Multi-Modal Investigation of Metabolism in Murine Breast Cancer Cell Lines Using Fluorescence Lifetime Microscopy and Hyperpolarized 13C-Pyruvate Magnetic Resonance Spectroscopy
"> Figure 1
<p>(<b>A</b>) The multi-modal bioreactor design uses a 3D collagen gel cell culture in the MRI-compatible bioreactor chamber with (<b>B</b>) a transparent portal in the base that can be placed on a fluorescence microscope stage for fluorescence lifetime imaging microscopy (FLIM setup). (<b>C</b>) The bioreactor system in the MRI setup adjacent to the volume coil was used for signal excitation and detection. (<b>D</b>) Culture media flow was constantly maintained (green arrow) with temperature control maintained by water bath flow around the culture volume (blue arrow). Hyperpolarized metabolic substrates (13C1-pyruvate in this case) were injected via bolus infusion into base of the culture chamber (red arrow).</p> "> Figure 2
<p>(<b>Top</b>) Example fluorescence lifetime imaging microscopy (FLIM) images showing mean lifetime (τ<sub>m</sub>) for NAD(P)H from non-metastatic (67NR), metastatically dormant (4T07), and metastatic (4T1) murine breast cancer cells (<b>upper images</b>). Cells were imaged at a 740 nm wavelength with a 450/70 nm filter. The color-coded images in the first row (<b>upper images</b>) show that the 67NR cells have a shorter τ<sub>m</sub> (more yellow in color) compared to the longer τ<sub>m</sub> in 4T07 and 4T1 cells (more blue in color). The cross hairs centered on specific cells in each panel indicate where fluorescence life time measurement is localized. (<b>Bottom</b>) Example photon lifetime distribution from a single pixel location as displayed by the SPCImage software (v8.0). Units are in nanoseconds (ns).</p> "> Figure 3
<p>Representative spectra from the 3 cell lines studied, 4T1 (<b>top</b>), 4T07 (<b>middle</b>), and 67NR (<b>bottom</b>), showing the pyruvate substrate (170 ppm, truncated), pyruvate hydrate (178 ppm) and lactate (182 ppm) components of hyperpolarized [1-13C] pyruvate metabolism. A vial of urea was included adjacent to the bioreactor chamber as a reference (163 ppm) for calibration. Elevated lactate is apparent for the 4T1 (highly metastatic) in contrast to the 4T07 and 67NR (non-metastatic) cell lines. Note that the small peak near the lactate frequency for the 67NR spectra was measured to be below the noise threshold by the jMRUI analysis software (v5.2).</p> "> Figure 4
<p>(<b>a</b>) An elevated lactate/pyruvate (Lac/Pyr) ratio was found for the 4T1 (highly metastatic) vs. 67NR (non-metastatic) cell lines, in contrast to (<b>b</b>) where the elevated redox ratio and (<b>c</b>) FAD intensity was found for 4T07 (metastatic-dormant) vs. both the 4T1 (highly metastatic) and 67NR (non-metastatic) cell lines. (<b>d</b>) NADH lifetimes trended higher for both 4T07 (metastatic-dormant) and 4T1 (highly metastatic) cell lines compared to the 67NR (non-metastatic) cell line. Three replicates were performed in parallel for each cell line, for a total of N = 9 data points per cell-line. * indicates statistical significance at <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, and ns stands for “not significant”.</p> ">
Abstract
:1. Introduction
2. Methods
2.1. Cell Culture in the Bioreactor
2.2. Fluorescence Lifetime Imaging Microscopy
2.3. Hyperpolarized Pyruvate MRS
2.4. Data and Statistical Analysis
3. Results
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- American Cancer Society, Breast Cancer Facts and Figures 2017–2018. Available online: www.cancer.org (accessed on 12 July 2024).
- Meltzer, A. Dormancy and breast cancer. J. Surg. Oncol. 1990, 43, 181–188. [Google Scholar] [CrossRef] [PubMed]
- Páez, D.; Labonte, M.J.; Bohanes, P.; Zhang, W.; Benhanim, L.; Ning, Y.; Wakatsuki, T.; Loupakis, F.; Lenz, H.J. Cancer dormancy: A model of early dissemination and late cancer recurrence. Clin. Cancer Res. 2012, 18, 645–653. [Google Scholar] [CrossRef]
- Brackstone, M.; Townson, J.L.; Chambers, A.F. Tumour dormancy in breast cancer: An update. Breast Cancer Res. 2007, 9, 208. [Google Scholar] [CrossRef] [PubMed]
- Cox, B.L.; Erickson-Bhatt, S.; Szulczewski, J.M.; Squirrell, J.M.; Ludwig, K.D.; Macdonald, E.B.; Swader, R.; Ponik, S.M.; Eliceiri, K.W.; Fain, S.B. A novel bioreactor for combined magnetic resonance spectroscopy and optical imaging of metabolism in 3D cell cultures. Magn. Reson. Med. 2019, 81, 3379–3391. [Google Scholar] [CrossRef]
- Warburg, O. On the origin of cancer cells. Science 1956, 123, 309–314. [Google Scholar] [CrossRef]
- Vander Heiden, M.G.; Cantley, L.C.; Thompson, C.B. Understanding the Warburg effect: The metabolic requirements of cell proliferation. Science 2009, 324, 1029–1033. [Google Scholar] [CrossRef] [PubMed]
- Dafni, H.; Ronen, S.M. Dynamic nuclear polarization in metabolic imaging of metastasis: Common sense, hypersense and compressed sensing. Cancer Biomark. 2010, 7, 189–199. [Google Scholar] [CrossRef]
- Brindle, K.M. Imaging metabolism with hyperpolarized (13)C-labeled cell substrates. J. Am. Chem. Soc. 2015, 137, 6418–6427. [Google Scholar] [CrossRef]
- Christensen, C.E.; Karlsson, M.; Winther, J.R.; Jensen, P.R.; Lerche, M.H. Non-invasive in-cell determination of free cytosolic [NAD+]/[NADH] ratios using hyperpolarized glucose show large variations in metabolic phenotypes. J. Biol. Chem. 2014, 289, 2344–2352. [Google Scholar] [CrossRef]
- Witney, T.H.; Kettunen, M.I.; Hu, D.E.; Gallagher, F.A.; Bohndiek, S.E.; Napolitano, R.; Brindle, K.M. Detecting Treatment Response in a Model of Human Breast Adenocarcinoma Using Hyperpolarised [1-13C]Pyruvate and [1,4-13C2]Fumarate. Br. J. Cancer 2010, 103, 1400–1406. [Google Scholar] [CrossRef]
- Harris, T.; Eliyahu, G.; Frydman, L.; Degani, H. Kinetics of Hyperpolarized 13C1-Pyruvate Transport and Metabolism in Living Human Breast Cancer Cells. Proc. Natl. Acad. Sci. USA 2009, 106, 18131–18136. [Google Scholar] [CrossRef] [PubMed]
- Macdonald, E.B.; Begovatz, P.; Barton, G.P.; Erickson-Bhatt, S.; Inman, D.R.; Cox, B.L.; Eliceiri, K.W.; Strigel, R.M.; Ponik, S.M.; Fain, S.B. Hyperpolarized 13C Magnetic Resonance Spectroscopic Imaging of Pyruvate Metabolism in Murine Breast Cancer Models of Different Metastatic Potential. Metabolites 2021, 11, 274. [Google Scholar] [CrossRef] [PubMed]
- Grashei, M.; Biechl, P.; Schilling, F.; Otto, A.M. Conversion of Hyperpolarized [1-13 C]Pyruvate in Breast Cancer Cells Depends on Their Malignancy, Metabolic Program and Nutrient Microenvironment. Cancers 2022, 14, 1845. [Google Scholar] [CrossRef] [PubMed]
- Sidani, M.; Wyckoff, J.; Xue, C.; Segall, J.E.; Condeelis, J. Probing the microenvironment of mammary tumors using multiphoton microscopy. J. Mammary Gland. Biol. Neoplasia 2006, 11, 151–163. [Google Scholar] [CrossRef] [PubMed]
- Provenzano, P.P.; Rueden, C.T.; Trier, S.M.; Yan, L.; Ponik, S.M.; Inman, D.R.; Keely, P.J.; Eliceiri, K.W. Nonlinear optical imaging and spectral-lifetime computational analysis of endogenous and exogenous fluorophores in breast cancer. J. Biomed. Opt. 2008, 13, 031220. [Google Scholar] [CrossRef]
- Conklin, M.W.; Provenzano, P.P.; Eliceiri, K.W.; Sullivan, R.; Keely, P.J. Fluorescence lifetime imaging of endogenous fluorophores in histopathology sections reveals differences between normal and tumor epithelium in carcinoma in situ of the breast. Cell Biochem. Biophys. 2009, 53, 145–157. [Google Scholar] [CrossRef]
- Li, L.Z.; Xu, H.N.; Ranji, M.; Nioka, S.; Chance, B. Mitochondrial redox imaging for cancer diagnostic and therapeutic studies. J. Innov. Opt. Health Sci. 2009, 2, 325–341. [Google Scholar] [CrossRef]
- Ostrander, J.H.; McMahon, C.M.; Lem, S.; Millon, S.R.; Brown, J.Q.; Seewaldt, V.L.; Ramanujam, N. Optical redox ratio differentiates breast cancer cell lines based on estrogen receptor status. Cancer Res. 2010, 70, 4759–4766. [Google Scholar] [CrossRef]
- Walsh, A.; Cook, R.S.; Rexer, B.; Arteaga, C.L.; Skala, M.C. Optical imaging of metabolism in HER2 overexpressing breast cancer cells. Biomed. Opt. Express 2012, 3, 75–85. [Google Scholar] [CrossRef]
- Xu, H.N.; Zheng, G.; Tchou, J.; Nioka, S.; Li, L.Z. Characterizing the metabolic heterogeneity in human breast cancer xenografts by 3D high resolution fluorescence imaging. Springerplus 2013, 2, 73. [Google Scholar] [CrossRef]
- Walsh, A.J.; Cook, R.S.; Sanders, M.E.; Aurisicchio, L.; Ciliberto, G.; Arteaga, C.L.; Skala, M.C. Quantitative optical imaging of primary tumor organoid metabolism predicts drug response in breast cancer. Cancer Res. 2014, 74, 5184–5194. [Google Scholar] [CrossRef] [PubMed]
- Cannon, T.M.; Shah, A.T.; Walsh, A.J.; Skala, M.C. High-throughput measurements of the optical redox ratio using a commercial microplate reader. J. Biomed. Opt. 2015, 20, 010503. [Google Scholar] [CrossRef] [PubMed]
- Sun, N.; Xu, H.N.; Luo, Q.; Li, L.Z. Potential Indexing of the Invasiveness of Breast Cancer Cells by Mitochondrial Redox Ratios. Adv. Exp. Med. Biol. 2016, 923, 121–127. [Google Scholar] [CrossRef] [PubMed]
- Alhallak, K.; Rebello, L.G.; Muldoon, T.J.; Quinn, K.P.; Rajaram, N. Optical redox ratio identifies metastatic potential-dependent changes in breast cancer cell metabolism. Biomed. Opt. Express. 2016, 7, 4364–4374. [Google Scholar] [CrossRef]
- Cannon, T.M.; Shah, A.T.; Skala, M.C. Autofluorescence imaging captures heterogeneous drug response differences between 2D and 3D breast cancer cultures. Biomed. Opt. Express 2017, 8, 1911–1925. [Google Scholar] [CrossRef]
- Hou, J.; Williams, J.; Botvinick, E.L.; Potma, E.O.; Tromberg, B.J. Visualization of Breast Cancer Metabolism Using Multimodal Nonlinear Optical Microscopy of Cellular Lipids and Redox State. Cancer Res. 2018, 78, 2503–2512. [Google Scholar] [CrossRef]
- Aslakson, C.J.; Miller, F.R. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res. 1992, 52, 1399–1405. [Google Scholar]
- Burkel, B.; Morris, B.A.; Ponik, S.M.; Riching, K.M.; Eliceiri, K.W.; Keely, P.J. Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo. J. Vis. Exp. 2016, 111, e53989. [Google Scholar] [CrossRef]
- Becker, W. Fluorescence lifetime imaging--techniques and applications. J. Microsc. 2012, 247, 119–136. [Google Scholar] [CrossRef]
- Ardenkjaer-Larsen, J.H.; Fridlund, B.; Gram, A.; Hansson, G.; Hansson, L.; Lerche, M.H.; Servin, R.; Thaning, M.; Golman, K. Increase in signal-to-noise ratio of >10,000 times in liquid-state NMR. Proc. Natl. Acad. Sci. USA 2003, 100, 10158–10163. [Google Scholar] [CrossRef]
- Rowland, I.J.; Peterson, E.T.; Gordon, J.W.; Fain, S.B. Hyperpolarized 13carbon MR. Curr. Pharm. Biotechnol. 2010, 11, 709–719. [Google Scholar] [CrossRef] [PubMed]
- Naressi, A.; Couturier, C.; Devos, J.M.; Janssen, M.; Mangeat, C.; de Beer, R.; Graveron-Demilly, D. Java-based graphical user interface for the MRUI quantitation package. MAGMA 2001, 12, 141–152. [Google Scholar] [CrossRef] [PubMed]
- Morris, B.A.; Burkel, B.; Ponik, S.M.; Fan, J.; Condeelis, J.S.; Aguirre-Ghiso, J.A.; Castracane, J.; Denu, J.M.; Keely, P.J. Collagen Matrix Density Drives the Metabolic Shift in Breast Cancer Cells. EBioMedicine 2016, 13, 146–156. [Google Scholar] [CrossRef] [PubMed]
- Xing, J.; Qi, L.; Liu, X.; Shi, G.; Sun, X.; Yang, Y. Roles of mitochondrial fusion and fission in breast cancer progression: A systematic review. World J. Surg. Oncol. 2022, 20, 331. [Google Scholar] [CrossRef] [PubMed]
- Zakic, T.; Kalezic, A.; Drvendzija, Z.; Udicki, M.; Ivkovic Kapicl, T.; Srdic Galic, B.; Korac, A.; Jankovic, A.; Korac, B. Breast Cancer: Mitochondria-Centered Metabolic Alterations in Tumor and Associated Adipose Tissue. Cells 2024, 13, 155. [Google Scholar] [CrossRef]
- Sabharwal, S.S.; Schumacker, P.T. Mitochondrial ROS in cancer: Initiators, amplifiers or an Achilles’ heel? Nat. Rev. Cancer 2014, 14, 709–721. [Google Scholar] [CrossRef]
NADH | FAD | |||||
---|---|---|---|---|---|---|
Cell Line | Lac/Pyr Ratio | Redox Ratio | Intensity | Lifetime (Tau Mean, ps) | Intensity | Lifetime (Tau Mean, ps) |
4T1 | 0.00293 | 0.299 | 613.58 | 757.8 | 250.56 | 751.8 |
4T1 | 0.00331 | 0.343 | 347.40 | 810.1 | 196.88 | 835.2 |
4T1 | 0.00662 | 0.340 | 416.14 | 844.2 | 238.73 | 975.8 |
Mean (SD) | 0.00429 (0.0020) | 0.327 (0.02) | 459.04 (138.2) | 804.0 (43.5) | 228.7 (28.2) | 854.3 (113.2) |
4T07 | 0.00333 | 0.424 | 432.05 | 1000.3 | 324.82 | 964.1 |
4T07 | 0.0011 | 0.344 | 520.70 | 671.9 | 288.71 | 910.8 |
4T07 | 0.00096 | 0.384 | 438.34 | 774.9 | 295.24 | 971.7 |
Mean (SD) | 0.00180 (0.0013) | 0.384 (0.04) | 463.70 (49.5) | 815.7 (168.0) | 302.9 (19.2) | 948.9 (33.2) |
67NR | 0 | 0.273 | 464.36 | 749.8 | 134.70 | 813.2 |
67NR | 0 | 0.263 | 414.45 | 339.5 | 160.55 | 396.9 |
67NR | 0 | 0.203 | 448.20 | 420.1 | 113.11 | 935.1 |
Mean (SD) | 0 (0) | 0.246 (0.04) | 442.33 (25.5) | 503.1 (217.4) | 136.1 (23.8) | 715.1 (282.2) |
Comparison HP-MRS Lac/Pyr Ratio vs. | Spearman Correlation Coefficient | p-Value |
---|---|---|
Redox Ratio | 0.63 | 0.071 + |
NADH Intensity | −0.25 | 0.51 |
NADH Lifetime | 0.86 | 0.0026 * |
FAD Intensity | 0.59 | 0.092 |
FAD Lifetime | 0.49 | 0.18 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Erickson-Bhatt, S.; Cox, B.L.; Macdonald, E.; Chacko, J.V.; Begovatz, P.; Keely, P.J.; Ponik, S.M.; Eliceiri, K.W.; Fain, S.B. Multi-Modal Investigation of Metabolism in Murine Breast Cancer Cell Lines Using Fluorescence Lifetime Microscopy and Hyperpolarized 13C-Pyruvate Magnetic Resonance Spectroscopy. Metabolites 2024, 14, 550. https://doi.org/10.3390/metabo14100550
Erickson-Bhatt S, Cox BL, Macdonald E, Chacko JV, Begovatz P, Keely PJ, Ponik SM, Eliceiri KW, Fain SB. Multi-Modal Investigation of Metabolism in Murine Breast Cancer Cell Lines Using Fluorescence Lifetime Microscopy and Hyperpolarized 13C-Pyruvate Magnetic Resonance Spectroscopy. Metabolites. 2024; 14(10):550. https://doi.org/10.3390/metabo14100550
Chicago/Turabian StyleErickson-Bhatt, Sarah, Benjamin L. Cox, Erin Macdonald, Jenu V. Chacko, Paul Begovatz, Patricia J. Keely, Suzanne M. Ponik, Kevin W. Eliceiri, and Sean B. Fain. 2024. "Multi-Modal Investigation of Metabolism in Murine Breast Cancer Cell Lines Using Fluorescence Lifetime Microscopy and Hyperpolarized 13C-Pyruvate Magnetic Resonance Spectroscopy" Metabolites 14, no. 10: 550. https://doi.org/10.3390/metabo14100550