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The Role of Oxygen in Avascular Tumor Growth.

Grimes DR, Kannan P, McIntyre A, Kavanagh A, Siddiky A, Wigfield S, Harris A, Partridge M - PLoS ONE (2016)

Bottom Line: These describe the basic rate of growth well, but do not offer an explicitly mechanistic explanation.The model is fitted to growth curves for a range of cell lines and derived values of OCR are validated using clinical measurement.Finally, we illustrate how changes in OCR due to gemcitabine treatment can be directly inferred using this model.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK/MRC Oxford Institute for Radiation Oncology, Gray Laboratories, University of Oxford, Old Road Campus, Oxford, OX3 7DQ, United Kingdom.

ABSTRACT
The oxygen status of a tumor has significant clinical implications for treatment prognosis, with well-oxygenated subvolumes responding markedly better to radiotherapy than poorly supplied regions. Oxygen is essential for tumor growth, yet estimation of local oxygen distribution can be difficult to ascertain in situ, due to chaotic patterns of vasculature. It is possible to avoid this confounding influence by using avascular tumor models, such as tumor spheroids, a much better approximation of realistic tumor dynamics than monolayers, where oxygen supply can be described by diffusion alone. Similar to in situ tumours, spheroids exhibit an approximately sigmoidal growth curve, often approximated and fitted by logistic and Gompertzian sigmoid functions. These describe the basic rate of growth well, but do not offer an explicitly mechanistic explanation. This work examines the oxygen dynamics of spheroids and demonstrates that this growth can be derived mechanistically with cellular doubling time and oxygen consumption rate (OCR) being key parameters. The model is fitted to growth curves for a range of cell lines and derived values of OCR are validated using clinical measurement. Finally, we illustrate how changes in OCR due to gemcitabine treatment can be directly inferred using this model.

No MeSH data available.


Related in: MedlinePlus

Representative images (maximum intensity projections) of five cell lines that were fluorescently labelled for estimating cell volume using 3D confocal microscopy.Merged images show the simultaneous staining of the nucleus (blue, Hoechst 33342, 3.2μM) and the cell membrane (green, PKH26, 2μM) in (A) HeLa cells and in (B) the MDA-468, HCT116, Ls-174T, and SCC25 cells. Scale bars represent 10μm.
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pone.0153692.g002: Representative images (maximum intensity projections) of five cell lines that were fluorescently labelled for estimating cell volume using 3D confocal microscopy.Merged images show the simultaneous staining of the nucleus (blue, Hoechst 33342, 3.2μM) and the cell membrane (green, PKH26, 2μM) in (A) HeLa cells and in (B) the MDA-468, HCT116, Ls-174T, and SCC25 cells. Scale bars represent 10μm.

Mentions: As cellular mass mc is related to the oxygen consumption rate by Eq 5, it was important to estimate this to facilitate comparison between OCR derived from theory and experiment. To obtain approximate values for cell mass, we estimated the volume of individual cells using 3D confocal microscopy. Cells (1 x 105 per well) from all lines were seeded onto glass coverslips in a 6-well plate and incubated at 37°C for 24 hours; all subsequent experiments were carried out at room temperature. Once cells had attached to the coverslip, they were washed in serum-free culture medium and then incubated for 5 min with PKH26 (2 μM; Sigma-Aldrich) prepared in Diluent C (Sigma-Aldrich) to fluorescently label the cell membrane. An equal volume of 100% fetal bovine serum was added for 1 min to stop the reaction. Cells were washed with regular culture medium, fixed for 10 min with 4% paraformaldehyde, washed with phosphate-buffered saline, and incubated with Hoechst 33342 (3.2 μM; Life Technologies) for 10 min to label nuclei. Coverslips were then washed in PBS and mounted using SlowFade® Gold Antifade Reagent (Life Technologies) onto glass slides. Five fields of view from each cell line were acquired using the 20x/0.87 M27 objective on a Zeiss LSM 710 microscope (Carl Zeiss AG). To obtain cell volume estimates, each field of view was scanned using the z-stack feature, with a 1 μm slice thickness, to obtain images for cell volume estimates as shown in Fig 2.


The Role of Oxygen in Avascular Tumor Growth.

Grimes DR, Kannan P, McIntyre A, Kavanagh A, Siddiky A, Wigfield S, Harris A, Partridge M - PLoS ONE (2016)

Representative images (maximum intensity projections) of five cell lines that were fluorescently labelled for estimating cell volume using 3D confocal microscopy.Merged images show the simultaneous staining of the nucleus (blue, Hoechst 33342, 3.2μM) and the cell membrane (green, PKH26, 2μM) in (A) HeLa cells and in (B) the MDA-468, HCT116, Ls-174T, and SCC25 cells. Scale bars represent 10μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835055&req=5

pone.0153692.g002: Representative images (maximum intensity projections) of five cell lines that were fluorescently labelled for estimating cell volume using 3D confocal microscopy.Merged images show the simultaneous staining of the nucleus (blue, Hoechst 33342, 3.2μM) and the cell membrane (green, PKH26, 2μM) in (A) HeLa cells and in (B) the MDA-468, HCT116, Ls-174T, and SCC25 cells. Scale bars represent 10μm.
Mentions: As cellular mass mc is related to the oxygen consumption rate by Eq 5, it was important to estimate this to facilitate comparison between OCR derived from theory and experiment. To obtain approximate values for cell mass, we estimated the volume of individual cells using 3D confocal microscopy. Cells (1 x 105 per well) from all lines were seeded onto glass coverslips in a 6-well plate and incubated at 37°C for 24 hours; all subsequent experiments were carried out at room temperature. Once cells had attached to the coverslip, they were washed in serum-free culture medium and then incubated for 5 min with PKH26 (2 μM; Sigma-Aldrich) prepared in Diluent C (Sigma-Aldrich) to fluorescently label the cell membrane. An equal volume of 100% fetal bovine serum was added for 1 min to stop the reaction. Cells were washed with regular culture medium, fixed for 10 min with 4% paraformaldehyde, washed with phosphate-buffered saline, and incubated with Hoechst 33342 (3.2 μM; Life Technologies) for 10 min to label nuclei. Coverslips were then washed in PBS and mounted using SlowFade® Gold Antifade Reagent (Life Technologies) onto glass slides. Five fields of view from each cell line were acquired using the 20x/0.87 M27 objective on a Zeiss LSM 710 microscope (Carl Zeiss AG). To obtain cell volume estimates, each field of view was scanned using the z-stack feature, with a 1 μm slice thickness, to obtain images for cell volume estimates as shown in Fig 2.

Bottom Line: These describe the basic rate of growth well, but do not offer an explicitly mechanistic explanation.The model is fitted to growth curves for a range of cell lines and derived values of OCR are validated using clinical measurement.Finally, we illustrate how changes in OCR due to gemcitabine treatment can be directly inferred using this model.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK/MRC Oxford Institute for Radiation Oncology, Gray Laboratories, University of Oxford, Old Road Campus, Oxford, OX3 7DQ, United Kingdom.

ABSTRACT
The oxygen status of a tumor has significant clinical implications for treatment prognosis, with well-oxygenated subvolumes responding markedly better to radiotherapy than poorly supplied regions. Oxygen is essential for tumor growth, yet estimation of local oxygen distribution can be difficult to ascertain in situ, due to chaotic patterns of vasculature. It is possible to avoid this confounding influence by using avascular tumor models, such as tumor spheroids, a much better approximation of realistic tumor dynamics than monolayers, where oxygen supply can be described by diffusion alone. Similar to in situ tumours, spheroids exhibit an approximately sigmoidal growth curve, often approximated and fitted by logistic and Gompertzian sigmoid functions. These describe the basic rate of growth well, but do not offer an explicitly mechanistic explanation. This work examines the oxygen dynamics of spheroids and demonstrates that this growth can be derived mechanistically with cellular doubling time and oxygen consumption rate (OCR) being key parameters. The model is fitted to growth curves for a range of cell lines and derived values of OCR are validated using clinical measurement. Finally, we illustrate how changes in OCR due to gemcitabine treatment can be directly inferred using this model.

No MeSH data available.


Related in: MedlinePlus