Publikationsrepositorium - Helmholtz-Zentrum Dresden-Rossendorf

Introduction
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 and prognostic marker for stratification and staging of patients [3].
In the present in vitro studies, we investigated the expression of the CB2R in HER2+ and TNBC models, as well as the potential of our novel radioligand [18F]JHU94620-d8 to assess the CB2R availability in TNBC models.

Methods
The colocalisation of CB2R with Iba1 (macrophages) and CD31 (blood vessels) in cryosections of mouse TNBC 4T1 tumours heterotopically implanted in both NMRI-nude and Balb/c mice was investigated by immunofluorescence staining (IF). The CB2R expression in 4T1, the human HER2+ cell lines HCC1954 (HCC and LCC2) and human TNBC cell lines MDA-MB-231 (MDA and BrM2) was determined by IF.
Competitive radioligand binding assays with the CB2R-specific ligands [3H]WIN55,212-2 and [3H]A-836339 were performed vs. 10 µM JHU94620-d8, GW405833 and WIN55,212-2, and A 836339 as competitor (n=1). Autoradiography with the CB2R-specific [18F]JHU94620-d8 vs. 10 µM competitor was performed with cryosections obtained from 4T1 tumours (n=3) as well as rat brains harbouring a local overexpression of the human CB2R (AAV-hCB2R, n=1) [4].

Results
A high correlation between the heterogeneously distributed CB2R and Iba1, and a weak correlation between CB2R and CD31 was found in 4T1 tumours. Colocalisation of CB2R and Iba1 (Balb/c: Pearson’s coefficient r=0.69±0.03, Manders’ coefficient M1: 0.7±0.12; NMRI-nude: r=0.7±0.12, M1=0.71±0.15) or CD31 (Balb/c: r=0.35±0.09, M1=0.15±0.02; NMRI-nude: r=0.35±0.11; M1=0.19±0.09) was independent of the mouse breed (CB2R/Iba1: pr=0.9, pM1=0.972; CB2R/CD31: pr=0.41, pM1=0.52).
By IF the expression of CB2R and HER2 was confirmed in HCC and LCC2, but absent in all TNBC cell lines.
The total binding of [3H]WIN55,212-2 in HCC (329.04±37.65 fmol/mg protein) and LCC (160.87±9.53 fmol/mg) homogenates could be reduced by homologues competition with WIN55,212 2 (HCC: 229.66±56.56 fmol/mg, -30.2%; LCC2: 117.73±18.49 fmol/mg, -26.82%) and GW405833 (LCC2: 99.41±2.81 fmol/mg, -38.20%) in contrast to JHU94620-d8. Moreover, a specific binding of [3H]A 836339 was not detectable.
In cryosections of AAV-hCB2R binding of [18F]JHU94620-d8 could be displaced by >80% in the target region with all competitors, however in 4T1 tumours a non-displaceable binding was found.

Conclusions
CB2R expression was detectable in Iba1 positive tumour-associated cells of 4T1 tumours cryosections. In HER2+ cells CB2R expression was cross-validated by IF and competitive binding studies. [18F]JHU94620-d8 binds target specific in the artificial AAV-hCB2R model, whereas heterogenous binding was non-displaceable in cryo-sections of mammary tumours.