The majority of breast cancer patients is treated with breast-conserving surgery (BCS) combined with adjuvant radiation therapy. Up to 40% of patients has a tumor-positive resection margin after BCS, which necessitates re-resection or additional boost radiation. Cathepsin-targeted near-infrared fluorescence imaging during BCS could be used to detect residual cancer in the surgical cavity and guide additional resection, thereby preventing tumor-positive resection margins and associated mutilating treatments. The cysteine cathepsins are a family of proteases that play a major role in normal cellular physiology and neoplastic transformation. In breast cancer, the increased enzymatic activity and aberrant localization of many of the cysteine cathepsins drive tumor progression, proliferation, invasion, and metastasis. The upregulation of cysteine cathepsins in breast cancer cells indicates their potential as a target for intraoperative fluorescence imaging. This review provides a summary of the current knowledge on the role and expression of the most important cysteine cathepsins in breast cancer to better understand their potential as a target for fluorescence-guided surgery (FGS). In addition, it gives an overview of the cathepsin-targeted fluorescent probes that have been investigated preclinically and in breast cancer patients. The current review underscores that cysteine cathepsins are highly suitable molecular targets for FGS because of favorable expression and activity patterns in virtually all breast cancer subtypes. This is confirmed by cathepsin-targeted fluorescent probes that have been shown to facilitate in vivo breast cancer visualization and tumor resection in mouse models and breast cancer patients. These findings indicate that cathepsin-targeted FGS has potential to improve treatment outcomes in breast cancer patients.

Background: Tumor-positive surgical margins during primary breast cancer (BCa) surgery are associated with a two-fold increase in the risk of local recurrence when compared with tumor-negative margins. Pathological microscopic evaluation of the samples only assesses about 1/10 of 1% of the entire volume of the removed BCa specimens, leading to margin under-sampling and potential local recurrence in patients with pathologically clean margins, i.e., false negative margins. In the case of tumor-positive margins, patients need to undergo re-excision and/or radiation therapy, resulting in increases in complications, morbidity, and healthcare costs. Development of a simple real-time imaging technique to identify residual BCa in the surgical cavity rapidly and precisely could significantly improve the quality of care. Methods: A small-molecule, fluorescently quenched protease-substrate probe, AKRO-QC-ICG, was tested as part of a thermosensitive imaging gel formulated for topical application and imaging of the BCa surgical cavity. Results: More than forty formulations of gel mixtures were investigated to enable easy fluid application and subsequent solidification once applied, preventing dripping and pooling in the surgical cavity. The final formulation was tested using human BCa orthotopic implants in nude and NSG patient-derived xenografts (PDX) mice. This formulation of Pluronic F-127/DMSO/AKRO-QC-ICG imaging gel was found to be a good solvent for the probe, with a desirable thermo-reversible solid–gel transition and mechanical strength for distribution of AKRO-QC-ICG on the surfaces of tissue. It demonstrated excellent ability to detect BCa tissue after 10 min exposure, with a high signal-to-noise ratio both in mouse xenografts and freshly excised human lumpectomy tissue. The in vivo efficacy of the AKRO-QC-ICG imaging gel to detect BCa revealed the levels of sensitivity/specificity = 0.92/1 in 12 nude mice, which was corroborated with the sensitivity/specificity = 0.94/1 in 10 PDX mice. Conclusions: Utilization of Pluronic F-127/DMSO/AKRO-QC-ICG imaging gel for topical application to detect BCa in the surgical cavity during surgery has the potential to reduce re-excisions, with consequent savings in healthcare costs and enhancement in patient quality of life.

Purpose: Cysteine cathepsins are proteases that play a role in normal cellular physiology and neoplastic transformation. Elevated expression and enzymatic activity of cathepsins in breast cancer (BCa) indicates their potential as a target for tumor imaging. In particular cathepsin B (CTSB), L (CTSL), and S (CTSS) are used as targets for near-infrared (NIR) fluorescence imaging (FI), a technique that allows real-time intraoperative tumor visualization and resection margin assessment. Therefore, this immunohistochemical study explores CTSB, CTSL, and CTSS expression levels in a large breast cancer patient cohort, to investigate in which BCa patients the use of cathepsin-targeted NIR FI may have added value. Procedures: Protein expression was analyzed in tumor tissue microarrays (TMA) of BCa patients using immunohistochemistry and quantified as a total immunostaining score (TIS), ranging from 0–12. In total, the tissues of 557 BCa patients were included in the TMA. Results: CTSB, CTSL, and CTSS were successfully scored in respectively 340, 373 and 252 tumors. All tumors showed CTSB, CTSL, and/or CTSS expression to some extent (TIS > 0). CTSB, CTSL, and CTSS expression was scored as high (TIS > 6) in respectively 28%, 80%, and 18% of tumors. In 89% of the tumors scored for all three cathepsins, the expression level of one or more of these proteases was scored as high (TIS > 6). Tumors showed significantly higher cathepsin expression levels with advancing Bloom-Richardson grade (p < 0.05). Cathepsin expression was highest in estrogen receptor (ER)-negative, human epidermal growth factor receptor 2(HER2)-positive and triple-negative (TN) tumors. There was no significant difference in cathepsin expression between tumors that were treated with neoadjuvant systemic therapy and tumors that were not. Conclusions: The expression of at least one of the cysteine cathepsins B, L and S in all breast tumor tissues tested suggests that cathepsin-activatable imaging agents with broad reactivity for these three proteases will likely be effective in the vast majority of breast cancer patients, regardless of molecular subtype and treatment status. Patients with high grade ER-negative, HER2-positive, or TN tumors might show higher imaging signals.