bph12803-sup-0001-si.pdf636 KB

Figure S1 CBD produces a dose‐dependent reduction of metastatic spread to the lung and increases survival. Lung metastases were generated in BALB/c mice by i.v. injection of 2 × 104 mouse 4T1 cells. (A) One day after the injection, the tumour‐bearing mice were injected i.p. once a day with vehicle or CBD (0.1–5 mg·kg−1) for 14 days and metastasis was evaluated. % metastasis = total tumour number of lung metastatic foci in drug‐treated group/total number of lung metastatic foci in vehicle‐treated group where the respective controls (vehicle‐treated mice) were set as 100%. (B) Lung metastases measured in mice treated with vehicle or CBD 1 mg·kg−1 included those with metastatic foci <2 and ≥2 mm. (C) Mice treated with vehicle or 1 mg·kg−1 CBD, starting 1 day after i.v. injection of 2 × 104 4T1 cells, were observed until they demonstrated signs of disease progression that necessitated killing. Survival between groups was compared using a log‐rank Mantel–Cox test. *P < 0.05, **P < 0.01 and #P < 0.001 indicate statistically significant differences from control. Figure S2 CBD reduces metastasis but not primary tumour growth. Primary tumours and subsequent secondary tumours (metastases) were generated in BALB/c mice by injection of 5 × 104 mouse 4T1 cells into the mammary fat pad between the second and third nipple. Treatment with CBD was initiated upon detection of the first palpable tumour (day 7). The tumour‐bearing mice were i.p. injected three times a week with vehicle or CBD (0.1–2.5 mg·kg−1) for 3 weeks. (A) To determine the tumour size in situ, the perpendicular largest diameters of the tumours were measured in millimetres as (L × W2)/2 based upon a modified ellipsoidal formula. (B) Visible lung metastases were counted and measured using a dissecting 4 microscope and % metastasis (total metastatic foci in treated/vehicle × 100) was calculated. (C) The number of metastatic foci ≥2 mm was also determined. *P < 0.05 and **P < 0.005 indicate statistically significant differences from control. Figure S3 CBD or Id1 knockdown produce comparable anti‐metastatic activity against human breast cancer cells. (A) To confirm the correlation between the effects of CBD and inhibition of Id1 expression in vivo, we established stable pooled populations of MDAMB231 cells expressing Id1 shRNA that showed a down‐regulation of Id1 protein levels (lower panel) comparable to parental cells treated with 2 μM CBD for 2 days (upper panel). (B) In culture, cell proliferation/viability (left panels) and invasion (right panels) rates were significantly reduced in parental MDA‐MB231 cells treated with CBD or in MDA‐MB231 cells expressing Id1 shRNA. (C) Left panel: lung metastases were generated in athymic nu/nu mice after i.v. injection of 5 × 105 MDA‐MB231. One day after the injection, the tumour‐bearing mice were injected i.p. once a day with vehicle or 0.5–1 mg·kg−1 CBD for 6 weeks. The percentage of metastatic foci (total metastatic foci in treated/vehicle × 100) was compared between vehicle and CBD‐treated groups. Right panel: the percentage of metastatic foci was compared between athymic nu/nu mice injected i.v. with 5 × 105 MDA‐MB231 cells stably expressing Ctl shRNA or Id1 shRNA. (D) Left panel: the number of lung metastatic foci ≥1 mm was compared between vehicle and CBD‐treated groups. Right panel: the number of lung metastatic foci ≥1 mm was compared between groups injected with cells stably expressing Ctl shRNA or Id1 shRNA. *P < 0.05 and **P < 0.01 indicate statistically significant differences from the control. Figure S4 The activity of various cannabinoids was compared for the down‐regulation of Id1 expression. (A) Structures of CBD, CP55940, THC, abnormal (Abn)‐CBD, O‐ 1918 and O‐1663. (B) Proteins from MDA‐MB231 cells treated with 1.5 μM of multiple cannabinoids for 3 days were extracted and analysed for Id1 by Western blot analysis. Normalization was carried out by stripping the blots and re‐probing with a monoclonal antitubulin antibody. Densitometry readings of the blots were taken and the percentage relative expression was calculated as the expression of Id1 in the treated cells/vehicle cells × 100. Figure S5 CB 2 receptor expression and CBD‐dependent generation of ROS in breast cancer cells. (A) Protein lysate from mouse spleen, mouse 4T1 cells or human MDAMB231 breast cancer cells was analysed for CB 2 receptor expression. (B) Mouse 4T1 cells (upper panel) were treated with 20 μM α‐tocopherol (TOC), 1 μM the CB 1 receptor antagonist (SR141716A – SR1) or 1μM the CB 2 receptor antagonist (SR144528 – SR2). Human MDAMB231 cells (lower panel) were treated with 20 μM α‐tocopherol (TOC), 4 μM the CB 1 receptor antagonist (SR141716A – SR1) or 4 μM the CB 2 receptor antagonist (SR144528 – SR2). Cell proliferation/viability was then evaluated using the MTT assay. (C) Human MDA‐MB231 cells were treated with vehicle or CBD (μM) for 2 days and the production of ROS was then measured using 2′‐7′dichloro‐dihydrofluorescein and cell flow cytometry. Figure S6 O‐1663 produces a significant inhibition of advanced stage breast metastasis. (A) Lung metastases were generated in BALB/c mice by i.v. injection of 2 × 104 4T1. One week after the injection of the cells, the tumour‐bearing mice were injected i.p. once a day with vehicle or O‐1663 for 14 days. In the treated group, 50% of the mice were still alive and 6 demonstrated no signs of disease progression at time of killing (2 months), few metastatic foci were observed when lungs were stained with India ink and visualized using a dissecting microscope. (B) Athymic nu/nu mice were treated with vehicle or O‐1663 (1 mg·kg−1) starting 18 days after i.v. injection of human MDA‐MB231‐luc‐D3H2LN breast cancer cells, a time point where the presence of lung tumours was confirmed using BLI. Fifteen minutes before imaging, mice were injected i.p. with 150 mg·kg−1 of luciferin. Mice were evenly distributed between vehicle and O‐1663‐treated groups before the initiation of treatment. Comparison of tumour progression over time was analysed between studies by comparing the absolute unit of radiance in photons per second per centimetre2 per steradian (p· s−1·cm−2·sr−1). (C) All athymic nu/nu mice where then imaged 1 week (left panel) and 10 days (right panel) later after the initiation of the study until animals in the vehicle group demonstrated signs of disease and began to be killed. (D) Regression of tumour burden, beyond the initial size the tumour when the treatment started, was observed in 25% of the athymic nu/nu mice. One mouse, where the tumour was regressing in the lung, was removed from the study to confirm the imaging results by visualizing the lung using an India ink stain and a dissecting microscope. (E) Athymic nu/nu mice treated with vehicle or CBD (1 mg·kg−1) starting 2 days after i.v. injection of MDA‐MB231‐luc‐D3H2LN cells were observed until they demonstrated signs of disease progression that necessitated killing. Table S1 CBD inhibits Id1 expression in lung metastatic foci. The data presented correspond to the number of metastatic foci in the lungs harvested from vehicle‐ and CBD‐treated mice, where immunohistochemical detection of Id1 was either negative, weakly positive or strongly positive. P‐value < 0.0004 was calculated using chi‐square test. Table S2 Inhibition of cAMP by O‐1663 and WIN55,212‐2 in CB 2 ‐transfected CHO cells (CHO‐CB 2 ). Experiments were carried out as previously described (Felder et al., 1995). Data represent the mean with corresponding confidence limits (CL) for three independent determinations.