# Fluorescence molecular imaging of high-grade gliomas and brain metastases using the RAS70 peptide targeting plasma membrane-bound Hsp70 on tumor cells

## TL;DR

A new peptide, RAS70, was used with a fluorescent dye to better detect brain tumors during surgery, showing higher accuracy than existing methods.

## Contribution

The RAS70 peptide conjugated with Cy7.5 provides a novel fluorescence imaging method for high-grade gliomas and brain metastases.

## Key findings

- RAS70-Cy7.5 showed significantly higher target-to-background ratios compared to a control peptide in various tumor zones.
- RAS70 fluorescence was more sensitive and specific than 5-ALA for detecting perifocal tumor zones.
- mHsp70 was consistently detected on tumor cell membranes in viable and perifocal zones.

## Abstract

One of the promising targets for fluorescence-guided surgery of malignant brain tumors is a 70 kDa heat shock protein (mHsp70), which has been found on the plasma membrane of tumor cells. In this study, we conjugated the RAS70 peptide, which targets mHsp70 on tumor cells, with the fluorophore Cy7.5 and used it for epifluorescence detection of mHsp70-positive glial tumors and brain metastases.

The study included adult patients with newly diagnosed glioblastoma (GBL, n = 7) and brain metastases (MTS, n = 8). Three hours prior to surgery, patients received an oral solution of 5-aminolevulinic acid (5-ALA, 20 mg/kg). During surgery, tumor samples were obtained from three zones: necrotic, viable tumor, and perifocal. Some samples were treated ex vivo with the RAS70-Cy7.5 peptide, while others were treated with the control peptide NGL-RGD-Cy7.5. Epifluorescence images were obtained using an operating microscope in FL400 and FL800 modes, image analysis was performed in ImageJ with calculation of the target-to-background ratio (TBR). The presence of mHsp70 on the tumor cell membrane was additionally determined using immunofluorescence microscopy.

Immunofluorescence staining revealed mHsp70-positive tumor cells in all studied samples, with preferential localization observed in the viable (contrast-enhancing) and perifocal tumor zones. TBRs for fluorescence imaging of RAS70-Cy7.5 peptide were significantly higher than those for the control peptide (p < 0.0001): necrotic zone 19.2 a.u. (15.5–21.0), viable tumor 9.0 a.u. (7.0–11.5), perifocal zone 8.9 a.u. (6.7–11.4), control – 1.2 a.u. (0.9–1.7). RAS70 exhibited 37.5% more visible fluorescence than 5-ALA in viable metastasis zones (p < 0.001). The RAS70 peptide was more sensitive and specific than 5-ALA for detecting perifocal zones in glioblastomas and brain metastases (100% vs. < 75% and 100% vs. < 85%, respectively).

The RAS70 peptide demonstrated high diagnostic accuracy for glioblastoma and metastases, suggesting potential applications in intraoperative image-guided tumor resection and the development of targeted drug delivery systems.