with PBS, the probe was incubated with the cell suspension with gentle shaking. Finally, the cells were washed with PBS and NH₄HCO₃ to remove the unbound probe. A greater fluorescence signal was observed for HER2-positive cells. Blue fluorescence represents the nucleus and green fluorescence represents the HER2 protein. The images were taken under a 40× objective and at a resolution of 1024 × 1024 with confocal microscopy. Scale bar: 20 μm.

Fig. 4. Fluorescence images, IHC images and the corresponding LC-MS/MS chromatograms of representative breast tumors classified as HER2 0, HER2 1+, HER2 2+ and HER2 3+. We selected four breast tumors with different scores identified by clinical pathologists and cut them into tissue sections. The tissues were first stained with DAPI. After washing the sample with PBS, the probe was added, and the mixture was incubated. Finally, the tissues were washed with PBS and NH₄HCO₃ to remove the excess probe. Fluorescence images were obtained immediately after incubation to avoid quenching. The blue fluorescence and green fluorescence represent the nucleus and the HER2 protein, respectively. These fluorescence signals also correspond to the blue and dark brown staining in IHC images. In order to confirm the consistency between the fluorescence images and traditional IHC images, we used adjacent tissue sections for both fluorescence imaging and IHC analysis. As a result, there was no significant difference between the fluorescence and IHC images. After fluorescence imaging, we performed mass spectrometric quantification of the HER2 protein. The peak area of the LC-MS/MS chromatogram represents the amount of protein expression, which was also in agreement with the score grade of tumors. The fluorescence images were taken under a 10× objective at a resolution of 1024 × 1024 by confocal microscopy. Scale bar for fluorescence image: 100 μm, scale bar for IHC: 100 μm. Three product ions m/z 171.2, m/z 419.2 and m/z 690.5 generated from the double charged precursor ion m/z 487.3 were used for quantification. The corresponding ion structures and MRM transitions were listed.

Fig. 5. Correlations between (A) FITC fluorescence of the probe vs. DAPI fluorescence and (B) FITC fluorescence intensity vs. dark brown staining intensity in IHC of breast tumors. We collected 42 breast tumors with different scores, including 7 tumors with a score of 3+, 8 tumors with a score of 2+, 13 tumors with a score of 1+, and 14 tumors with a score of 0. The FITC fluorescence intensity increased with the scores. The clusters of HER2 IHC subgroups can be easily discerned. Then, we selected 20 breast tumors (5 tumors with each score) to confirm the consistency of fluorescence staining and IHC staining. Additionally, both the fluorescence intensity and IHC chromogenic intensity increased with increasing score. In addition, the levels of fluorescence were in good agreement with those of dark brown staining.

Fig. 6. HER2 expression levels in 42 matched pairs of breast tissue samples. IHC testing gave a score of HER2 0 to 3+ for each sample, and these scoring groups had 14, 13, 8 and 7 samples (numbers in brackets), respectively. Among the HER2 2+ group, FISH was positive in 3 cases and negative in 5 cases. A Mann–Whitney test showed significant differences among the IHC subgroups. In particular, the HER2 expression level of the IHC 2+/FISH− group was different from that of the IHC 2+/FISH+ group (P = 0.014). However, there was no significant difference between the IHC 2+/FISH+ group and the IHC 3+ group. The table lists the expression levels of HER2 in each HER2 group. The mean (±standard deviation (SD)) and the 95% confidence interval (CI) of each group were also provided. The samples were classified according to the 2018 ASCO/CAP updated guideline. *P < 0.05, **P < 0.01, ***P < 0.001.