P05.09.A RAS70 PEPTIDE-MEDIATED FLUORESCENCE IMAGING OF HSP70-EXPRESSING HIGH-GRADE GLIOMAS AND BRAIN METASTASES

Abstract BACKGROUND The 70kDa heat shock protein (Hsp70) exhibits a unique expression pattern, being prominently displayed on the plasma membrane of tumor cells while remaining absent from normal cells. This differential expression makes mHsp70 an attractive target for selective tumor imaging. In this study, a 17-mer RAS70 peptide, designed to recognize the membrane-bound mHsp70 on cancer cells, was linked to Cy7.5 fluorophore. This conjugate was then employed for epifluorescent detection of mHsp70-positive brain tumors and metastases. METHODS Adult patients with glioblastoma (n=6) and brain metastases (n=6) were included in the pilot study between November 2024 and April 2025. Histologically confirmed tumor specimens were obtained from three tumor regions based on preoperative MRI scanning: necrotic, the contrast-enhanced, and peritumoral zones. Specimens were ex vivo sprayed with Cy7.5-conjugated RAS70 peptide (exposure time of 5 min), followed by an intensive rinsing of the sample with PBS. Cy7.5-conjugated scramble peptide was used as a control agent. RAS70 was excited by an 800 nm light source on an operating microscope (Leica M720 OH5, Germany). Images were analyzed using ImageJ software with IBRs estimated as the Ratio of fluorescence signal intensity of Interest regions (ROIs, n=102) and the mean signal intensity of black Background. RESULTS Сomparison of IBRs in samples from different tumor areas revealed significantly higher IBRs for RAS70 in samples compared to control scramble-peptide (p<0.0001), constituting: necrotic zone (18.7 ± 6.1 a.u), contrast-enhanced zone (9.7 ± 5.1 a.u.), peritumoral zone (9.3 ± 5.0 a.u), control (1.9 ± 1.1 a.u). The mean fluorescence intensities did not significantly differ between glioblastoma and metastasis specimens. According to ROC analysis, the sensitivity and specificity of the RAS70 peptide for diagnosing the contrast-enhanced zone were 95.56% and 88.89% (threshold >3.05 a.u., AUC 0.9790, p<0.0001), for the peritumoral zone 95.0% and 88.89% (threshold >3.05 a.u., AUC 0.9971, p<0.0001), for the necrotic zone 94.74% and 94.44% (threshold >5.60 a.u., AUC 0.9971, p<0.0001). CONCLUSION The RAS70 peptide, exhibiting targeting properties for brain tumors and metastases, holds promise not only for fluorescence-guided surgery but also as a potential drug delivery vehicle.