Detecting Doxorubicin Induced Surface Expression of Calreticulin Using a 68Ga-Radiolabeled Peptide

Introduction: Early detection of cancer therapy response is vital for positive patient outcomes. Immunogenic cell death (ICD) is a pathway that cancer cells may take when initiating cell death in response to a treatment. ICD involves a change in cell surface composition resulting in signals that lead to immune system activation and ultimately cell death. Trafficking of the endoplasmic reticular protein calreticulin (CALR) to the cell surface is an indicator of ICD and has been shown to be induced by chemotherapeutics like doxorubicin. Detecting surface CALR could be an important prognostic biomarker to guide cancer immunotherapy. We aimed to use a ⁶⁸Ga labeled peptide as a novel PET imaging agent to detect doxorubicin-induced surface CALR in CT26 colon cancer cells.

Methods: The CALR targeting peptide sequence (KLGFFKR) was synthesized with addition of the chelator DOTA (DOTA-SCN-_βAD_βA_βAKLGFFKR) for binding of the radioisotope ⁶⁸Ga. 30 µg of peptide was radiolabeled with 1 mCi of ⁶⁸Ga while shaking at 1200 rpm for 15 minutes at 95 °C. For binding studies, 10⁶ CT26 cells were plated on 6-well plates for 24 hours to adhere to the plate. The cells were incubated at 37 °C for either 2 or 4 hours with 50 µM of doxorubicin in complete media while control cells were incubated with complete media only. The media was removed and 1 µM of [⁶⁸Ga]DOTA-SCN-_βAD_βA_βAKLGFFKR in PBS was added to each well and was incubated for 1 hour at 37 °C. After incubation, PBS with the radiolabeled peptide was removed and the cells were washed with ice cold PBS in triplicate. Sodium hydroxide was added to the wells to detach the cells, which were transferred into microcentrifuge tubes to measure the radioactivity in each well on a gamma counter. Uptake per well was normalized to total protein concentration as determined by BCA assay.

Results: The [⁶⁸Ga]DOTA-SCN-_βAD_βA_βAKLGFFKR peptide was synthesized in a yield of 93% resulting in a specific activity of 31 µCi/µg. The radiolabeled peptide showed significantly higher percent activity per ng of protein in cells incubated with doxorubicin at two hours (0.48±0.09 versus 0.26±0.05, p<0.05) and at four hours (0.69±0.20 versus 0.48±0.06, p<0.05) compared to control cells. There was also significantly greater binding to the cells with four hours of doxorubicin incubation compared to two hours (0.69±0.20 and 0.48±0.09, p<0.05, respectively).

Conclusions: [⁶⁸Ga]DOTA-SCN-_βAD_βA_βAKLGFFKR shows potential to be used as an imaging agent to detect surface CALR. Future studies will investigate imaging of tumor-bearing mice treated with doxorubicin for increased CALR expression. Developing a real-time biomarker for ICD has the clinical potential to significantly inform cancer immunotherapy decisions across a wide range of malignancies

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