Although optical microscopy techniques have been invaluable for diagnosing cancer, they do not routinely detect pathological features at the nanoscale level. This ability may be critical for understanding how malignancies vary both between patients and within individual tumors. Mapping heterogeneity at the level of single molecules can, in turn, deliver more personalized diagnoses and guide individual therapeutic strategies. While such subcellular structures cannot be detected with diffraction-limited optical techniques, this limitation has been circumvented with super-resolution microscopy methods. Here, we employed quantitative single molecule localization microscopy (qSMLM) to probe molecular signatures of HER2-positive breast cancer. To detect HER2 receptors in both cultured cell lines and patient tissue samples, we used fluorescently labeled trastuzumab and pertuzumab; both are clinically administered antibodies against HER2. When trastuzumab was used as a probe, trastuzumab-resistant cultured cells (BT-474<sup>R</sup>, JIMT1) had lower detected density, smaller clusters, and lower HER2 clustering compared to respective trastuzumab-sensitive cultured cells (BT-474, SKBR3). When pertuzumab was used as a probe, the main difference was significantly reduced detected HER2 density in JIMT1 compared to SKBR3 cultured cells. Additionally, in preliminary results from six patients using trastuzumab as the probe, we detected low HER2 density and clustering in patients who were poor responders to neoadjuvant chemotherapy combined with trastuzumab. HER2 clustering for these patients was lower than that of women who achieved complete pathological responses. Altogether, qSMLM may complement standard diagnostics with molecular information. Our long-term goal is to identify an early signature of therapeutic resistance and help guide personalized medicine.

Molecular assessment of HER2 to identify signatures associated with therapy resistance in breast cancer

ACS Spring 2022

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