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The efficacy of cetuximab in a tissue-engineered three-dimensional in vitro model of colorectal cancer.

Magdeldin T, López-Dávila V, Villemant C, Cameron G, Drake R, Cheema U, Loizidou M - J Tissue Eng (2014)

Bottom Line: However, a considerable amount of evidence demonstrates that two-dimensional cell culture does not accurately reflect the three-dimensional in vivo tumour microenvironment, specifically with regard to gene expression profiles, oxygen and nutrient gradients and pharmacokinetics.Epidermal growth factor receptor expression levels revealed a twofold and threefold increase in three-dimensional cultures for both HT29 and HCT116 cells in comparison to two-dimensional monolayers, respectively (p < 0.05; p < 0.01).Cetuximab efficacy was significantly lower in HT29 three-dimensional cultures in comparison to two-dimensional monolayers, whereas HCT116 cells in both two-dimension and three-dimension were non-responsive to treatment in agreement with their KRAS mutant status.

View Article: PubMed Central - PubMed

Affiliation: Cancer Nanotechnology Group, UCL Division of Surgery and Interventional Science, London, UK ; Tissue Repair and Engineering Centre, Institute of Orthopaedics and Musculoskeletal Sciences, UCL Division of Surgery and Interventional Science, Stanmore, UK.

ABSTRACT
The preclinical development process of chemotherapeutic drugs is often carried out in two-dimensional monolayer cultures. However, a considerable amount of evidence demonstrates that two-dimensional cell culture does not accurately reflect the three-dimensional in vivo tumour microenvironment, specifically with regard to gene expression profiles, oxygen and nutrient gradients and pharmacokinetics. With this objective in mind, we have developed and established a physiologically relevant three-dimensional in vitro model of colorectal cancer based on the removal of interstitial fluid from collagen type I hydrogels. We employed the RAFT™ (Real Architecture For 3D Tissue) system for producing three-dimensional cultures to create a controlled reproducible, multiwell testing platform. Using the HT29 and HCT116 cell lines to model epidermal growth factor receptor expressing colorectal cancers, we characterized three-dimensional cell growth and morphology in addition to the anti-proliferative effects of the anti-epidermal growth factor receptor chemotherapeutic agent cetuximab in comparison to two-dimensional monolayer cultures. Cells proliferated well for 14 days in three-dimensional culture and formed well-defined cellular aggregates within the concentrated collagen matrix. Epidermal growth factor receptor expression levels revealed a twofold and threefold increase in three-dimensional cultures for both HT29 and HCT116 cells in comparison to two-dimensional monolayers, respectively (p < 0.05; p < 0.01). Cetuximab efficacy was significantly lower in HT29 three-dimensional cultures in comparison to two-dimensional monolayers, whereas HCT116 cells in both two-dimension and three-dimension were non-responsive to treatment in agreement with their KRAS mutant status. In summary, these results confirm the use of a three-dimensional in vitro cancer model as a suitable drug-screening platform for in vitro pharmacological testing.

No MeSH data available.


Related in: MedlinePlus

Growth kinetics of HT29 and HCT116 cells in 3D culture. (a) The proliferation profile of HT29 and HCT116 colorectal cancer cells cultured in 3D over a 14-day period measured by the alamarBlue assay and (b) morphological analysis of HT29 and HCT116 3D cultures over a 14-day period. Single cells are seeded and migrate together over time to form cellular aggregates (scale bar − 50 µm).3D: three-dimensional.
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fig2-2041731414544183: Growth kinetics of HT29 and HCT116 cells in 3D culture. (a) The proliferation profile of HT29 and HCT116 colorectal cancer cells cultured in 3D over a 14-day period measured by the alamarBlue assay and (b) morphological analysis of HT29 and HCT116 3D cultures over a 14-day period. Single cells are seeded and migrate together over time to form cellular aggregates (scale bar − 50 µm).3D: three-dimensional.

Mentions: To assess the metabolic activity of HT29 and HCT116 cells in 3D culture, the alamarBlue assay was used. Since the assay is non-toxic, multiple readings over a timescale were measured for each well. Increases in metabolic activity, taken as an indicator of cell proliferation, and therefore increases in cell number were determined over 14 days for both cell lines (Figure 2(a)). HT29 cells displayed a steady increase in cell number that appeared to plateau at day 14. Morphologically, the cellular aggregates appeared to grow in size consistently over the 14-day period, in line with the proliferation measurements. On the other hand, although HCT116 cultures exhibited an overall growth pattern over 14 days, there was a significant drop in the proliferation readings at day 7 indicating a quiescent state. However, phase-contrast images of HCT116 cellular aggregates showed no reduction or a slower rate of apparent growth between day 3 and day 7 (Figure 2(b)); therefore, any state of biochemical quiescence did not result in a concomitant morphological change.


The efficacy of cetuximab in a tissue-engineered three-dimensional in vitro model of colorectal cancer.

Magdeldin T, López-Dávila V, Villemant C, Cameron G, Drake R, Cheema U, Loizidou M - J Tissue Eng (2014)

Growth kinetics of HT29 and HCT116 cells in 3D culture. (a) The proliferation profile of HT29 and HCT116 colorectal cancer cells cultured in 3D over a 14-day period measured by the alamarBlue assay and (b) morphological analysis of HT29 and HCT116 3D cultures over a 14-day period. Single cells are seeded and migrate together over time to form cellular aggregates (scale bar − 50 µm).3D: three-dimensional.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2 - License 3
Show All Figures
getmorefigures.php?uid=PMC4221936&req=5

fig2-2041731414544183: Growth kinetics of HT29 and HCT116 cells in 3D culture. (a) The proliferation profile of HT29 and HCT116 colorectal cancer cells cultured in 3D over a 14-day period measured by the alamarBlue assay and (b) morphological analysis of HT29 and HCT116 3D cultures over a 14-day period. Single cells are seeded and migrate together over time to form cellular aggregates (scale bar − 50 µm).3D: three-dimensional.
Mentions: To assess the metabolic activity of HT29 and HCT116 cells in 3D culture, the alamarBlue assay was used. Since the assay is non-toxic, multiple readings over a timescale were measured for each well. Increases in metabolic activity, taken as an indicator of cell proliferation, and therefore increases in cell number were determined over 14 days for both cell lines (Figure 2(a)). HT29 cells displayed a steady increase in cell number that appeared to plateau at day 14. Morphologically, the cellular aggregates appeared to grow in size consistently over the 14-day period, in line with the proliferation measurements. On the other hand, although HCT116 cultures exhibited an overall growth pattern over 14 days, there was a significant drop in the proliferation readings at day 7 indicating a quiescent state. However, phase-contrast images of HCT116 cellular aggregates showed no reduction or a slower rate of apparent growth between day 3 and day 7 (Figure 2(b)); therefore, any state of biochemical quiescence did not result in a concomitant morphological change.

Bottom Line: However, a considerable amount of evidence demonstrates that two-dimensional cell culture does not accurately reflect the three-dimensional in vivo tumour microenvironment, specifically with regard to gene expression profiles, oxygen and nutrient gradients and pharmacokinetics.Epidermal growth factor receptor expression levels revealed a twofold and threefold increase in three-dimensional cultures for both HT29 and HCT116 cells in comparison to two-dimensional monolayers, respectively (p < 0.05; p < 0.01).Cetuximab efficacy was significantly lower in HT29 three-dimensional cultures in comparison to two-dimensional monolayers, whereas HCT116 cells in both two-dimension and three-dimension were non-responsive to treatment in agreement with their KRAS mutant status.

View Article: PubMed Central - PubMed

Affiliation: Cancer Nanotechnology Group, UCL Division of Surgery and Interventional Science, London, UK ; Tissue Repair and Engineering Centre, Institute of Orthopaedics and Musculoskeletal Sciences, UCL Division of Surgery and Interventional Science, Stanmore, UK.

ABSTRACT
The preclinical development process of chemotherapeutic drugs is often carried out in two-dimensional monolayer cultures. However, a considerable amount of evidence demonstrates that two-dimensional cell culture does not accurately reflect the three-dimensional in vivo tumour microenvironment, specifically with regard to gene expression profiles, oxygen and nutrient gradients and pharmacokinetics. With this objective in mind, we have developed and established a physiologically relevant three-dimensional in vitro model of colorectal cancer based on the removal of interstitial fluid from collagen type I hydrogels. We employed the RAFT™ (Real Architecture For 3D Tissue) system for producing three-dimensional cultures to create a controlled reproducible, multiwell testing platform. Using the HT29 and HCT116 cell lines to model epidermal growth factor receptor expressing colorectal cancers, we characterized three-dimensional cell growth and morphology in addition to the anti-proliferative effects of the anti-epidermal growth factor receptor chemotherapeutic agent cetuximab in comparison to two-dimensional monolayer cultures. Cells proliferated well for 14 days in three-dimensional culture and formed well-defined cellular aggregates within the concentrated collagen matrix. Epidermal growth factor receptor expression levels revealed a twofold and threefold increase in three-dimensional cultures for both HT29 and HCT116 cells in comparison to two-dimensional monolayers, respectively (p < 0.05; p < 0.01). Cetuximab efficacy was significantly lower in HT29 three-dimensional cultures in comparison to two-dimensional monolayers, whereas HCT116 cells in both two-dimension and three-dimension were non-responsive to treatment in agreement with their KRAS mutant status. In summary, these results confirm the use of a three-dimensional in vitro cancer model as a suitable drug-screening platform for in vitro pharmacological testing.

No MeSH data available.


Related in: MedlinePlus