In vitro assessment of [¹⁸F]JHU94620-d8 to determine the expression of cannabinoid receptor 2 – a prognostic biomarker in breast cancer

The cannabinoid receptor 2 (CB2R) is involved in inflammatory processes [1], whereby an increased expression correlates with malignancy in various cancer types like human epidermal growth receptor 2 positive (HER2+) or triple negative breast cancer (TNBC) [2]. Hence, the CB2R is suggested as a pharmacological target, as well as a prognostic biomarker for the stratification and staging of patients [3]. In this study, we evaluated the potential of our novel CB2R-specific radioligand [¹ ⁸F]JHU94620-d8 for the assessment of the CB2R expression in HER2+ breast cancer and TNBC models in vitro.
The KD value of [¹ ⁸F]JHU94620-d8 was determined by autoradiography on cryosections of rat and mouse spleen, as well as on rat brains harbouring a local overexpression of the hCB2R (AAV-hCB2R) [4]. The CB2R status was investigated by competitive radioligand binding assays (CRBA) in cell membranes of CHO cells overexpressing the human CB2R (CHOhCB2R), human breast cancer cell lines HCC1954 (HER2+) and MDA-MB-231 (TNBC) with 3.1±0.4 nM [³H]WIN55,212-2 using 10 µM of WIN55,212-2, GW405833 and JHU94620-d8 (each n=3) as agonistic competitors. CB2R expression was validated by immunofluorescence microscopy (IFM). On cryosections of 4T1 tumors the CB2R specific binding of [¹ ⁸F]JHU94620-d8 was investigated by CRBA and the colocalisation of CB2R with Iba1 (macrophages) and CD31 (blood vessels) by IFM.
We determined KD values for [¹ ⁸F]JHU94620-d8 of 30 nM in mouse spleen, of 1.0 nM in rat spleen, and of 42 nM in AAV-hCB2R. The cell membrane binding of [³H]WIN55,212-2 was comparable in all used cell lines between 20 ± 1 and 30 ± 11 fmol/106 cells. Competition by JHU94620-d8 decreased the total binding by 57 % (p<0.01) only in CHOhCB2R cells, WIN55,212-2 by 37 % (p=0.01) and 77 % (p<0.01) and GW405833 by 42 % (p<0.01) and 75 % (p<0.01) in HCC1954 and CHOhCB2R cells, respectively, however in MDA MB 231 cells binding was not affected by these compounds (Fig. 1A). The expression of CB2R was confirmed by IFM (Fig. 1B). IFM analysis of murine 4T1 tumours revealed a high correlation between the heterogeneously distributed CB2R and Iba1 (Pearson´s coefficient r=0.69±0.03), and a weak correlation between CB2R and CD31 (r=0.35±0.09), however autoradiography studies revealed a non-displaceable binding of [¹ ⁸F]JHU94620-d8 (Fig. 2).
The potential of [¹ ⁸F]JHU94620-d8 as radioindicator to assess the CB2R status of tumours as a prognostic imaging biomarker should be investigated in vivo in PET studies. As shown in this study, the apparently species depended CB2R binding affinity and cell type specific (tumour cells and tumour associated macrophages) CB2R expression should be considered.

References
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Acknowledgement
The human mammary cell lines were provided by Dr. Joan Massagué (MSKCC, NY, USA).
This research was funded by the Deutsche Forschungsgemeinschaft (DFG), grant number MO2677/4-1.