Effects of novel somatostatin-dopamine chimeric drugs in 2D and 3D cell culture models of neuroendocrine tumors

in Endocrine-Related Cancer
Correspondence should be addressed to L J Hofland: l.hofland@erasmusmc.nl

(M D Culler is now at Culler Consulting, LLC, Cambridge, Massachusetts, USA)

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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.

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  • 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 &#x00B1; 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 &#x00B1; 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

 

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    BON-1 and QGP-1 cell growth in 2D- (monolayer) and 3D- (spheroids) cultures in different medium conditions. Cell growth (DNA content per well) was evaluated in untreated control cells at day zero (0) and after three (3) and seven (7) days of incubation. Panels A (2D) and B (3D) cultures of BON-1 cells; panels C (2D) and D (3D) represent cultures of QGP-1 cells. Dotted lines represent cell cultures in medium containing 10% FCS and the continuous black lines represent cell cultures in medium containing 0.1% BSA. Values represent the mean ± s.e.m. and are shown as a percentage of control at day 0. Asterisks: *P < 0.05; **P < 0.01; ***P  < 0.001.

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    DNA fragmentation (apoptosis) in 2D and 3D cultures in BON-1 and QGP-1 cell lines in different medium conditions. Panels A (2D) and B (3D) represent BON-1 cultures; panels C (2D) and D (3D) represent QGP-1 cultures. White bars represent cell cultures in medium containing 10% FCS and the gray bars represent cell cultures in medium containing 0.1% BSA. Apoptosis was higher in BON-1 spheroids after seven days (absolute values of 0.0041 ± 0.0009–0.003 ± 0.001 in optimal and serum-deprived conditions respectively) than in QGP-1 spheroids (absolute values of 0.001 ± 0–0.001 ± 0 in optimal and serum-deprived conditions respectively). Values represent the mean ± s.e.m. and are shown as a percentage of cells cultured in 10%FCS. Asterisks: *P < 0.05; ***P < 0.001.

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    Cell proliferation and spheroid size in BON-1 and QGP-1 cells grown as 3D spheroids in different medium conditions. Cells were followed by imaging using a Zeiss Axiovert 200/M-based phase-contrast microscope using a 5× objective. Representative images of BON-1 and QGP-1 spheroids after their formation (day 0) and after three and seven days culture are depicted in the right panels. Left panels: Increase in BON-1 spheroid cell growth and spheroid size in medium containing 10% FCS (A) and 0.1% BSA (B); Increase in QGP-1 spheroid cell growth and spheroid size in medium containing 10% FCS (C) and 0.1% BSA (D). Continuous black lines represent cell growth (DNA content), dotted lines represent spheroid size. Values represent mean ± s.e.m. and are shown as a percentage of control at day 0. Asterisks: *P < 0.05; **P < 0.01; ***P < 0.001.

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    mRNA expression profile of somatostatin receptor subtypes and dopamine type 2 receptor in BON-1 and QGP-1 cell lines using 2D and 3D culture systems. Relative mRNA relative expression (normalized to HPRT) in 2D (monolayer) cultures of BON-1 (A) and QGP-1 (B) cell lines and relative mRNA relative expression in 3D (spheroid) BON-1 (C) and QGP-1 (D) spheroids. Receptor expression after 3 and 7 days was compared to day 0. ND: not detectable. Asterisks: *P < 0.05; **P < 0.01; ***P < 0.001, compared to monolayer (2D) or spheroid (3D) at day 0.

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    Effect of cabergoline, BIM065, BIM23A760 and octreotide on growth of BON-1 and QGP-1 cells cultured in 2D (monolayer) or 3D (spheroids) in medium with 0.1% BSA. 2D cultures of BON-1 cells after three days of incubation (A); 3D cultures of BON-1 cells after three (B) or seven (7) days of incubation (C). 2D cultures of QGP-1 cells after 3 days of incubation (D); 3D cultures of QGP-1 cells after 3 (E) or 7 (F) days of incubation with the indicated drugs. Cell growth (DNA content) is expressed as the percentage of untreated control (mean ± SEM).

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    Effect of cabergoline, BIM065, BIM23A760 and octreotide on chromogranin A and serotonin secretion in 2D (monolayer) cultures of BON-1 and QGP-1 cells. Cells were incubated with the indicated drugs during three days in medium with 0.1% BSA. Chromogranin A (CgA) release in BON-1 cells (A); serotonin release in BON-1 cells (B); serotonin release in QGP-1 cells (C). Values represent mean ± s.e.m. and are shown as a percentage of untreated control. Asterisks: *P < 0.05; **P < 0.01; ***P < 0.001, compared to untreated controls (serotonin absolute values in controls of BON-1: 521.5 ± 57.8; QGP-1: 192.2 ± 16.3 pg/mL; CgA absolute values of BON-1 controls: 721.6 ± 38.8 ng/mL).

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    Effect of cabergoline, BIM065, BIM23A760 and octreotide on chromogranin A and serotonin secretion on 3D (spheroid) cultures of BON-1 and QGP-1 cells. 3D spheroids were incubated during seven days with the indicated drugs inmedium with 0.1% BSA. Chromogranin A (CgA) release in BON-1 cells (A); serotonin release in BON-1 cells (B); serotonin release in QGP-1 cells (C). Values represent mean ± s.e.m. and are shown as a percentage of untreated control. Asterisks: *P < 0.05; **P < 0.01; ***P < 0.001, compared to untreated controls (serotonin absolute values in BON-1 controls: 238.21.5 ± 21.6; QGP-1: 13.67 ± 1.8 pg/mL; CgA absolute values of BON-1 controls: 297.6 ± 14.89 ng/mL).

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