Opposing Effects of Cannabidiol in Patient-derived Neuroendocrine Tumor, Pheochromocytoma/Paraganglioma Primary Cultures

Abstract

Context: Treatment options for advanced neuroendocrine tumors (NETs), pheochromocytomas and paragangliomas (PPGLs) are still limited. In recent years, antitumor effects of cannabinoids have been reported; however, there are only very limited data available in NETs or PPGLs.

Objective: Investigation of the effects of cannabidiol (CBD) on patient-derived human NET/PPGL primary cultures and on NET/PPGL cell lines.

Methods: We established primary cultures derived from 46 different patients with PPGLs (n = 35) or NETs (n = 11) who underwent tumor resection at 2 centers. Treatment of patient primary cultures with clinically relevant doses (5 µM) and slightly higher doses (10 µM) of CBD was performed.

Results: We found opposing effects of 5 µM CBD: significant antitumor effects in 5/35 (14%) and significant tumor-promoting effects in 6/35 (17%) of PPGL primary cultures. In terms of antitumor effects, cluster 2-related PPGLs showed significantly stronger responsivity to CBD compared to cluster 1-related PPGLs (P = .042). Of the cluster 2-related tumors, NF1 PPGLs showed the strongest responsivity (4/5 PPGL primary cultures with a significant decrease in cell viability were NF1-mutated). We also found opposing effects of 10 µM CBD in PPGLs and NETs: significant antitumor effects in 9/33 of PPGL (27%) and 3/11 of NET (27%) primary cultures and significant tumor-promoting effects in 6/33 of PPGL (18%) and 2/11 of NET (18%) primary cultures.

Conclusion: We suggest a potential novel treatment option for some NETs/PPGLs but also provide evidence for caution when applying cannabinoids as supportive therapy for pain or appetite management to cancer patients and possibly as health supplements.

Keywords: CBD; cannabidiol; cannabinoids; neuroendocrine tumor; pheochromocytoma and paraganglioma.

Figure 1.

Simplified scheme of molecular anticancer pathways mediated by cannabinoids through cannabinoid receptors. ↑ activation; ⊥ inhibition. Created with Biorender.com.

Figure 2.

Cell viability results of different PPGL and NET cell lines and MPC cell spheroid data. (A) Cell viability of PPGL cell lines MPC, MTT, and NET cell lines BON1, QGP-1, NCI-H727, GOT1 after incubation with 10 µM CBD for 72 hours and 144 hours. (B) CBD reduced spheroid growth in a pseudohypoxic model. MPC cell spheroids generated from cells expressing HIF-2α (MPCmCherry H2A) or control cells (MPCmCherry EV) were treated with 5 µM or 10 µM CBD or DMSO as control (single treatment: day 4; fractionated treatment: day 4, 8, 11, 15). Spheroid diameter was determined 18 days after seeding. Four independent experiments (n = 12). Average ± SEM, one-way ANOVA with Bonferoni correction, vs MPCmCherry crtl, DMSO control **P < .01, vs DMSO control of the corresponding cell line #P < .05, ##P < .01. Abbreviations: CBD, cannabidiol; DMSO, dimethyl sulphoxide; NET, neuroendocrine tumor; PPGL, pheochromocytoma and paraganglioma.

Figure 3.

Cell viability of NET primary cultures after 72 hours incubation with (A) CBD 5 µM (n = 12) and (B) CBD 10 µM (n = 11). Primary cultures 1.1 and 1.2 are from the same patient. Small intestinal NETs in light green; pancreatic NETs in dark green. Functionally active NETs: Carcinoid syndromes in striped light green (tumors 8, 10); insulinoma in patterned dark green (tumor 2). Each cell viability experiment comprised 3 to 8 samples per drug concentration and patient. The mean values ± SD are shown. Green asterisk: significant decrease of cell viability P < .05. Red asterisk: significant increase of cell viability P < .05. Abbreviations: CBD, cannabidiol; NET, neuroendocrine tumor.

Figure 4.

Cell viability of different human PPGL primary cultures (n = 35, n = 2 from the same patient) after incubation with 5 μM CBD for 72 hours (A) listed in numerical order and (B) listed in order of responsivity to CBD (significant decrease of cell viability, significant increase in cell viability, no significant decrease or increase of cell viability). Primary cultures 13.1 and 13.2 are from the same patient. Cluster 1-related PVs in red: SDHB (tumors 1, 32), SDHB and VUS in EPAS (tumor 2), SDHC (tumor 24), SDHD (tumors 11, 15), VUS in VHL (tumor 12). Cluster 2-related PVs in blue: NF1 (tumors 3, 4, 6, 7, 21; patterned blue), RET (tumors 13.1, 13.2, 17), HRAS (tumor 18), MAX (tumor 30). Other PVs in gold: BRCA (tumor 34), ATRX (tumor 35). Metastatic tumors 2, 35 framed in green. Germline and somatic testing was negative in tumors 8, 9, 19 (light grey). Tumors with negative germline testing and not available somatic status in black. Tumors without available germline and somatic status (unknown status) in dark grey. The detailed listing of the germline/somatic status of the individual PPGL patients can be found in Supplementary Table S3 (50). Each cell viability experiment comprised 3 to 8 samples per drug concentration and patient. The mean values ± SD are shown. Green asterisk: significant decrease of cell viability P < .05. Red asterisk: significant increase of cell viability P < .05. Abbreviations: CBD, cannabidiol; PPGL, pheochromocytoma and paraganglioma; PV, pathogenic variant; VUS, variant of unknown significance.

Figure 5.

Boxplot of PPGL primary cultures after 72 hours incubation with 5 µM CBD: comparison between cluster 1-related (red) and cluster 2-related (blue) tumors. PPGLs with PVs in NF1 are shown in light blue. Additionally, there is a comparison of cluster 1-related and cluster 2-related tumors with tumors without germline PV (and without cluster 1/2 somatic PV or with unknown somatic status) (black). Each cell viability experiment comprised 3 to 8 samples per drug concentration and patient. The mean values are shown as 1 dot. This figure was created with the open-source statistics software R (Version 4.3.1, R Foundation for Statistical Computing, Vienna, Austria). *P < .05. Abbreviations: CBD, cannabidiol; ns, nonsignificant; PPGL, pheochromocytoma and paraganglioma; PV, pathogenic variant.

Figure 6.

Cell viability of different human PPGL primary cultures (n = 33, n = 2 from the same patient) after incubation with 10 μM CBD for 72 hours (A) listed in numerical order and (B) listed in order of responsivity to CBD (significant decrease of cell viability, significant increase of cell viability, no significant increase or decrease of cell viability). Primary cultures 13.1 and 13.2 are from the same patient. Cluster 1-related PVs in red: SDHB (tumors 1, 32), SDHB and VUS in EPAS (tumor 2), SDHC (tumor 24), SDHD (tumor 15), VUS in VHL (tumor 12). Cluster 2-related PVs in blue: NF1 (tumors 3, 4, 6, 21; patterned blue), RET (tumors 13.1, 13.2, 17), HRAS (tumor 18), MAX (tumor 30). Other PVs in gold: BRCA (tumor 34), ATRX (tumor 35). Metastatic tumors 2, 35 framed in green. Germline and somatic testing was negative in tumors 8, 19 (light grey). Tumors with negative germline testing and not available somatic status in black. Tumors without available germline and somatic status (unknown status) in dark grey. The detailed listing of the germline/somatic status of the individual PPGL patients can be found in Supplementary Table S3 (50). Each cell viability experiment comprised 3 to 8 samples per drug concentration and patient. The mean values ± SD are shown. Green asterisk: significant decrease of cell viability P < .05. Red asterisk: significant increase of cell viability P < .05. Abbreviations: CBD, cannabidiol; PPGL, pheochromocytoma and paraganglioma; PV, pathogenic variant.