Control of symptoms related to hormonal hypersecretion by functioning neuroendocrine tumors (NETs) is challenging. New therapeutic options are required. Since novel in vitro tumor models seem to better mimic the tumor in vivo conditions, we aimed to study the effect of somatostatin and dopamine receptor agonists (octreotide and cabergoline, respectively) and novel somatostatin-dopamine chimeric multi-receptor drugs (BIM-065, BIM-23A760) using 2D (monolayer) and 3D (spheroids) cultures. Dose–response studies in 2D and 3D human pancreatic NET cell cultures (BON-1 and QGP-1) were performed under serum-containing and serum-deprived conditions. Cell proliferation, somatostatin and dopamine receptor expression (SSTs and D2R), apoptosis, lactate dehydrogenase, as well as serotonin and chromogranin A (CgA) release were assessed. The following results were obtained. 3D cultures of BON-1/QGP-1 allowed better cell survival than 2D cultures in serum-deprived conditions. SSTs and D2R mRNA levels were higher in the 3D model vs 2D model. Octreotide/cabergoline/BIM-065/BIM-23A760 treatment did not affect cell growth or spheroid size. In BON-1 2D-cultures, only BIM-23A760 significantly inhibited CgA release –this effect being more pronounced in 3D cultures. In BON-1 2D cultures, cabergoline/BIM-065/BIM-23A760 treatment decreased serotonin release (maximal effect up to 40%), being this effect again more potent in 3D cultures (up to 67% inhibition; with BIM-23A760 having the most potent effects). In QGP-1, cabergoline/BIM-065 treatment decreased serotonin release only in the 3D model. In conclusion, cultures of NET 3D spheroids represent a promising method for evaluating cell proliferation and secretion in NET cell-line models. Compared to 2D models, 3D models grow relatively serum independent. In 3D model, SST-D2R multi-receptor targeting drugs inhibit CgA and serotonin secretion, but not NET cell growth.
Supplemental Figure 1: mRNA expression profile of somatostatin receptors and dopamine type 2 receptor in BON-1 and QGP-1 cells. Relative mRNA expression (normalized to HPRT) in BON-1 (A) and QGP-1 (B) cells. Legend: ND: non-detectable.
Supplemental Figure 2: mRNA expression profile of somatostatin receptors and dopamine type 2 receptor in BON-1 and QGP-1 cells in 2D (monolayer) or 3D (spheroids) cultures. Relative mRNA expression (normalized to HPRT) in BON-1 (A) and QGP-1 (B) cells. Receptor expression was compared after 0, 3 and 7 days. Legend: ND: not detectable. Asterisks: *, p<0.05; **, p<0.01; ***, p<0.001.
Supplemental Figure 3: Effect of CAB, BIM065, BIM23A760 and OCT on cell growth in 2D monolayer cultures of BON-1 and QGP-1 in medium containing 10% FCS. Panels A-B: BON-1 cells after three and seven days of incubation respectively; Panels C-D: QGP-1 cells after three and seven days respectively. Values represent mean ± SEM and are shown as a percentage of untreated control.
Supplemental Figure 4: Effect of cabergoline, BIM065, BIM23A760 and OCT on spheroid size in BON-1 and QGP-1 cells in medium with 0.1% BSA. Change in spheroid size in BON-1 cultures after three (A) or seven days (B); change in spheroid size in QGP-1 cultures after three (C) or seven days (D). Values represent mean ± SEM and are shown as a percentage of untreated control.
Supplementary Table 1: BIM065 and BIM23A760 binding affinity (in nM) (Culler 2011; Halem H 2016)
Supplementary Table 2: Primer-probe sequences for SSTs and D2R
Supplementary Table 3: LDH release in BON-1 and QGP-1 monolayer cultures