Short-term 3D culture systems of various complexity for treatment optimization of colorectal carcinoma

Three-dimensional (3D) cultures have the potential to increase the predictive value of pre-clinical drug research and bridge the gap towards anticipating clinical outcome of proposed treatments. However, their implementation in more advanced drug-discovery programs is still in its infancy due to the lack of reproducibility and low time- and cost effectiveness. HCT116, SW620 and DLD1 cells, cell lines with distinct mutations, grade and origin, were co-cultured with fibroblasts and endothelial cells (EC) in 3D spheroids. Clinically relevant drugs, i.e. 5-fluorouracil (5−FU), regorafenib and erlotinib, were administered individually to in CRC cell cultures. In this study, we established a robust, low-cost and reproducible short-term 3D culture system addressing the various complexities of the colorectal carcinoma (CRC) microenvironment. We observed a dose-dependent increase of erlotinib sensitivity in 3D (co-)cultures compared to 2D cultures. Furthermore, we compared the drug combination efficacy and drug-drug interactions administered in 2D, 3D and 3D co-cultures. We observed that synergistic/additive drug-drug interactions for drug combinations administered at low doses shifted towards additive and antagonistic when applied at higher doses in metastatic CRC cells. The addition of fibroblasts at various ratios and EC increased the resistance to some drug combinations in SW620 and DLD1 cells, but not in HCT116. Retreatment of SW620 3D co-cultures with a low-dose 3-drug combination was as active (88% inhibition, relative to control) as 5-FU treatment at high dose (100 μM). Moreover, 3D and 3D co-cultures responded variably to the drug combination treatments, and also signalling pathways were differently regulated, probably due to the influence of fibroblasts and ECs on cancer cells. The short-term 3D co-culture system developed here is a powerful platform for screening (combination) therapies. Understanding of signalling in 3D co-cultures versus 3D cultures and the responses in the 3D models upon drug treatment might be beneficial for designing anti-cancer therapies.


Immunohistochemistry
To unmask cellular targeted markers glass slides were heated in a beaker with TRIS/EDTA until boiling and continued to be heated for an additional 15 min on the defrost program after which they were left to cool. Before blocking, slides were washed with PBS and PBS supplemented with 0.2% Triton-X (PBST-X). To perform blocking, slides were covered with 80 µL PBST-X and 1% BSA blocking buffer and incubated for 20 min at RT. Following, cells were stained overnight at 4 °C with primary antibodies, i.e. Ki67 Rabbit mAb (9027, Cell Signaling) or CleavedCaspase3 Rabbit mAb (9664,Cell Signaling). Slides were washed twice with PBST-X and once with PBS and incubated for 1 hour at RT in the dark with the secondary antibody AF488 Donkey-α-Rabbit (A-21206, ThermoFisher Scientific) and DAPI. Slides were washed twice with PBST-X, washed once with PBS and mounted with Dako fluorescence mounting medium (S3023, Agilent) for fluorescence imaging.

Supplementary Figure S4
Supplemantary Figure S4. Optimization of 3D spheroid growth conditions. A Metabolic activity of HCT116, SW620 and DLD1 3D co-cultures consisting of tumor cells with 30%, 50% or 70% fibroblasts and an additinal 5% of endothelial cells relative to the 3D co-cultures with 50% fibroblasts at 100%, tested in various media compositions (DMEM, RPMI and/or EMEM), as indicated. Error bars represent the standard deviation of n = 3. B Spheroid size of HCT116, SW620 and DLD1 3D cultures at 1000 or 500 c/w and 3D co-cultures with various percentages of fibroblasts at the start of treatment at day 2 and day 5. Error bars represent the standard error of the mean of n = 9. Significances of *p < 0.05, **p < 0.01 and ***p < 0.001 define the differences between the CRC 3D and 3D co-culture conditions as determined with a two-way ANOVA with post-hoc Tukey`s multiple comparisons test.

Supplementary Figure S5
Supplemantary Figure S5. Responsiveness of 3D co-culture spheroids to different treatment schedules. Metabolic activity of HCT116 (A), SW620 (B) and DLD1 (C) 3D cultures at 1000 or 500 c/w after treatment with the low dose (LD) and max plasma concentration (MPC) drug combinations in two treatment schedules from day 2-5 and day 4-7. Error bars represent the standard deviation of n = 9-12. Significances of *p < 0.05, **p < 0.01 and ***p < 0.001 represent the comparison between the 3D cultures and treatment schedules as determined with a two-way ANOVA with post-hoc Sidak`s multiple comparisons test. Figure S7. SW620 spheroid formation with methylcellulose supplement. Representative images of CRC cells seeded in low-attachment plates supplemented with Methocel methylcellulose at 0.01%, 0.1%, 0.25% and 0.5%. Scale bar represents 300 µm.

Supplementary Figure S10
Supplementary Figure S10. Full length unprocessed western blot results of Pl3K-AKT-mTOR and RAS-RAF-MAPK signaling and extracellular matrix in 3D and 3D co-cultures for three CRC cell lines. Western blot analysis of various proteins and phosphorylated (p-) proteins in the Pl3K-AKT-mTOR and RAS-RAF-MAPK pathway of HCT116, SW620 and DLD1 3D cultures (seeded 1000 c/w) and 3D co-cultures (3D-CC, seeded with 50% fibroblasts). Cultures were treated with a 3-drug combination at maximum plasma concentration (MPC) or with CTRL. A,C,E Western blots for βactin (45 kDa), Akt (60 kDa) and p-rpS6 (S235/236, 32 kDa). B,D,F Western blots for α-tubulin (52 kDa), MAPK and p-MAPK (T202/Y204, 42 and 44 kDa) proteins. G,H,I Western blots for β-actin (45 kDa), fibronectin (285 kDa) and laminin (210 and 400 kDa) proteins. Cropped parts of the gels are presented in Figure 7. The blots images were prepared in compliance with the digital image and integrity policies of the journal. Images were obtained with the Licor Odyssey CLx scanner at one default exposure setting.

Supplementary Tables
Supplementary Table S1. Morphological characteristics of the 3D cultures

Cell line
Morphology observed as of day two DLD1 Circular, compact core and defined periphery Over time evolves into heterogeneous and lobular shapes SW620 Spherical, clear and uncompact, no defined periphery Over time increased compactness of the core HCT116 Circular, compact core and fully defined periphery Over time increased compactness of the core with loose cells surrounding the clearly defined rim LS174T Spherical form with defined periphery. Increased compactness of the core over time and increased definition of the periphery in slightly lobular shapes HT29 Circular, compact core and defined periphery. Spheroid disintegration starting at day five Caco2 Round, loose with varying shapes and defined periphery. Over time increased compactness of the core CCD841 CoN Circular, clear and uncompact with aligned periphery at day one. From day 2 decreased size and compactness with cells sprouting at the periphery * Circularity: the closer to a circularity of 1, the more symmetrical the spheroid.