Introduction. Breast cancer (BCa) is the most common cancer in women. For 2023, breastcancer.org projected 297,790 new cases of invasive BCa and 55,720 new cases of non-invasive (in situ) BCa, projecting 43,700 deaths. Majority of US BCa surgeries are currently breast-conserving surgery (BCS)¹. Among patients treated by BCS and radiation, positive margins are associated with a 2-fold increase in the risk of local recurrence when compared with negative margins². Pathological analysis of post-operative specimens takes 3-5 days and if margins are positive requires patients to return for further surgery. Local disease recurrence is experienced by 5%-16% of patients with negative surgical margins, likely from false negatives due to under sampling during assessment. Thus, there remains a significant unmet clinical need to improve both surgical techniques and imaging probes for identifying tumor margins and complete removal of BCa during BCS.

Goal.  To expand on the utility  of a current fluorescence image guided surgery (FIGS) probe, AKRO-6qc³, as a theranostic photothermal therapy (PTT) agent. The AKRO-6qc (aka 6QC or 6qc-ICG), belongs to a well-characterized cathepsin-targeted family^3,4 of ICG contrast⁵ imaging agents^3,5-7 with excellent cellular retention leading to increased ICG signal strength and durability in solid tumors³. Here we test AKRO-6qc for FIGS followed by adjuvant PTT to ablate  any inoperative/non-detectable microscopic disease in a single surgical procedure.

Methods. Mouse BCa 4T1 cells were pretreated with AKRO-6qc for 24-h and then harvested, washed, and then irradiated (800-nm, up to 1-Watt) as pellets in tubes using a clinically approved ML7710-laser (Modulight) to record temperature changes in response to PTT. Nude female mice were inoculated with 4T1 luciferase-expressing cells in the flank. When tumor size reached 100-mm³ mice were divided into four groups: 1) intact control; 2) white light surgery (WLS); 3) FIGS; and 4) FIGS, followed by PTT (FIGS+PTT). AKRO-6qc was i.v. administered to the FIGS and FIGS+PTT mice and 24-hr later presence of both ICG fluorescence and/or bioluminescence in the tumor tissue was confirmed (IVIS-Spectrum). Then WLS, FIGS under live-image control (Curadel-FLARE), and FIGS followed by PTT (1.0-Watt once to the surgical cavities with 800-nm fiber-optic, Modulight) were provided under assessment of temperature kinetics using a photothermal camera (FLIR-One-PRO). Next, all surgical wounds were closed, and animals’ tumor size, lung metastasis, and survival were monitored for 90-days.

Results. In vitro data revealed: 1) Positive correlation between PTT-induced temperature changes and i) power of laser source, ii) distance from the optical fiber to the cell pellet for light, and iii) dose of AKRO-6qc; 2) PTT-induced temperature increase in BCa cells pre-treated with AKRO-6qc up to 55⁰C (ΔT30⁰C) in 5-min as compared to controls (no probe), leading to 100% killing after 10-min of irradiation. In vivo data showed that: 1) Tumor specific bio-luminescence signals correlate with AKRO-6qc fluorescence in tumor-bearing mice; 2) 24-36-hr after delivery of AKRO-6qc may be a suitable time-window for PTT; 3) PTT induced temperature increases only in the surgical cavities of AKRO-6qc-treated mice that had BCa surgically removed, demonstrating utility of combined FIGS+PTT; 4) Combination of FIGS and PTT may extend animal survival, abolish tumor recurrence, and delay/eliminate lung metastasis.

Conclusion. Combination of AKRO-6qc FIGS followed by PTT of the surgical cavity in this mouse model of 4T1 aggressive triple-negative BCa extended animal survival, abolished tumor regrowth, and delayed/abolished 4T1 metastasis to lungs in the host animals. AKRO-6qc probe has a potential to be a theranostic for solid cancers when it is used for FIGS and adjuvant PTT, and likely will improve the outcome of resection of tumor masses.

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