The Role of Plasma Membrane Viscosity in the Response and Resistance of Cancer Cells to Oxaliplatin
<p>Plasma membrane viscosity in cultured HCT116 cells during development of chemoresistance to oxaliplatin. (<b>a</b>) Representative FLIM images of parental cells (upper row) and cells adapted to the indicated drug doses (bottom row). Oxaliplatin was removed from the culture medium 48 h prior to the viscosity imaging. BODIPY 2 (4.5 μM) was used as a viscosity-sensitive probe. Ex. 850 nm, reg. 500–550 nm. Molecular rotor demonstrated a monoexponential fluorescence decay in plasma membrane. Bar is 40 µm, applicable to all images. (<b>b</b>) Quantification of viscosity of plasma membranes in HCT116 cells. Mean ± SD, <span class="html-italic">n</span> = 20–30 cells for each drug dose. Statistical significance was determined by ANOVA with Bonferroni post-hoc test. *, <span class="html-italic">p</span> ≤ 0.05 with control.</p> "> Figure 2
<p>ToF-SIMS analysis of plasma membrane lipid composition of cultured HCT116 cells adapted to different oxaliplatin doses. (<b>a</b>) Phosphatidylcholine (PC) (<span class="html-italic">m/z</span> 224), sphingomyelin (SM) (<span class="html-italic">m/z</span> 264) and cholesterol (<span class="html-italic">m/z</span> 385) ion yields obtained in positive ions. Data normalized to corresponding lipid intensities of a control sample. (<b>b</b>) Saturated, mono- and polyunsaturated fatty acids ratio in control cells and cells adapted to the indicated doses of oxaliplatin. Data normalized to the saturated fatty acids signal of corresponding sample. *, <span class="html-italic">p</span> ≤ 0.05 with control.</p> "> Figure 3
<p>Viscosity of HCT116 and HCT116-OXAR cell membranes recorded during the treatment with oxaliplatin. (<b>a</b>) Phase-contrast microscopy of HCT116 and HCT116-OXAR cells. Bar is 100 µm. (<b>b</b>) % viability in the presence of oxaliplatin (MTT-assay). (<b>c</b>) The doubling time. Mean ± SD. For characteristics of HCT116 cells adapted to different doses of oxaliplatin see <a href="#app1-cancers-13-06165" class="html-app">Figure S1</a>. (<b>d</b>) Representative FLIM images of sensitive and resistant HCT116 cells incubated with 2.0 µM oxaliplatin for 1 h and 24 h and untreated controls. Staining with fluorescent molecular rotor BODIPY 2 (4.5 μM). Ex. 850 nm, reg. 500–550 nm. BODIPY 2 demonstrated a monoexponential fluorescence decay in plasma membrane. Bar is 40 µm, applicable to all images. (<b>e</b>) Quantification of viscosity of plasma membranes in HCT116 and HCT116-OXAR cells. Mean ± SD, <span class="html-italic">n</span> = 20–30 cells. Statistical significance was determined by ANOVA with Bonferroni post-hoc test. *, <span class="html-italic">p</span> ≤ 0.05 with control.</p> "> Figure 4
<p>ToF-SIMS analysis of plasma membrane lipid composition of cultured HCT116 and HCT116-OXAR cells under the action of 2.0 µM oxaliplatin. (<b>a</b>) Phosphatidylcholine (PC) (<span class="html-italic">m/z</span> 224), sphingomyelin (SM) (<span class="html-italic">m/z</span> 264) and cholesterol (<span class="html-italic">m/z</span> 385) ion yields obtained in positive ions. Data normalized to corresponding lipid intensities of a HCT116 control sample. (<b>b</b>) Saturated, mono- and polyunsaturated fatty acids ratio. Data normalized to the saturated fatty acids signal of corresponding sample. *, <span class="html-italic">p</span> = 0.001 with HCT116 control cells without oxaliplatin incubation. #, <span class="html-italic">p</span> = 0.001 with HCT116-OXAR cells without oxaliplatin incubation.</p> "> Figure 5
<p>Principal component analysis of ToF-SIMS results demonstrating the differences between HCT116 and HCT116-OXAR cells in membrane lipid composition. The data from untreated and oxaliplatin-treated (2.0 µM, 1 or 24 h incubation) cells were analyzed. (<b>a</b>) Scores plots on PC1 and PC2 resulting from PCA in positive ion mode. (<b>b</b>) Scores plots on PC1 and PC2 resulting from PCA in negative ion mode. A 95% confidence limit for each region was defined by an ellipse with the same color to the corresponding region clusters. Each data point represents an individual field of view. Different samples are color coded.</p> "> Figure 6
<p>Membrane viscosity in oxaliplatin-sensitive HCT116 and resistant HCT116-OXAR tumors. (<b>a</b>) Dynamics of growth of untreated HCT116 and HCT116-OXAR tumors and tumors treated with oxaliplatin (7.5 mg/kg, seven doses for two weeks). Tumor volumes were normalized to the values of 10th day. Mean ± SEM, <span class="html-italic">n</span> = 4 tumors.*, <span class="html-italic">p</span> ≤ 0.05 with untreated HCT116 tumors. (<b>b</b>) Photographs of tumors. (<b>c</b>) Histopathology of HCT116 and HCT116-OXAR tumors on 23 day of growth after treatment with oxaliplatin and untreated controls. H&E-staining. Bar is 100 µm, applicable to all images. Initial magnification is 40×. (<b>d</b>) IHC for Ki-67 (red) overlapped with DAPI (blue) staining. Bar is 100 µm, applicable to all images. Initial magnification is 40×. (<b>e</b>) Representative FLIM images of HCT116 and HCT116-OXAR tumors in vivo after chemotherapy with oxaliplatin and untreated controls. Imaging was performed after i.v. injection of BODIPY 2 at 3 mg/kg. Ex. 850 nm, reg. 500–550 nm. Bar is 40 µm, applicable to all images. (<b>f</b>) Quantification of viscosity of the tumors. Mean ± SEM, <span class="html-italic">n</span> = 4 tumors. Measurements were done in 20–30 cells in each field of view. *, <span class="html-italic">p</span> = 0.0002 with untreated HCT116 tumors.</p> "> Figure 6 Cont.
<p>Membrane viscosity in oxaliplatin-sensitive HCT116 and resistant HCT116-OXAR tumors. (<b>a</b>) Dynamics of growth of untreated HCT116 and HCT116-OXAR tumors and tumors treated with oxaliplatin (7.5 mg/kg, seven doses for two weeks). Tumor volumes were normalized to the values of 10th day. Mean ± SEM, <span class="html-italic">n</span> = 4 tumors.*, <span class="html-italic">p</span> ≤ 0.05 with untreated HCT116 tumors. (<b>b</b>) Photographs of tumors. (<b>c</b>) Histopathology of HCT116 and HCT116-OXAR tumors on 23 day of growth after treatment with oxaliplatin and untreated controls. H&E-staining. Bar is 100 µm, applicable to all images. Initial magnification is 40×. (<b>d</b>) IHC for Ki-67 (red) overlapped with DAPI (blue) staining. Bar is 100 µm, applicable to all images. Initial magnification is 40×. (<b>e</b>) Representative FLIM images of HCT116 and HCT116-OXAR tumors in vivo after chemotherapy with oxaliplatin and untreated controls. Imaging was performed after i.v. injection of BODIPY 2 at 3 mg/kg. Ex. 850 nm, reg. 500–550 nm. Bar is 40 µm, applicable to all images. (<b>f</b>) Quantification of viscosity of the tumors. Mean ± SEM, <span class="html-italic">n</span> = 4 tumors. Measurements were done in 20–30 cells in each field of view. *, <span class="html-italic">p</span> = 0.0002 with untreated HCT116 tumors.</p> "> Figure 7
<p>ToF-SIMS analysis of lipid composition of oxaliplatin-sensitive HCT116 and resistant HCT116-OXAR tumors treated with oxaliplatin and untreated controls. Oxaliplatin was injected at 7.5 mg/kg, seven doses for two weeks. Phosphatidylcholine (PC) (<span class="html-italic">m/z</span> 224), sphingomyelin (SM) (<span class="html-italic">m/z</span> 264) and cholesterol (<span class="html-italic">m/z</span> 385) ion yields were obtained in positive ions. Mean ± SEM, <span class="html-italic">n</span> = 4 tumors. *, <span class="html-italic">p</span> ≤ 0.05 with untreated HCT116 tumors.</p> ">
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Establishment of Chemoresistant Cell Line
2.3. MTT Assay
2.4. Cell Proliferation
2.5. Tumor Xenografts
2.6. Viscosity Measurements and FLIM
2.7. ToF-SIMS
2.8. Histopathology and Immunohistochemistry
2.9. Statistics
3. Results
3.1. Effects of Oxaliplatin on the Plasma Membrane of Cultured Cancer Cells
3.1.1. Changes of Viscosity and Lipid Profile during the Development of Chemoresistance
3.1.2. Effects of Oxaliplatin on Viscosity and Lipid Profile of Chemosensitive and Resistant Cells
3.2. In Vivo Effects of Oxaliplatin on Cell Membranes in Tumor Xenografts
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Viscosity | PC | SM | Chol | MUFA | PUFA | |
---|---|---|---|---|---|---|
Development of chemoresistance in vitro | ||||||
OXA 0.1 µM | ↑ | ↓ | ↑ * | ↑ * | ↓ | = |
OXA 1.0 µM | ↑ * | ↓ * | ↓ * | ↑ * | ↓ * | = |
OXA 2.0 µM | ↑ * | ↑ * | ↑ * | ↑ * | ↓ | ↑ |
OXA 8.0 µM | = | ↓ * | = | ↑ * | ↓ | ↑ |
Treatment with oxaliplatin in vitro | ||||||
HCT116 + OXA, 1 h | ↓ * | ↓ * | ↓ * | ↑ * | ↓ | ↓ |
HCT116 + OXA, 24 h | ↑ * | ↓ * | ↓ * | ↑ * | ↓ * | ↓ |
HCT116-OXAR + OXA, 1 h | = | = | = | ↓ * | = | = |
HCT116-OXAR + OXA, 24 h | = | = | = | = | = | = |
Treatment with oxaliplatin in vivo | ||||||
HCT116 + OXA | ↑ * | ↓ * | ↓ * | ↑ | N/A | |
HCT116-OXAR + OXA | = | ↑ * | = | ↑ * |
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Shimolina, L.; Gulin, A.; Ignatova, N.; Druzhkova, I.; Gubina, M.; Lukina, M.; Snopova, L.; Zagaynova, E.; Kuimova, M.K.; Shirmanova, M. The Role of Plasma Membrane Viscosity in the Response and Resistance of Cancer Cells to Oxaliplatin. Cancers 2021, 13, 6165. https://doi.org/10.3390/cancers13246165
Shimolina L, Gulin A, Ignatova N, Druzhkova I, Gubina M, Lukina M, Snopova L, Zagaynova E, Kuimova MK, Shirmanova M. The Role of Plasma Membrane Viscosity in the Response and Resistance of Cancer Cells to Oxaliplatin. Cancers. 2021; 13(24):6165. https://doi.org/10.3390/cancers13246165
Chicago/Turabian StyleShimolina, Liubov, Alexander Gulin, Nadezhda Ignatova, Irina Druzhkova, Margarita Gubina, Maria Lukina, Ludmila Snopova, Elena Zagaynova, Marina K. Kuimova, and Marina Shirmanova. 2021. "The Role of Plasma Membrane Viscosity in the Response and Resistance of Cancer Cells to Oxaliplatin" Cancers 13, no. 24: 6165. https://doi.org/10.3390/cancers13246165