Near infrared-light treatment alters mitochondrial homeostasis to induce senescence in breast cancer cells

Abstract

The application of near infrared (NIR)-light to living systems has been suggested as a potential method to enhance tissue repair, decrease inflammation, and possibly mitigate cancer therapy-associated side effects. In this study, we examined the effect of exposing three cell lines: breast cancer (MCF7), non-cancer breast cells (MCF10A), and lung fibroblasts (IMR-90), to 734 nm NIR-light for 20 minutes per day for six days, and measuring changes in cellular senescence. Positive senescent populations were induced using doxorubicin. Flow cytometry was used to assess relative levels of senescence together with mitochondria-related variables. Exposure to NIR-light significantly increased the level of senescence in MCF7 cells (13.5%; P<0.01), with no observable effects on MCF10A or IMR-90 cell lines. NIR-induced senescence was associated with significant changes in mitochondria homeostasis, including raised ROS level (36.0%; P<0.05) and mitochondrial membrane potential (14.9%; P<0.05), with no changes in mitochondrial Ca ²⁺ . These results suggest that NIR-light exposure can significantly arrest the proliferation of breast cancer cells via inducing senescence, while leaving non-cancerous cell lines unaffected.

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This review resulted from the graduate-level course "How to Read and Evaluate Scientific Papers and Preprints" from the University of São Paulo, which aimed to provide students with the opportunity to review scientific articles, develop critical and constructive discussions on the endless frontiers of knowledge, and understand the peer review process.

General Remark:

This manuscript presents an interesting study investigating the impact of Near Infrared-Light on mitochondrial homeostasis, specifically its role in inducing senescence across various cell lines. The authors explored the effects of infrared irradiation on breast cancer cells (MCF7) and non-cancerous cells (MCF10A and IMR-90). Remarkably, they found that MCF7 cells exposed to 734 nm NIR-light for 20 minutes per day over a six-day period increased the level of senescence, a phenomenon absents in MCF10A/IMR-90 cells. The authors thoughtfully employed a pharmacological inducer, doxorubicin (Dox), to compare the observed senescence.

The authors' proposed mechanism suggests that NIR-induced senescence is linked to perturbations in mitochondrial homeostasis, notably characterized by alterations in reactive oxygen species (ROS) levels and mitochondrial membrane potential (MMP). The manuscript presents a compelling proposal, bolstered by a well-designed set of control experiments. In my assessment, several modifications could benefit the manuscript further, and these suggestions are outlined below:

Suggestions:

Results and methods:

- In the context of a study on photobiomodulation and photochemistry, it's customary to begin with a LED characterization (including λ_Max) and its impact on the temperature of the media. Therefore, Figure 2 could potentially be presented as Figure 1, illustrating the absence of temperature variations due to LED and establishing that the solely variable responsible for the reported effect is irradiation. Within the same Figure 2, it might be advisable to clarify the lack of effect on IMR-90 + IMR-90_LED (Figure 2B).

- It could be beneficial to combine Figures 1 and 3, allowing for a concise comparison and quantification of pharmacologically-induced senescence (using Dox) versus NIR-light induced senescence for each cell type. This fusion would entail presenting the results for MCF7, followed by a distinct graph for MCF10A, and another for IMR-90, akin to the style of Figures 4 and 5.

- The result showed in Figure 3 demonstrates that NIR-light doesn't induce senescence in MCF10A and IMR-90 cells, it could be valuable to investigate the impact on MMP, ROS, and Ca²⁺ levels in those cells. Such an exploration would confirm the author´s hypothesis and shed light on the potential application of NIR-light for more specific breast cancer treatment, or its implications for surrounding cells.

- In the pre-print, DCFDA was used to quantify ROS, revealing increased levels in senescent cells due to Dox and NIR-light. However, DCFDA is sensitive to not only ROS but also other types of radicals, and its final product (DCF) can be photo-excited, leading to non-zero quantification and possibly generation of singlet oxygen (¹O₂). To mitigate these concerns, the authors could consider an alternative, non-fluorescent technique for quantifying reactive species. For instance, markers of oxidative stress in DNA, lipids, or proteins could indirectly reflect ROS effects. A helpful resource for selecting an appropriate probe is the review paper by Murphy et al., 2022. The authors can find a summary which offers recommendations for ROS measurement, available in Figure 1 of the paper.

Murphy, M.P., Bayir, H., Belousov, V. et al. Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. Nat Metab 4, 651–662 (2022). https://doi.org/10.1038/s42255-022-00591-z.

- In the experiment involving Rhod2 to quantify Ca²⁺ levels and correlate them with the senescence induced by Dox and NIR-light, the absence of significant changes in Ca²⁺ levels after NIR exposure in both Dox-treated and Dox-untreated populations suggests an interesting avenue for further exploration. To enhance the control and insights, an alternative approach could be implemented. For instance, elevating cytoplasmic Ca²⁺ (such as through endoplasmic reticulum release) or chelating Ca²⁺ (to reduce cytoplasmic levels) could provide additional context. This control would help confirm whether the response is influenced by Ca²⁺ itself. Thapsigargin or BAPTA-AM could be employed to elegantly perform this experiment.

- Regarding the measurement of mitochondrial membrane potential, MMP, using TMRE dye, it was shown a significant increase in MMP after chronic NIR-light treatment, in Dox-untreated and Dox-treated populations. The experiments were performed using the TMRE stain in live-intact cells, which is crucial at the moment to understand the real dynamic. To warrant appropriate subsequent interpretations the authors should take into account that:

1. TMRE is a cationic fluorescent probe, and it's important to note that these probes tend to lack quantitative accuracy unless calibrated, preferably with isolated mitochondria or permeabilized cells (possibly using digitonin). The use of cationic compounds may influence membrane transport, especially in intact cells.

2. It is worth considering the potential impact of multi-drug resistant transporters, which can vary between cells and influence indicator accumulation. These transporters might lead to different accumulation levels based on their presence and functionality.

3. Cells have both a plasma membrane potential and an MMP, distinct from each other. Plasma membrane potential can influence measurements, and excessive probe concentration could affect transport and oxidative phosphorylation, possibly leading to alterations unrelated to the treatments (Dox and NIR-light).

4. Flow Cytometry might not be the optimal technique for MMP quantification, particularly with intact cells. The methodology requires careful control and consideration. Cytometry should be compared with microscopy and selecting between quenching or non-quenching modes which could yield different implications. To enhance the reliability, quantifying MMP in permeabilized cells using a different technique could be beneficial. For this purpose, I'm sharing a recommended paper that provides insights on MMP measurement by Kowaltowski, et al., (2002). The paper includes a relatively straightforward and cost-effective protocol with methods for probe calibration in permeabilized cells:

Kowaltowski AJ, Cosso RG, Campos CB, Fiskum G. Effect of Bcl-2 overexpression on mitochondrial structure and function. J Biol Chem. 2002 Nov 8;277(45):42802-7. doi: 10.1074/jbc.M207765200. Epub 2002 Aug 30. PMID: 12207028.

Conclusions:

- In the conclusions, it is possible to be more precise and specific. When the authors says: "Our study demonstrates that a selective effect of low-dose PBM at NIR wavelength induces senescence in a cancer cell line without affecting non-cancer cell lines.". MMP, Ca²⁺ and ROS quantifications were not performed for non-cancer cell lines. The pre-print does not provide evidence that PBM or NIR-light have no effect in non-cancer cells. It shows that these treatments do not induce senescence.

Bibliography:

The reference of Golovynska et al., 2023 has a format mistake:

Change

Golovynska, I., Golovynskyi, S., & Qu, J., 2023. Comparing the Impact of <scp>NIR</scp> , Visible and <scp>UV</scp> Light on <scp>ROS</scp> Upregulation via Photoacceptors of Mitochondrial Complexes in Normal, Immune and Cancer Cells. Photochemistry and Photobiology, 99(1), 106–119. https://doi.org/10.1111/php.13661

for

Golovynska I, Golovynskyi S, Qu J. Comparing the Impact of NIR, Visible and UV Light on ROS Upregulation via Photoacceptors of Mitochondrial Complexes in Normal, Immune and Cancer Cells. Photochem Photobiol. 2023 Jan;99(1):106-119. doi: 10.1111/php.13661. Epub 2022 Jul 4. PMID: 35689798.

Final remarks:

- The methodology predominantly relies on Flow Cytometry. To comprehensively uncover the mechanisms underlying NIR-light and senescence, exploring alternative methods that could provide additional insights.

- The concept of investigating mitochondrial homeostasis and photobiomodulation is impressive and holds significant potential for photomedical applications and photobiology. Your research not only enhances our understanding of cellular responses to stimuli and environmental factors but also has the potential to advance the battle against cancer – a primary objective of this study.

- The pre-print exemplifies exceptional research that integrates a broad spectrum of literature, including papers, books, and reviews.

- If the paper has not yet to be submitted to a journal, the authors could consider the Journal of Photochemistry and Photobiology. This journal appears to be a fitting platform for publishing your impactful findings:

https://www.sciencedirect.com/journal/journal-of-photochemistry-and-photobiology

Competing interests:

The author declares that they have no competing interests.

Competing interests

The author declares that they have no competing interests.

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Near infrared-light treatment alters mitochondrial homeostasis to induce senescence in breast cancer cells

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