Comparative Analysis of Commercial and Home-Made Media on RSPO1/S6R Axis in Organoids with Different Wnt Backgrounds: A Methodological Guide for the Selection of Intestinal Patient-Derived Organoids Culture Media

Abstract

WNT3A is an intestinal ligand triggering the Wnt/β-catenin (Wnt) pathway, which can be enhanced by R-spondin 1 (RSPO1) through the RSPO1-LGR axis or antagonized by the adenomatous polyposis coli (APC) protein supporting β-catenin-degradation. Wnt interplays with several pathways including PI3K/mTOR (mTOR). In this study, we evaluated the influence of WNT3A-commercial and home-made culture media and RSPO1 protein on the Wnt and mTOR interplay in non-APC and APC-mutated intestinal patient-derived organoids (PDOs). Normal mucosa (NM) of sporadic CRC and FAP PDOs were cultured with: WNT3A-lacking/containing commercial (A/A+B) or home-made (BASAL/WNT3A-conditioned medium (CM)±RSPO1) media. In non-APC-mutated-PDOs (CRC-NM), WNT3A-CM, over commercial A+B, strongly activated Wnt-target-genes CCND1 and c-MYC. Most importantly, the addition of RSPO1 to home-made WNT3A-CM or A+B led to the downregulation of the mTOR-downstream-effector phospho-S6 ribosomal protein (p-S6R), highlighting the activation of the RSPO1-pS6R in both non-APC (CRC-NM) and APC-mutated (FAP-NM) PDOs, independently from LGR5 gene expression modulation. Our work demonstrates that home-made WNT3A-CM strongly impacts the crosstalk between Wnt and mTOR over commercial media, and proposes RSPO1 as a key regulator of the RSPO1-p-S6R axis in both non-APC and APC-mutated PDOs. Together, these findings represent an important methodological guide for scientists working in these fields to select the most appropriate intestinal PDO media.

Keywords: R-spondin 1 (RSPO1); colorectal cancer (CRC); intestinal patient-derived organoid (PDO); organoids culture media; phospho-S6 ribosomal protein (p-S6R).

Figure 1

Canonical Wnt/β-catenin pathway activation in the HEK 293 STF cell reporter cell line in commercial and home-made-cultured non-APC mutated CRC NM PDOs. (A) Luciferase assay of HEK 293 STF cells cultured for 24 h with different intestinal organoid culture media (n = 4). (B) Luciferase-based quality control test of WNT3A-CM in HEK 293 STF cells cultured for 24 h with freshly thawed medium (termed 0) or 2/7/14-days-thawed medium (n = 3). Experiments were independently repeated at least three times and the values are reported as the mean ± SD. Data were analyzed with the parametric Student’s t-test according to Shapiro–Wilk normality test results. * p < 0.05, ** p  < 0.01, *** p  < 0.001, **** p < 0.0001. NC = negative control; PC1 = positive control 1; PC2 = positive control 2; A = WNT3A-lacking commercial medium composed of DMEMF12 and Component A in a 1:1 ratio; A+B = WNT3A-containing commercial medium composed of Component A and Supplement B in a 1:1 ratio (exact formulation relative to other factors present is not known); BASAL = WNT3A-lacking home-made basal medium; WNT3A-CM = WNT3A-containing complete home-made medium, with or without R-spondin1 (+/−RSPO1).

Figure 2

Different activation of Wnt/β-catenin in commercial and home-made-cultured non-APC mutated CRC NM PDOs. (A) RNA expression of Wnt/β-catenin target genes AXIN2, CCND1, and c-MYC of CRC NM PDOs cultured for 48 h with the indicated media (n = 8, of which PT10 = 6, PT13 = 1, PT16 = 1). (B) WB analysis of non-p(Active)/total β-catenin protein ratio in CRC NM PDOs cultured for 48 h with the specified media as a marker of Wnt activation (n = 7, of which PT10 = 5, PT13 = 1, PT16 = 1). (C) Representative WB imaging of non-p(Active)-β-catenin, total β-catenin, p-S6R, and GAPDH (n = 7); GAPDH was used as the loading control (cropped blots). Experiments were independently repeated at least three times and the values are reported as mean ± SD. Data were analyzed with the parametric Student’s t-test (CCND1 and non-p(Active)/total β-catenin) and non-parametric Mann–Whitney test (AXIN2 and c-MYC), according to the Shapiro–Wilk normality test results. * p < 0.05, ** p  < 0.01 and *** p  < 0.001. A = WNT3A-lacking commercial medium composed of DMEMF12 and Component A in a 1:1 ratio; A+B = WNT3A-containing commercial medium composed of Component A and Supplement B in a 1:1 ratio (exact formulation relative to other factors present is not known); BASAL = WNT3A-lacking home-made basal medium; WNT3A-CM = WNT3A-containing complete home-made medium, with or without R-spondin1 (+/−RSPO1).

Figure 3

Different activation of intestinal stemness/differentiation markers and PI3K/mTOR in commercial and home-made-cultured non-APC mutated CRC NM PDOs. (A) RNA expression of stemness (LGR5) and differentiation (KRT20) intestinal markers in 48 h-cultured CRC NM PDOs (n = 8, of which PT10 = 6, PT13 = 1, PT16 = 1). (B) WB analysis of mTOR effector p-S6R protein in CRC NM PDOs cultured for 48 h with the indicated media (n = 7, of which PT10 = 5, PT13 = 1, PT16 = 1). (C) Representative WB imaging of non-p(Active)-β-catenin, total β-catenin, p-S6R, and GAPDH (n = 7); GAPDH was used as the loading control (cropped blots). Experiments were independently repeated at least three times and the values are reported as mean ± SD. Data were analyzed with the parametric Student’s t-test (LGR5 and KRT20) and non-parametric Mann–Whitney test (p-S6R), according to the Shapiro–Wilk normality test results. * p < 0.05, ** p  < 0.01, *** p  < 0.001, **** p < 0.0001. A = WNT3A-lacking commercial medium composed of DMEMF12 and Component A in a 1:1 ratio; A+B = WNT3A-containing commercial medium composed of Component A and Supplement B in a 1:1 ratio (exact formulation relative to other factors present is not known); BASAL = WNT3A-lacking home-made basal medium; WNT3A-CM = WNT3A-containing complete home-made medium, with or without R-spondin1 (+/−RSPO1).

Figure 4

Effect of commercial and home-made media on the viability and morphology of non-APC-mutated CRC NM PDOs. (A) Representative images of CRC NM PDOs cultured with the indicated media at 48 h (40× magnification, scale bar = 100 µm) (n = 6); representative pictures were captured through an EVOS M5000 Microscope. (B) Analysis of CRC NM PDO viability cultured for 48 h with the different culture media (n = 6, of which PT10 = 4, PT13 = 1, PT16 = 1). (C) Morphological analysis of the total object area and darkness of CRC NM PDOs in different culture media at the 48 h time point (n = 6, of which PT10 = 4, PT13 = 1, PT16 = 1). (D) Time-lapse morphological analysis of cultured-CRC NM PDOs. Morphology (total object area and darkness) was monitored every 8 h for 48 h (n = 6, of which PT10 = 4, PT13 = 1, PT16 = 1). (E,F) WB analysis and representative imaging of SURVIVIN protein in CRC NM PDOs cultured for 48 h with the specified media (n = 6, of which PT10 = 4, PT13 = 1, PT16 = 1); GAPDH was used as the loading control (cropped blots). Experiments were independently repeated six times and values are shown as the mean ± SD. Data were analyzed with the parametric Student’s t-test (viability assay and morphological analysis) and non-parametric Mann–Whitney test (SURVIVIN), according to the Shapiro–Wilk normality test results. A = WNT3A-lacking commercial medium composed of DMEMF12 and Component A in a 1:1 ratio; A+B = WNT3A-containing commercial medium composed of Component A and Supplement B in a 1:1 ratio (exact formulation relative to other factors present is not known); BASAL = WNT3A-lacking home-made basal medium; WNT3A-CM = WNT3A-containing complete home-made medium, with or without R-spondin1 (+/−RSPO1).

Figure 5

A+B-cultured-APC-mutated FAP NM PDOs induce lower LGR5 activation without affecting p-S6R downregulation. (A) RNA expression analysis of the Wnt/β-catenin target genes AXIN2, CCND1, and c-MYC in CRC NM and FAP NM PDOs cultured for 48 h in A and A+B (n = 5 of which, FAP NM-01 = 2, FAP NM-03 = 3, PT10 CRC NM = 3, PT13 CRC NM = 1, PT16 CRC NM = 1). (B) RNA expression analysis of stemness (LGR5) and differentiation (KRT20) intestinal markers in CRC NM and FAP NM PDOs cultured for 48 h in A and A+B (n = 5 of which, FAP NM-01 = 2, FAP NM-03 = 3, PT10 CRC NM = 3, PT13 CRC NM = 1, PT16 CRC NM = 1). (C,D) WB analysis of non-p(Active)/total β-catenin protein ratio and p-S6R in CRC NM and FAP NM PDOs cultured for 48 h with A and A+B (n = 5 of which, FAP NM-01 = 2, FAP NM-03 = 3, PT10 CRC NM = 3, PT13 CRC NM = 1, PT16 CRC NM = 1). (E) Representative WB imaging of non-p(Active)-β-catenin, total β-catenin, p-S6R, and GAPDH in CRC NM and FAP NM PDOs cultured for 48 h with A and A+B (n = 5 of which, FAP NM-01 = 2, FAP NM-03 = 3, PT10 CRC NM = 3, PT13 CRC NM = 1, PT16 CRC NM = 1); Representative WB imaging of FAP NM (A/A+B) and CRC NM (A/A+B) (cropped blots). GAPDH was used as the loading control. Experiments were independently repeated five times and values are shown as mean ± SD. Data were analyzed with the parametric Student’s t-test (AXIN2, c-MYC, CCND1, LGR5, non-p(Active)/total β-catenin and p-S6R) and non-parametric Mann–Whitney test (KRT20), according to the Shapiro–Wilk normality test results. * p < 0.05, ** p  < 0.01. A = WNT3A-lacking commercial medium composed of DMEMF12 and Component A in a 1:1 ratio; A+B = WNT3A-containing commercial medium composed of Component A and Supplement B in a 1:1 ratio (exact formulation relative to other factors present is not known).

Figure 6

RSPO1 guides p-S6R downregulation in APC-mutated FAP NM PDOs independently from a boosted activation of LGR5. (A,B) Morphological analysis of the total object area and darkness of FAP NM PDOs cultured with the indicated media for 48 h (n = 3, FAP NM-03 = 3). (C–F) RNA expression analysis of the AXIN2, CCND1, c-MYC Wnt-target genes and LGR5 intestinal marker in FAP NM PDOs cultured for 48 h with A, A+B, and A+B+RSPO1 (n = 4, FAP NM-03 = 4). (G,H) WB analysis of non-p(Active)/total β-catenin protein ratio and p-S6R in FAP NM PDOs cultured for 48 h with the indicated media (n = 4, FAP NM-03 = 4). (I) Representative WB imaging of non-p(Active)-β-catenin, total β-catenin, p-S6R, and GAPDH in FAP NM PDOs cultured for 48 h with the indicated media (n = 4, FAP NM-03 = 4); GAPDH was used as the loading control (cropped blots). Experiments were independently repeated at least three times and values are shown as the mean ± SD. Data were analyzed with the parametric Student’s t-test (morphological analysis, LGR5, non-p(Active)/total β-catenin and p-S6R), according to the Shapiro–Wilk normality test results. * p < 0.05, ** p  < 0.01, **** p < 0.0001. A = WNT3A-lacking commercial medium composed of DMEMF12 and Component A in a 1:1 ratio; A+B = WNT3A-containing commercial medium composed of Component A and Supplement B in a 1:1 ratio (exact formulation relative to other factors present is not known); A+B+RSPO1 = WNT3A-containing commercial medium composed of Component A and Supplement B in a 1:1 ratio, with RSPO1 3.