HER2 overexpression is associated with Breast Cancer (BC) poor prognosis, due to increased metastasis and angiogenesis, and decreased apoptosis. HER2 expression status is commonly assessed by immunohistochemistry, however, this technique requires tedious sample manipulation, since antibodies have poor penetration, therefore thin fixed samples (3-5 mm) are used, and labelling interpretation is based on subjective algorithms. Consequently, lacks accuracy and reproducibility, and could lead to misdiagnosis. Therefore, we aimed to develop a 3D confocal imaging detection method of HER2 using the small recognition Affibody (Aff) molecules conjugated with Quantum Dots (QD), and ratiometric analysis (RMA). Aff anti-HER2 and Aff negative control were conjugated by the maleimide reaction with QD605 and QD545, respectively (where numbers refer to emission wavelength, and both were exited at 488 nm). Fixed BC spheroids of HCC1954 (HER2+) and MCF-7 (HER2-) cell lines were incubated with a mixture (1:1) of both Aff-QD probes, and confocal image stacks were recorded in the z-axis. Images were processed by RMA (AffantiHER2-QD605/Affneg-QD545 fluorescence), and (%) of HER2+ cells, as well as HER2 density were assessed in the stack. We found in HCC1954 spheroids, that Aff-QDs could be optimally resolved up to 50 mm depth, and the AffantiHER2-QD605 signal was significantly higher than that of Affneg-QD545 (due to unspecific accumulation). After unspecific signal removal by RMA, the HER2+ expression status was objectively quantified. In MCF-7 spheroids, Aff-QDs could be resolved up 80 mm, however, no significant differences between AffantiHER2-QD605 and Affneg-QD545 signals were found. Therefore, after RMA almost all unspecific AffantiHER2-QD605 fluorescence was removed, and no false HER2+ cells (%) or cellular HER2 labeling were found. The results demonstrate that conjugated Aff-QDs could efficiently penetrate in spheroids, as 3D BC models, with minimal sample manipulation. Importantly, RMA, efficiently removed unspecific fluorescence, allowing objective HER2 quantification in relatively thick samples.

3D imaging detection method of HER2: Application of conjugated affibody-quantum dots probes and ratiometric analysis

254th National Meeting (2017)

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