Abstract
Background/Aim: Breast-cancer metastasis to the brain is an intractable disease. To discover improved therapy for this disease, we developed a precise non-invasively-imageable orthotopic nude-mouse model, using very-narrow-band-width laser fluorescence excitation. Materials and Methods: Female nu/nu nude mice, aged 4-8 weeks, were inoculated through the midline of the skull with triple-negative human MDA-MB-231 breast cancer cells (5×105) expressing red fluorescent protein (RFP). The mice were imaged with the Analytik Jena UVP Biospectrum Advanced at 520 nm excitation with peak emission at 605 nm. Results: Three weeks after injection of MDA-MB-231-RFP cells in the brain, non-invasive fluorescence images of the breast tumor growing on the brain were obtained. The images of the tumor were very bright, with well-defined margins with no detectable skin autofluorescence background. Images obtained at various angles showed that the extent of the tumor margins could be precisely measured. A skin flap over the skull confirmed that the tumor was growing on the surface of the brain which is a frequent occurrence in breast cancer. Conclusion: A precise orthotopic model of RFP-expressing breast-cancer metastasis to the brain was developed that could be non-invasively imaged with very-narrow-band-width laser excitation, resulting in an ultra-bright, ultra-low-background signal. The model will be useful in discovering improved therapeutics for this recalcitrant disease.
- Breast cancer
- brain metastasis
- red fluorescent protein
- orthotopic
- nude mouse
- non-invasive imaging
- laser
- excitation
- low background
Breast cancer metastasis to the brain is a recalcitrant disease (1, 2). Radiation and chemotherapy are generally ineffective (3). Especially recalcitrant is triple-negative breast cancer characterized by the loss of receptors for estrogen, progesterone, and human epidermal growth factor receptor-2 (HER-2) (4). Current mouse models of breast cancer are either subcutaneous or orthotopic in the mammary fat pad (5-9), neither of which addresses the problem of breast-cancer brain metastasis. Our laboratory pioneered in vivo imaging with fluorescent proteins beginning in the last century (10-18). In the present study, we used the human triple-negative breast cancer cell line MDA-MB-231 expressing high levels of red fluorescent protein (RFP) (19) and the high-power imaging capability of the Analytik Jena UVP Biospectrum Advanced system (Analytik Jena US LLC, Upland, CA, USA) to develop a precise model of brain-metastatic of breast cancer. The very-narrow-band-width laser excitation of the UVP Biospectrum Advanced gave an ultra- bright signal with no skin autofluorescence background, essential for non-invasive imaging. Previously, the field of small-animal fluorescence imaging was confounded and severely set back by misinformation stating that autofluorescence from the skin interfered with fluorescent-protein imaging (20, 21). The present report demonstrates that skin autofluorescence can be reduced to essentially zero with proper instrumentation comprising very-narrow-band-width laser excitation.
Materials and Methods
Cell line. The human triple negative breast cancer cell-line MDA-MB-231 was stably transfected with DsRed2 as previously described (13). The cells were cultured in DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum.
Mice. Nude nu/nu mice aged 4-6 weeks were used (AntiCancer Inc, San Diego, CA, USA). Mice were bred and maintained in a barrier facility using HEPA filters. All mouse studies were approved by the AntiCancer Institutional Animal Care and Use Committee. All experiments were conducted according to Animal Research: Reporting of In Vivo Experiments (ARRIVE) 2.0 criteria (20).
Model development. The number of inoculated MDA-MB-231 RFP cells was adjusted to 5×105 cells/5 μl using phosphate buffered saline (PBS) and Matrigel™ (Becton Dickinson, Bedford, MA, USA). For injection into the brain, a HAMILTON syringe with a 27 G needle was used. The needle was covered with a 1-10 μl tip for pipetting (Figure 1A). The distance between the needle top and tip’s end was set to 3 mm using a gum cap (Figure 1B and C). After making an incision of approximately 5 mm in the parietal skin, 5×105 cells/5 μl were injected into the right caudate nucleus at the following coordinates; 0.3 mm anterior to the bregma, 2.0 mm right lateral, and 3.0 mm below the surface of the dura over 3 min (Figure 1D-F).
Cell-injection procedure into the brain. A) HAMILTON syringe with a 27 G needle which was covered with a 1-10 μl tip for pipetting. B, C) The distance between the needle top and tip’s end was set at 3 mm using a gum cap. D) Opening of the skin of the head. E, F) 5×105 cells/5 μl were injected over 3 minutes into the right caudate nucleus at the coordinates 0.3 mm anterior to the bregma, 2.0 mm right lateral, and 3.0 mm below the surface of the dura.
Imaging. A UVP Biospectrum Advanced system (Analytik Jena US LLC, Upland, CA, USA) was used in the present study for imaging with excitation at 520 nm and peak emission at 605 nm.
Results
Non-invasive images from the Biospectrum Advanced from the RFP-expressing breast cancer in the brain showed a very bright and well delineated tumor with no skin autofluorescence background (Figure 2A). Images obtained from the right and left sides of the head showed that the tumor was on the brain (Figure 2B and C).
Non-invasive RFP fluorescence images of breast cancer growing in the brain. A) Front side. B) Right side. C) Left side.
A skin flap was made at the top of the head to image through the translucent skull which showed that the tumor was growing on the brain (Figure 3A and B).
RFP fluorescence images of the breast-cancer brain metastases acquired through the skull exposed by a skin flap. A) Bright-field image. B) Fluorescence image.
Discussion
There is an urgent need for effective therapeutics for breast-cancer brain metastasis, especially triple-negative breast cancer, which is a fatal disease. The present study demonstrates the establishment of a precise model of breast cancer on the surface of the brain, which reflects the clinical condition of brain-metastatic breast cancer. The precise orthotopic model was generated using breast-cancer cells expressing high levels of RFP (19) and the imaging capability of the Analytik Jena UVP Biospectrum Advanced system. The very narrow-band-width laser excitation enabled very bright images of breast-cancer brain metastasis with no skin autofluorescence background. Using this system, the tumor was located on the surface of the brain. Exceedingly rapid image acquisition (50 milli seconds) will enable rapid screening of novel therapies for improved efficacy of breast-cancer brain metastasis.
Acknowledgements
This article is dedicated to the memory of A. R. Moossa, MD, Sun Lee, MD, Gordon H. Sato, PhD, Professor Li Jiaxi, Masaki Kitajima, MD, Shigeo Yagi, PhD, Jack Geller, MD, Joseph R Bertino, MD, and J.A.R. Mead, PhD. The Robert M. Hoffman Foundation for Cancer Research provided funds for this study.
Footnotes
Authors’ Contributions
YK, YA, AW, NC, ST, and SG performed experiments. YK and RMH wrote the article. TT reviewed the article.
Conflicts of Interest
AW, NC, ST and SG are employees of Analytik Jena. YA, YT, NM, KO, SM and RMH are non-salaried associates of AntiCancer Inc. AntiCancer Inc. uses mouse models of cancer for contract research.
- Received June 14, 2023.
- Revision received October 19, 2023.
- Accepted October 20, 2023.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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