Blue light irradiation inhibits the activation of cancer-associated macrophages in colon cancer

Blue light irradiation inhibits the activation of cancer-associated macrophages in colon cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Blue light irradiation inhibits the activation of cancer-associated macrophages in colon cancer Toshiaki Yoshimoto, Masaaki Nishi, Shohei Okikawa, Kozo Yoshikawa, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3951809/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Recent studies have shown that blue light-emitting diode (LED) light has anti-tumor effects, suggesting the possibility of using visible light in cancer therapy. However, the effects of blue light irradiation on cells in the tumor microenvironment, including tumor-associated macrophages (TAMs), are unknown. Here, THP-1 cells were cultured in the conditioned medium (CM) of HCT-116 cells to prepare TAMs. TAMs were divided into LED-irradiated and control groups. Then, the effects of blue LED irradiation on TAM activation were examined. Expression levels of M2 macrophage markers CD163 and CD206 expression were significantly decreased in LED-irradiated TAMs compared with the control group. While control TAM-CM could induce HCT-116 cell migration, these effects were not observed in cells cultured in TAM-CM with LED irradiation. Vascular endothelial growth factor (VEGF) secretion was significantly suppressed in LED-exposed TAMs. PD-L1 expression was upregulated in HCT-116 cells cultured with TAM-CM but attenuated in cells cultured with LED-irradiated TAM-CM. In an in vivo model, protein expression levels of F4/80 and CD163, which are TAM markers, were reduced in the LED-exposed group. These results indicate that blue LED light may have an inhibitory effect on TAMs, as well as anti-tumor effects on colon cancer cells. Blue light colorectal cancer light-emitting diode Opsin3 tumor-associated macrophage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Irradiating cells and living organisms with light-emitting diodes (LEDs) with specific wavelengths of light has a variety of effects. Photodynamic therapy (PDT), which combines light and photosensitizers, is attracting attention as a new minimally invasive alternative to chemoradiotherapy. PDT is being clinically applied to treat esophageal cancer, lung cancer, brain tumors, and other cancer types. Several in vitro and in vivo reports have also demonstrated the efficacy of PDT for colorectal cancer (1,2). However, recent studies have demonstrated that using blue LED light alone is effective for suppressing the growth of some cancer cell types. Specific mechanisms for the phototoxic and anti-proliferative effects of blue light have been reported, including the induction of autophagy (3), necroptosis (4), apoptosis (5), and cell cycle arrest (6). Blue light irradiation can also result in the production of reactive oxygen species and DNA damage in colon cancer cells (7). In a previous report, we showed that using 465 nm blue light irradiation (30 mW/cm 2 , 30 minutes) could inhibit colon cancer cell growth via inducing autophagy (8). Furthermore, we revealed that the blue photoreceptor Opsin3 (Opn3), which is expressed in colon cancer cells, can promote the cytotoxic effects of blue light. The opsin family of proteins is composed of G-protein-coupled receptors (GPCRs) that are expressed on photoreceptor cells in the retina. Opn3 regulates GPCR signaling and acts as a receptor for blue light (11). Additionally, Opn3 activates the G i/o subtype of G proteins with light at a wavelength of 460 to 470 nm, inhibits adenylate cyclase, and decreases cyclic adenosine monophosphate (cAMP) (11, 12). Recent studies have shown that Opn3 is also widely expressed in organs unrelated to vision, such as the respiratory epithelium, liver, kidney, and heart, and is called non-visual opsin (9, 10). One report indicates that Opn3 acts as a photoreceptor in malignant melanocytes and contributes to phototherapy (13), and our findings indicate that Opn3 is a potential target for phototherapy as a photoreceptor in colon cancer cells (8). Regulation of the tumor microenvironment (TME), which is composed of various cell types including fibroblasts, immune cells, and tumor cells, is crucial for cancer therapy. We recently reported that irradiation of cancer-associated fibroblasts (CAFs) with blue light can decrease CAF activity and interleukin (IL)-6 secretion, which can support decreased tumor malignancy of colon cancer cells (14). However, the effect of blue light on the TME, particularly tumor-associated macrophages (TAMs), is unknown. In this study, we focused on TAMs, mainly M2 macrophages, which are involved in tumor growth and metastasis through the secretion of cytokines, such as VEGF and PD-L1. We investigated the impact of blue LED irradiation on TAMs to examine its effects on the TME. 2. Materials and methods 2.1 Cell culture The human colon cancer cell line HCT-116 (KAC Co., Ltd., Kyoto, Japan, Cat. No.: EC91091005-F0) was used for in vitro and in vivo experiments. The cell line was confirmed to be negative for mycoplasma. Cells were maintained and cultured in accordance with international guidelines for proper cell culture practices. Cells were cultured in RPMI 1640 medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in a 37°C humidified incubator with 5% CO 2 . 2.2 Preparation of conditioned medium (CM) and TAMs To obtain CM, HCT-116 cells were cultured in 100 mm culture dishes until 80% confluent. The cells were washed twice with pre-warmed phosphate-buffered saline (PBS) and incubated in fresh medium without FBS. After incubation at 37°C for 48 hours, the supernatant was collected, centrifuged at 800´g for 5 minutes at 22°C, and filtered through a 0.2-µm sterile filter. To obtain TAMs, M0 macrophages were treated with CM derived from HCT-116 cells. CM was added to the culture medium at a 1:1 ratio and cells were incubated at 37°C for 48 hours. To obtain TAM-CM, the TAMs were treated with blue LED light (465 nm, 30 mW/cm 2 , 30 minutes) (irradiation group) or left untreated. The medium was changed once and the supernatant (TAM-CM) was collected. A schematic for these processes is shown in Figure 1. 2.3 Animals Four-week-old female BALB/c mice were purchased from Charles River Japan, Inc. (Kanagawa, Japan). Animals were caged under controlled conditions and provided water and standard laboratory rations for at least seven days prior to use. Before and after surgery, the animals had free access to tap water and food. The experiments and procedures in this study were approved by the Animal Welfare and Use Committee of the University of Tokushima (Approval No. T2021-42). All animal experiments were performed in accordance with the U.K. Animals (Scientific Procedures) Act 1986 and associated guidelines, the EU Directive 2010/63/EU for animal experiments, and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). 2.4 Orthotopic model and LED irradiation CT-29 cells were harvested with 1 mM ethylenediaminetetraacetic acid (EDTA) in PBS, washed three times with PBS, and resuspended at a density of 1×10 7 cells/mL in PBS containing 500 mg/mL of Matrigel (Becton Dickinson Labware, Bedford, MA, USA). Animals (n=8) were anesthetized with isoflurane (3% to 4% for induction and 1% to 2% for maintenance). A 7-mm incision was made in the anterior rectal wall to prevent colonic obstruction caused by rectal tumor progression. CT-29 cells (1×10 6 ) were injected into the submucosa of the posterior wall of the rectum using a 29-gauge needle. Seven days after cell implantation, the mice developed rectal cancer. The mice were randomized into two groups (n=4 mice per group) and treated as follows: (i) the LED irradiation group (465 nm, 30 mW/cm 2 , 30 minutes, once) and (ii) the control group (untreated). The mice were sacrificed by cervical spinal cord dissection at 2 weeks after cell implantation. A humane endpoint was set at the stage when the tumor size increased by more than 10% of the mice’s body weight, but this did not apply to any of the mice. A 465-nm blue LED (NCSB119, NICHIA Corporation, Tokushima, Japan) was used as a light source. An LED irradiation device (Department of Electrical and Electronic Engineering, Faculty of Engineering, The University of Tokushima, Tokushima, Japan) was used for irradiation experiments. 2.5 Immunohistochemistry (IHC) The tissues resected from the orthotopic model or cultured cells were fixed in 10% formalin (Wako 066-03847) for 24 hours at room temperature, paraffin-embedded, and sliced into 4-μm-thick sections. The samples were blocked in 3% BSA (MACS ®︎ BSA Stock Solution 130-091-376) for 1 hour at room temperature. An anti-F4/80 antibody (15361-1-AP; Proteintech, Rosemont, IL, USA), anti-CD163 antibody (SAB2700986; Sigma-Aldrich), and anti-PD-L1 antibody (17952-1-AP; Proteintech) were used for IHC staining following the manufacturer’s instructions. The fluorescence area was measured using Image J software (ver. 1.53, National Institutes of Health, Bethesda, MD, USA). 2.6 Quantitative real-time PCR (qRT-PCR) analysis Total RNA was isolated from samples using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Tokyo, Japan). Then, qRT-PCR was performed using a 7500 real-time PCR system, TaqMan gene expression assay on demand, and TaqMan Universal Master Mix (Applied Biosystems). The following TaqMan assays were used: Opn3 (Hs00173892_m1), CD163 (Hs00174705_m1), CD206 (Hs00267207_m1), and PD-L1 (Hs00204257_m1). GAPDH (4326317E) was used as an internal control for mRNA expression. The thermal cycler conditions were as follows: 2 minutes at 50°C, 10 minutes at 95°C, and then 40 cycles of 15 s at 95°C and 1 minute at 60°C. Amplification data were analyzed using the Prism 7500 Sequence Detection System ver. 1.3.1 (Applied Biosystems). qRT-PCR was performed using a Step One Plus Real-Time PCR System (Applied Biosystems). The relative target transcript expression levels were assessed and normalized to the expression levels of GAPDH mRNA as an internal control using the 2 -ΔΔCq method (15). 2.7 Migration and scratch assays For migration assays, Transwell inserts (Corning, Corning, NY, USA) with a pore size of 8 µm were used. CT-116 cells (2´10 4 ) were seeded into the upper chamber. After cell attachment, the medium was discarded and fresh medium containing 1% FBS was added to the upper chamber. CM containing 10% FBS was added to the lower chamber. After incubation for 24 hours, cells at the bottom of the Transwell inserts were fixed in 4% paraformaldehyde, then stained with 0.2% crystal violet. The stained cells in three random microscopic fields (100×) were counted. For scratch assays, HCT-116 cells were seeded at a density of 2×10 4 cells/well in 6-well plates. After the cells became 100% confluent, a plastic pipette tip was used to scrape the center of the well, creating a 1-mm-wide scratch. The medium was discarded and fresh DMEM containing 1% FBS was added, followed by CM at a 1:1 ratio. The final FBS concentration was 0.5%. The cells were then incubated at 37°C for 24 hours. Images of the wound were taken using a phase-contrast microscope (40× magnification; DP22-CU; Olympus Corporation) at 0 and 24 hours after scratching. 2.8 Statistical analysis All statistical analyses were performed using Stat View version 5.0 software (SAS Institute, Cary, NC, USA). The Mann-Whitney U -test and Wilcoxon signed-rank test were used for statistical comparisons. P -values less than 0.05 were considered statistically significant. 3. Results 3.1 Opn3 expression in M0 macrophages and TAMs We examined Opn3 mRNA expression levels in TAMs generated by culturing M0 macrophages with the CM of HCT-116 colon cancer cells, which were then exposed to LED or left untreated. LED-irradiated TAMs showed higher Opn3 expression levels compared with the untreated control group ( P <0.05; Fig. 2). 3.2 Blue LED irradiation suppressed the polarization of macrophages The mRNA expression levels of M2 macrophage markers CD163 and CD206 were significantly upregulated in TAMs (M0 macrophages cultured in HCT-116 cell CM) compared with those of M0 macrophages ( P <0.05; Fig. 3). In contrast, CD163 and CD206 mRNA expression levels were decreased in TAMs treated with blue LED light irradiation, suggesting that blue LED light could inhibit the polarity change to M2 macrophages ( P <0.05; Fig. 3). 3.3 Migration and scratch assays for HCT-116 cells cultured in TAM-CM HCT-116 cells were cultured in TAM-CM, then migration (Fig. 4A) and scratch assays (Fig. 4B) were performed. Culturing the cells in TAM-CM significantly promoted the migration of HCT-116 cells ( P <0.05). However, TAM-CM derived from TAMs irradiated with blue light did not promote the migration of HCT-116 cells ( P <0.05). 3.4 Blue LED irradiation suppressed TAM secretion of VEGF As shown in Figure 5, the levels of VEGF secretion were significantly higher in TAMs compared with M0 macrophages ( P <0.05). However, in LED-irradiated TAMs, VEGF secretion was significantly suppressed ( P <0.05). 3.5 PD-L1 expression in HCT-116 cells cultured in TAM-CM In HCT-116 cells cultured with TAM-CM, PD-L1 mRNA expression levels were upregulated ( P <0.05; Fig. 6). However, the degree of PD-L1 mRNA expression induction was attenuated in HCT-116 cells cultured with TAM-CM and irradiated with blue LED ( P <0.05; Fig. 6). 3.6 Effects of blue LED irradiation in an in vivo model Colon cancer tumor growth in a mouse model was significantly suppressed by blue LED irradiation ( P <0.05; Fig. 7A), as indicated by reduced tumor size. PD-L1 expression levels were downregulated in tumors exposed to blue LED irradiation (Fig. 7B). Furthermore, IHC staining showed that tumors irradiated with blue LED showed fewer cells positive for F4/80 and CD163 protein compared with the control samples (Fig. 8). The CD163-positive cell areas in five randomly selected fields of view were compared, with a significant reduction observed in the irradiated group compared with the control group ( P <0.05). 4. Discussion Recent studies have demonstrated that cells in the TME, including fibroblasts and immune cells, are extremely important for the efficacy of anti-cancer therapies. We previously reported that blue LED light can act in an inhibitory manner on not only tumor cells, but also on CAFs, resulting in reduced tumor malignancy (14). In the present study, we examined the impact of blue LED on the TME by investigating its effects on TAMs. Our results showed that blue light irradiation could decrease the expression of M2 macrophage markers CD163 and CD206, suggesting that it could suppress macrophage polarization to the M2 phenotype. In addition, migration assays showed that HCT-116 cell migration was promoted in the presence of CM from non-LED-irradiated TAMs but was significantly reduced with CM from LED-irradiated TAMs. We previously reported that VEGF secretion by TAMs plays a significant role in tumor malignancy in hepatocellular carcinoma (16). In the present study, VEGF secretion was observed in TAMs without LED irradiation, but this was significantly suppressed in TAMs irradiated with LED. In addition, HCT-116 cells cultured in TAM-CM showed elevated PD-L1 expression levels, whereas cells cultured with LED-irradiated TAM-CM showed attenuated induction of PD-L1 expression. The transcription factor STAT3 can reportedly regulate expression of the M2 macrophage markers CD163 and CD206 (17). Additionally, STAT3 has been implicated in elevated tumor malignancy and PD-L1 expression through VEGF in tumor cells (18). Therefore, STAT3 may play a crucial role in the mechanism through which blue light irradiation can affect both TAMs and tumor cells. Recently, PDT targeting of TAMs has been reported (19). We have also reported the effect of blue LEDs on CAFs, suggesting the future possibility of phototherapy targeting the TME. Furthermore, Opn3 is expressed in TAMs, suggesting the possibility that blue LEDs may suppress TAMs via photoreceptors. In our previous experiments, similar anti-tumor effects were observed with daily 5-minute irradiation and a single 30-minute irradiation (8). Therefore, we used a 30-minute irradiation in the current study. Subsequent investigations should focus on evaluating the impact of short-duration fractionated irradiation on TAMs, as well as the effects of frequent 30-minute irradiation sessions. The major limitation of this study is that the significance of Opn3 expression in tumors and macrophages has not been fully investigated. Future studies involving Opn3 expression knocked down in macrophages or with Opn3 knockout mice are required. Although further studies are required to fully elucidate the mechanism of action of blue LED irradiation on tumors and the TME, this study demonstrated significant effects of blue LED irradiation on colon cancer cells and TAMs. In summary, blue light could suppress macrophage polarization to TAMs and decreased the levels of VEGF secretion. Additionally, blue light attenuated TAM-mediated tumor malignancy and decreased PD-L1 expression levels. The blue light receptor Opn3 is expressed in TAMs, and macrophage polarization and activity were reduced by blue light irradiation. Taken together, these findings suggest that blue light may regulate TAMs in tumors and the TME (Fig. 9). Abbreviations LED: light-emitting diode TAM: tumor-associated macrophage CM: conditioned medium Opn3: Opsin 3 cAMP: cyclic adenosine monophosphate GPCR: G protein-coupled receptor PDT: photodynamic therapy Declarations Funding This study was partly supported by the Grant-in-Aid for Scientific Research (Grant no. 22K16465). This study was also funded by Taiho Pharmaceutical Co., Ltd. (Tokyo, Japan). Availability of data and materials The datasets generated in the present study are available from the corresponding author upon reasonable request. Authors' contributions TY, SO, KY, MN, TT and MS conceived and designed the study. TY, SO, HK and YW performed the experiments and collected the data. TY, SO, TNa, TNo and CT analyzed and interpreted the data. TY and SO wrote the paper. TT, TNa, TNo and MN reviewed the data and information and edited the manuscript. KY and MS revised the paper critically for important intellectual content. KY, TY and TT confirmed the authenticity of all the raw data. All authors agreed to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript. Ethics approval and consent to participate The research was conducted in compliance with the Division for Animal Research Resources, Institute of Health Biosciences, University of Tokushima, Japan. The experiments and procedures were approved by the Animal Care and Use Committee of the University of Tokushima, Japan. Patient consent for publication Not applicable. Competing interests The authors declare no competing interests. Conflicts of interest None. References Hatakeyama T, Murayama Y, Komatsu S, et al. Efficacy of 5-aminolevulinic acid-mediated photodynamic therapy using light-emitting diodes in human colon cancer cells. Oncology reports. 2013;29(3):911-6. Hodgkinson N, Kruger CA, Abrahamse H. Targeted photodynamic therapy as potential treatment modality for the eradication of colon cancer and colon cancer stem cells. Tumor Biology. 2017;39(10):1010428317734691. Oh P-S, Hwang H, Jeong H-S, et al. Blue light emitting diode induces apoptosis in lymphoid cells by stimulating autophagy. The international journal of biochemistry & cell biology. 2016;70:13-22. del Olmo-Aguado S, Núñez-Álvarez C, Osborne NN. Blue light action on mitochondria leads to cell death by necroptosis. Neurochemical research. 2016;41(9):2324-35. Oh P-S, Na KS, Hwang H, et al. Effect of blue light emitting diodes on melanoma cells: involvement of apoptotic signaling. Journal of Photochemistry and Photobiology B: Biology. 2015;142:197-203. Sparsa A, Faucher K, Sol V, et al. Blue light is phototoxic for B16F10 murine melanoma and bovine endothelial cell lines by direct cytocidal effect. Anticancer research. 2010;30(1):143-7. Yan G, Zhang L, Feng C, et al. Blue light emitting diodes irradiation causes cell death in colorectal cancer by inducing ROS production and DNA damage. The international journal of biochemistry & cell biology. 2018;103:81-8. Yoshimoto T, Morine Y, Takasu C, et al. Blue light‐emitting diodes induce autophagy in colon cancer cells by Opsin 3. Annals of gastroenterological surgery. 2018;2(2):154-61. Koyanagi M, Terakita A. Diversity of animal opsin-based pigments and their optogenetic potential. Biochimica et Biophysica Acta (BBA)-Bioenergetics. 2014;1837(5):710-6. Jiao J, Hong S, Zhang J, et al. Opsin3 sensitizes hepatocellular carcinoma cells to 5-fluorouracil treatment by regulating the apoptotic pathway. Cancer letters. 2012;320(1):96-103. Koyanagi M, Takada E, Nagata T, et al. Homologs of vertebrate Opn3 potentially serve as a light sensor in nonphotoreceptive tissue. Proceedings of the National Academy of Sciences. 2013;110(13):4998-5003. Terakita A, Nagata T. Functional properties of opsins and their contribution to light-sensing physiology. Zoological science. 2014;31(10):653-9. de Assis L, Moraes M, da Silveira Cruz-Machado S, et al. The effect of white light on normal and malignant murine melanocytes: a link between opsins, clock genes, and melanogenesis. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 2016;1863(6):1119-33. Yoshimoto T, Shimada M, Tokunaga T, et al. Blue light irradiation inhibits the growth of colon cancer and activation of cancer associated fibroblasts. Oncology Reports. 2022 May 1;47(5):1-9. Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real‑time quantitative PCR and the 2(‑Delta Delta C(T)) method. Methods 25: 402‑408, 2001. Okikawa S, Morine Y, Saito Y, et al. Inhibition of the VEGF signaling pathway attenuates tumor‑associated macrophage activity in liver cancer. Oncology reports. 2022 Apr 1;47(4):1-1. Gharibi, T., Babaloo, Z., Hosseini, A., et al. Targeting STAT3 in cancer and autoimmune diseases. European journal of pharmacology, 2020, 878: 173107. De Beule, N., De Veirman, K., Maes, K., et al. Tumour‐associated macrophage‐mediated survival of myeloma cells through STAT3 activation. The Journal of Pathology, 2017, 241.4: 534-546. Soyama, T., Sakuragi, A., Oishi, D., et al. Photodynamic therapy exploiting the anti-tumor activity of mannose-conjugated chlorin e6 reduced M2-like tumor-associated macrophages. Translational oncology, 2021, 14.2: 101005. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 30 Mar, 2024 Reviews received at journal 28 Mar, 2024 Reviewers agreed at journal 27 Feb, 2024 Reviews received at journal 27 Feb, 2024 Reviewers agreed at journal 27 Feb, 2024 Reviewers invited by journal 23 Feb, 2024 Editor invited by journal 15 Feb, 2024 Editor assigned by journal 13 Feb, 2024 Submission checks completed at journal 13 Feb, 2024 First submitted to journal 12 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3951809","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":272763642,"identity":"3aeb0d9c-2aab-407e-a211-bf7a8e3e102f","order_by":0,"name":"Toshiaki Yoshimoto","email":"data:image/png;base64,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","orcid":"","institution":"Tokushima University Graduate School","correspondingAuthor":true,"prefix":"","firstName":"Toshiaki","middleName":"","lastName":"Yoshimoto","suffix":""},{"id":272763643,"identity":"60e61b77-c070-4c49-ad4a-f087e9746579","order_by":1,"name":"Masaaki Nishi","email":"","orcid":"","institution":"Tokushima University Graduate School","correspondingAuthor":false,"prefix":"","firstName":"Masaaki","middleName":"","lastName":"Nishi","suffix":""},{"id":272763644,"identity":"f6924d54-9278-42d3-9631-a875f0159a64","order_by":2,"name":"Shohei Okikawa","email":"","orcid":"","institution":"Tokushima University Graduate School","correspondingAuthor":false,"prefix":"","firstName":"Shohei","middleName":"","lastName":"Okikawa","suffix":""},{"id":272763645,"identity":"10175183-f04b-48e1-8345-2091ddcf8f85","order_by":3,"name":"Kozo Yoshikawa","email":"","orcid":"","institution":"Tokushima University Graduate School","correspondingAuthor":false,"prefix":"","firstName":"Kozo","middleName":"","lastName":"Yoshikawa","suffix":""},{"id":272763646,"identity":"fcedbac3-5a25-419b-ad92-e117c18726ca","order_by":4,"name":"Takuya Tokunaga","email":"","orcid":"","institution":"Tokushima University Graduate School","correspondingAuthor":false,"prefix":"","firstName":"Takuya","middleName":"","lastName":"Tokunaga","suffix":""},{"id":272763647,"identity":"f7fcf29c-b0d6-4334-b8dc-f07af35f5f17","order_by":5,"name":"Toshihiro Nakao","email":"","orcid":"","institution":"Tokushima University Graduate School","correspondingAuthor":false,"prefix":"","firstName":"Toshihiro","middleName":"","lastName":"Nakao","suffix":""},{"id":272763648,"identity":"f9b13adf-e6f3-4ea5-a7f9-0e39c22901fd","order_by":6,"name":"Chie Takasu","email":"","orcid":"","institution":"Tokushima University Graduate School","correspondingAuthor":false,"prefix":"","firstName":"Chie","middleName":"","lastName":"Takasu","suffix":""},{"id":272763649,"identity":"ac8f07df-6a23-4ca5-95ad-b4b8e6e9f014","order_by":7,"name":"Hideya Kashihara","email":"","orcid":"","institution":"Tokushima University Graduate School","correspondingAuthor":false,"prefix":"","firstName":"Hideya","middleName":"","lastName":"Kashihara","suffix":""},{"id":272763650,"identity":"1223ed54-161b-4ce8-8c90-b0b8dfb076d3","order_by":8,"name":"Yuma Wada","email":"","orcid":"","institution":"Tokushima University Graduate School","correspondingAuthor":false,"prefix":"","firstName":"Yuma","middleName":"","lastName":"Wada","suffix":""},{"id":272763651,"identity":"08aade63-23e5-41a4-af15-dcedc00ea916","order_by":9,"name":"Takayuki Noma","email":"","orcid":"","institution":"Tokushima University Graduate School","correspondingAuthor":false,"prefix":"","firstName":"Takayuki","middleName":"","lastName":"Noma","suffix":""},{"id":272763652,"identity":"4cd4dc4c-57e0-4a60-94bb-6e35f65f918f","order_by":10,"name":"Mitsuo Shimada","email":"","orcid":"","institution":"Tokushima University Graduate School","correspondingAuthor":false,"prefix":"","firstName":"Mitsuo","middleName":"","lastName":"Shimada","suffix":""}],"badges":[],"createdAt":"2024-02-12 19:46:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3951809/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3951809/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51193585,"identity":"c593bd90-b4c3-47fd-87fa-77d4a7f5daa8","added_by":"auto","created_at":"2024-02-15 17:47:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":879495,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProcedures for the generation of tumor-associated macrophages (TAMs), conditioned medium (CM), and irradiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM0 macrophages (from PMA-treated THP-1 cells) were cultured in the CM of HCT-116 colon cancer cells to generate TAMs. The TAMs were either untreated (control group) or irradiated as indicated with blue light. The medium was changed once, and the supernatant (TAM-CM) was collected. The TAM-CM from the irradiated and control groups was used for subsequent experiments.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3951809/v1/c0951b0cfc4bccac986c6779.jpg"},{"id":51193586,"identity":"ccd82435-a531-4b4d-9a54-d6c09336d6b0","added_by":"auto","created_at":"2024-02-15 17:47:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":302264,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOpsin 3 (Opn3) mRNA expression levels in M0 macrophages and tumor-associated macrophages (TAMs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOpn3 mRNA expression levels were examined in TAMs with or without blue LED irradiation.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3951809/v1/c32c5906b273a091a002def6.jpg"},{"id":51193588,"identity":"4f606874-d7a1-4d7f-978c-9a03e48b24a4","added_by":"auto","created_at":"2024-02-15 17:47:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":754793,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects on blue LED irradiation on tumor-associated macrophage (TAM) polarization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression levels of CD163 and CD206 were examined in M0 macrophages, TAMs, and blue LED-irradiated TAMs.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3951809/v1/217fdb927ffdead9e53fa26a.jpg"},{"id":51193591,"identity":"56d2f8bf-9af6-4398-b63e-89e204a9a99c","added_by":"auto","created_at":"2024-02-15 17:47:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1736400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMigration and scratchassays for HCT-116 cells cultured in tumor-associated macrophage conditioned medium (TAM-CM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Migration assay results for HCT-116 cells cultured in TAM-CM from untreated or blue LED-irradiated TAMs. (B) Scratch assay results for HCT-116 cells cultured in TAM-CM from untreated or blue LED-irradiated TAMs.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3951809/v1/7f7058b0f4ea6d214f22a570.jpg"},{"id":51193592,"identity":"0a4053f3-214c-4aec-9d04-af7614722424","added_by":"auto","created_at":"2024-02-15 17:47:17","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":436239,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of blue LED irradiation on vascular endothelial growth factor (VEGF) secretion by tumor-associated macrophages (TAMs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLevels of VEGF secretion were measured in M0 macrophages, TAMs, and blue LED-irradiated TAMs.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3951809/v1/69a761829b0e65c437ce99a9.jpg"},{"id":51193589,"identity":"be474be9-a645-4f35-94f2-1a5477cdec77","added_by":"auto","created_at":"2024-02-15 17:47:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":342690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePD-L1 expression in HCT-116 cells cultured in tumor-associated macrophage conditioned medium (TAM-CM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePD-L1 expression levels were measured in HCT-116 cells cultured in TAM-CM from untreated or blue LED-irradiated TAMs.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3951809/v1/24df73d940c36d0119b054f5.jpg"},{"id":51193593,"identity":"2e71ace7-b9ed-4d06-9154-03d66d35d39d","added_by":"auto","created_at":"2024-02-15 17:47:17","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3420730,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTumor suppressive effects of blue LED irradiation in an orthotopic model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The \u003cem\u003ein vivo\u003c/em\u003e models were irradiated with blue LED once a week and tumor size was measured 2 weeks after tumor injection. (B) Immunohistochemistry assays showed attenuated PD-L1 protein expression in LED-irradiated tumors.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3951809/v1/1b263e70c7aedf0e2822e55b.jpg"},{"id":51193717,"identity":"a022fc81-6f45-4e4e-833f-ca7738708d0e","added_by":"auto","created_at":"2024-02-15 17:55:17","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1768784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC) results of tumor-associated macrophage (TAM) marker expression in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIHC assays were used to examine cells that were positive for F4/80 or CD163 protein expression within tumors.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3951809/v1/57004924c13373d6590efca1.jpg"},{"id":51193594,"identity":"d11967b9-c5dc-4282-b36a-651608886892","added_by":"auto","created_at":"2024-02-15 17:47:17","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":4445670,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSummarized mechanisms of the effects of blue LED irradiation on tumor-associated macrophages (TAMs)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3951809/v1/ea8acb0b6814877c34af3089.jpg"},{"id":51194122,"identity":"4bbd0134-858b-4e5c-b89e-6908aee6fad6","added_by":"auto","created_at":"2024-02-15 18:03:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1279301,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3951809/v1/640a34f9-2cba-4ad0-8fe0-117def6c4526.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Blue light irradiation inhibits the activation of cancer-associated macrophages in colon cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIrradiating cells and living organisms with light-emitting diodes (LEDs) with specific wavelengths of light has a variety of effects. Photodynamic therapy (PDT), which combines light and photosensitizers, is attracting attention as a new minimally invasive alternative to chemoradiotherapy. PDT is being clinically applied to treat esophageal cancer, lung cancer, brain tumors, and other cancer types. Several \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e reports have also demonstrated the efficacy of PDT for colorectal cancer (1,2). However, recent studies have demonstrated that using blue LED light alone is effective for suppressing the growth of some cancer cell types. Specific mechanisms for the phototoxic and anti-proliferative effects of blue light have been reported, including the induction of autophagy (3), necroptosis (4), apoptosis (5), and cell cycle arrest (6). Blue light irradiation can also result in the production of reactive oxygen species and DNA damage in colon cancer cells (7).\u0026nbsp;In a previous report, we showed that using 465 nm blue light irradiation (30 mW/cm\u003csup\u003e2\u003c/sup\u003e, 30 minutes) could inhibit colon cancer cell growth via inducing autophagy (8). Furthermore, we revealed that the blue photoreceptor Opsin3 (Opn3), which is expressed in colon cancer cells, can promote the cytotoxic effects of blue light.\u003c/p\u003e\n\u003cp\u003eThe opsin family of proteins is composed of G-protein-coupled receptors (GPCRs) that are expressed on photoreceptor cells in the retina. Opn3 regulates GPCR signaling and acts as a receptor for blue light (11). Additionally, Opn3 activates the G\u003csub\u003ei/o\u003c/sub\u003e subtype of G proteins with light at a wavelength of 460 to 470 nm, inhibits adenylate cyclase, and decreases cyclic adenosine monophosphate (cAMP) (11, 12). Recent studies have shown that Opn3 is also widely expressed in organs unrelated to vision, such as the respiratory epithelium, liver, kidney, and heart, and is called non-visual opsin (9, 10).\u0026nbsp;One report indicates that Opn3 acts as a photoreceptor in malignant melanocytes and contributes to phototherapy (13), and our findings indicate that Opn3 is a potential target for phototherapy as a photoreceptor in colon cancer cells (8).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegulation of the tumor microenvironment (TME), which is composed of various cell types including fibroblasts, immune cells, and tumor cells, is crucial for cancer therapy. We recently reported that irradiation of cancer-associated fibroblasts (CAFs) with blue light can decrease CAF activity and interleukin (IL)-6 secretion, which can support decreased tumor malignancy of colon cancer cells (14). However, the effect of blue light on the TME, particularly\u0026nbsp;tumor-associated macrophages\u0026nbsp;(TAMs), is unknown.\u003c/p\u003e\n\u003cp\u003eIn this study, we focused on TAMs, mainly M2 macrophages, which are involved in tumor growth and metastasis through the secretion of cytokines, such as VEGF and PD-L1. We investigated the impact of blue LED irradiation on TAMs to examine its effects on the TME.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.1 Cell culture\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human colon cancer cell line HCT-116\u0026nbsp;(KAC Co., Ltd., Kyoto, Japan, Cat. No.:\u0026nbsp;EC91091005-F0)\u0026nbsp;was used for \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments. The cell line was confirmed to be negative for mycoplasma. Cells were maintained and cultured in accordance with international guidelines for proper cell culture practices. Cells were cultured in RPMI 1640 medium\u0026nbsp;(Wako, Osaka, Japan)\u0026nbsp;supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in a 37°C humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.2 Preparation of conditioned medium (CM) and TAMs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo obtain CM, HCT-116 cells were cultured in 100 mm culture dishes until 80% confluent. The cells were washed twice with pre-warmed phosphate-buffered saline (PBS) and incubated in fresh medium without FBS. After incubation at 37°C for 48 hours, the supernatant was collected, centrifuged at 800´g for 5 minutes at 22°C, and filtered through a 0.2-µm sterile filter.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo obtain TAMs, M0 macrophages were treated with CM derived from HCT-116 cells. CM was added to the culture medium at a 1:1 ratio and cells were incubated at 37°C for 48 hours.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo obtain TAM-CM, the TAMs were treated with blue LED light (465 nm, 30 mW/cm\u003csup\u003e2\u003c/sup\u003e, 30 minutes) (irradiation group) or left untreated. The medium was changed once and the supernatant (TAM-CM) was collected. A schematic for these processes is shown in Figure 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.3 Animals\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour-week-old female BALB/c mice were purchased from Charles River Japan, Inc. (Kanagawa, Japan). Animals were caged under controlled conditions and provided water and standard laboratory rations for at least seven days prior to use. Before and after surgery, the animals had free access to tap water and food. The experiments and procedures in this study were approved by the Animal Welfare and Use Committee of the University of Tokushima (Approval No. T2021-42). All animal experiments were performed in accordance with the U.K. Animals (Scientific Procedures) Act 1986 and associated guidelines, the EU Directive 2010/63/EU for animal experiments, and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.4 Orthotopic model and LED irradiation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCT-29 cells were harvested with 1 mM ethylenediaminetetraacetic acid (EDTA) in PBS, washed three times with PBS, and resuspended at a density of 1×10\u003csup\u003e7\u003c/sup\u003e cells/mL in PBS containing 500 mg/mL of Matrigel (Becton Dickinson Labware, Bedford, MA, USA). Animals (n=8) were anesthetized with isoflurane (3% to 4% for induction and 1% to 2% for maintenance). A 7-mm incision was made in the anterior rectal wall to prevent colonic obstruction caused by rectal tumor progression. CT-29 cells (1×10\u003csup\u003e6\u003c/sup\u003e) were injected into the submucosa of the posterior wall of the rectum using a 29-gauge needle.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeven days after cell implantation, the mice developed rectal cancer. The mice were randomized into two groups (n=4 mice per group) and treated as follows: (i) the LED irradiation group (465 nm, 30 mW/cm\u003csup\u003e2\u003c/sup\u003e, 30 minutes, once) and (ii) the control group (untreated). The mice were sacrificed by cervical spinal cord dissection at 2 weeks after cell implantation.\u0026nbsp;A humane endpoint was set at the stage when the tumor size increased by more than 10% of the mice’s body weight, but this did not apply to any of the mice.\u003c/p\u003e\n\u003cp\u003eA 465-nm blue LED (NCSB119, NICHIA Corporation, Tokushima, Japan) was used as a light source. An LED irradiation device (Department of Electrical and Electronic Engineering, Faculty of Engineering, The University of Tokushima, Tokushima, Japan) was used for irradiation experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.5 Immunohistochemistry (IHC)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tissues resected from the orthotopic model or cultured cells were fixed in 10% formalin (Wako 066-03847) for 24 hours at room temperature, paraffin-embedded, and sliced into 4-μm-thick sections. The samples were blocked in 3% BSA (MACS\u003csup\u003e®︎\u0026nbsp;\u003c/sup\u003eBSA Stock Solution 130-091-376) for 1 hour at room temperature. An anti-F4/80 antibody (15361-1-AP; Proteintech, Rosemont, IL, USA), anti-CD163 antibody (SAB2700986; Sigma-Aldrich), and anti-PD-L1 antibody (17952-1-AP; Proteintech) were used for IHC staining following the manufacturer’s instructions. The fluorescence area was measured using Image J software (ver. 1.53, National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.6 Quantitative real-time PCR (qRT-PCR) analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from samples using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Tokyo, Japan). Then, qRT-PCR was performed using a 7500 real-time PCR system, TaqMan gene expression assay on demand, and TaqMan Universal Master Mix (Applied Biosystems). The following TaqMan assays were used: Opn3 (Hs00173892_m1), CD163 (Hs00174705_m1), CD206 (Hs00267207_m1), and PD-L1 (Hs00204257_m1). GAPDH (4326317E) was used as an internal control for mRNA expression. The thermal cycler conditions were as follows: 2 minutes at 50°C, 10 minutes at 95°C, and then 40 cycles of 15 s at 95°C and 1 minute at 60°C. Amplification data were analyzed using the Prism 7500 Sequence Detection System ver. 1.3.1 (Applied Biosystems). qRT-PCR was performed using a Step One Plus Real-Time PCR System (Applied Biosystems). The relative target transcript expression levels were assessed and normalized to the expression levels of GAPDH mRNA as an internal control using the 2\u003csup\u003e-ΔΔCq\u003c/sup\u003e method (15).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.7 Migration and scratch assays\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor migration assays, Transwell inserts (Corning, Corning, NY, USA) with a pore size of 8 µm were used. CT-116 cells (2´10\u003csup\u003e4\u003c/sup\u003e) were seeded into the upper chamber. After cell attachment, the medium was discarded and fresh medium containing 1% FBS was added to the upper chamber. CM containing 10% FBS was added to the lower chamber. After incubation for 24 hours, cells at the bottom of the Transwell inserts were fixed in 4% paraformaldehyde, then stained with 0.2% crystal violet. The stained cells in three random microscopic fields (100×) were counted.\u003c/p\u003e\n\u003cp\u003eFor scratch assays, HCT-116 cells were seeded at a density of 2×10\u003csup\u003e4\u003c/sup\u003e cells/well in 6-well plates. After the cells became 100% confluent, a plastic pipette tip was used to scrape the center of the well, creating a 1-mm-wide scratch. The medium was discarded and fresh DMEM containing 1% FBS was added, followed by CM at a 1:1 ratio. The final FBS concentration was 0.5%. The cells were then incubated at 37°C for 24 hours. Images of the wound were taken using a phase-contrast microscope (40× magnification; DP22-CU; Olympus Corporation) at 0 and 24 hours after scratching.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.8 Statistical analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed using Stat View version 5.0 software (SAS Institute, Cary, NC, USA). The Mann-Whitney \u003cem\u003eU\u003c/em\u003e-test and\u0026nbsp;Wilcoxon signed-rank test\u0026nbsp;were used for statistical comparisons. \u003cem\u003eP\u003c/em\u003e-values less than 0.05 were considered statistically significant.\u0026nbsp;\u003c/p\u003e"},{"header":"3.\tResults","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.1 Opn3 expression in M0 macrophages and TAMs\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe examined Opn3 mRNA expression levels in TAMs generated by culturing M0 macrophages with the CM of HCT-116 colon cancer cells, which were then exposed to LED or left untreated. LED-irradiated TAMs showed higher Opn3 expression levels compared with the untreated control group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05;\u0026nbsp;Fig. 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.2\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eBlue LED irradiation suppressed the polarization of macrophages\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mRNA expression levels of M2 macrophage markers CD163 and CD206 were significantly upregulated in TAMs (M0 macrophages cultured in HCT-116 cell CM) compared with those of M0 macrophages\u0026nbsp;(\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; Fig. 3). In contrast, CD163 and CD206 mRNA expression levels were decreased in TAMs treated with blue LED light irradiation, suggesting that blue LED light could inhibit the polarity change to M2 macrophages (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; Fig. 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.3\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eMigration and scratch assays for HCT-116 cells cultured in TAM-CM\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHCT-116 cells were cultured in TAM-CM, then migration (Fig. 4A) and scratch assays (Fig. 4B) were performed. Culturing the cells in TAM-CM significantly promoted the migration of HCT-116 cells (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). However, TAM-CM derived from TAMs irradiated with blue light did not promote the migration of HCT-116\u0026nbsp;cells (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.4\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eBlue LED irradiation suppressed TAM secretion of VEGF\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 5, the levels of VEGF secretion were significantly higher in TAMs compared with M0 macrophages (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). However, in LED-irradiated TAMs, VEGF secretion was significantly suppressed (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.5\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ePD-L1 expression in HCT-116 cells cultured in TAM-CM\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn HCT-116 cells cultured with TAM-CM, PD-L1 mRNA expression levels were upregulated (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05;\u0026nbsp;Fig. 6). However,\u0026nbsp;the degree of PD-L1 mRNA expression induction was attenuated in HCT-116 cells cultured with TAM-CM and irradiated with blue LED (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05;\u0026nbsp;Fig. 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.6 Effects of\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eblue LED irradiation in an in vivo model\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eColon cancer tumor growth in a mouse model was significantly suppressed by blue LED irradiation (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; Fig. 7A), as indicated by reduced tumor size. PD-L1 expression levels were downregulated in tumors exposed to blue LED irradiation (Fig. 7B). Furthermore, IHC staining showed that tumors irradiated with blue LED showed fewer cells positive for F4/80 and CD163 protein compared with the control samples\u0026nbsp;(Fig. 8).\u0026nbsp;The CD163-positive cell areas in five randomly selected fields of view were compared, with a significant reduction observed in the irradiated group compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e"},{"header":"4.\tDiscussion","content":"\u003cp\u003eRecent studies have demonstrated that cells in the TME, including fibroblasts and immune cells, are extremely important for the efficacy of anti-cancer therapies.\u0026nbsp;We previously reported that blue LED light can act in an inhibitory manner on not only tumor cells, but also on CAFs, resulting in reduced tumor malignancy (14). In the present study, we examined the impact of blue LED on the TME by investigating its effects on TAMs.\u003c/p\u003e\n\u003cp\u003eOur results showed that blue light irradiation could decrease the expression of M2 macrophage markers CD163 and CD206, suggesting that it could suppress macrophage\u0026nbsp;polarization\u0026nbsp;to the M2 phenotype. In addition, migration assays showed that HCT-116 cell migration was promoted in the presence of CM from non-LED-irradiated TAMs but was significantly reduced with CM from LED-irradiated TAMs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe previously reported that VEGF secretion by TAMs plays a significant role in tumor malignancy in hepatocellular carcinoma (16). In the present study, VEGF secretion was observed in TAMs without LED irradiation, but this was significantly suppressed in TAMs irradiated with LED. In addition, HCT-116 cells cultured in TAM-CM showed elevated PD-L1 expression levels, whereas cells cultured with LED-irradiated TAM-CM showed attenuated induction of PD-L1 expression. The transcription factor STAT3 can reportedly regulate expression of the M2 macrophage markers CD163 and CD206 (17). Additionally, STAT3 has been implicated in elevated tumor malignancy and PD-L1 expression through VEGF in tumor cells (18). Therefore, STAT3 may play a crucial role in the mechanism through which blue light irradiation can affect both TAMs and tumor cells.\u003c/p\u003e\n\u003cp\u003eRecently, PDT targeting of TAMs has been reported (19). We have also reported the effect of blue LEDs on CAFs, suggesting the future possibility of phototherapy targeting the TME. Furthermore, Opn3 is expressed in TAMs, suggesting the possibility that blue LEDs may suppress TAMs via photoreceptors.\u003c/p\u003e\n\u003cp\u003eIn our previous experiments, similar anti-tumor effects were observed with daily 5-minute irradiation and a single 30-minute irradiation (8). Therefore, we used a 30-minute irradiation in the current study. Subsequent investigations should focus on evaluating the impact of short-duration fractionated irradiation on TAMs, as well as the effects of frequent 30-minute irradiation sessions.\u003c/p\u003e\n\u003cp\u003eThe major limitation of this study is that the significance of Opn3 expression in tumors and macrophages has not been fully investigated. Future studies involving Opn3 expression knocked down in macrophages or with Opn3 knockout mice are required. Although further studies are required to fully elucidate the mechanism of action of blue LED irradiation on tumors and the TME, this study demonstrated significant effects of blue LED irradiation on colon cancer cells and TAMs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn summary, blue light could suppress macrophage polarization to TAMs and decreased the levels of VEGF secretion. Additionally, blue light attenuated TAM-mediated tumor malignancy and decreased PD-L1 expression levels. The blue light receptor Opn3 is expressed in TAMs, and macrophage polarization and activity were reduced by blue light irradiation. Taken together, these findings suggest that blue light may regulate TAMs in tumors and the TME (Fig. 9).\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eLED:\u0026nbsp;light-emitting diode\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTAM: tumor-associated macrophage\u003c/p\u003e\n\u003cp\u003eCM: conditioned medium\u003c/p\u003e\n\u003cp\u003eOpn3: Opsin 3\u003c/p\u003e\n\u003cp\u003ecAMP: cyclic adenosine monophosphate\u003c/p\u003e\n\u003cp\u003eGPCR: G protein-coupled receptor\u003c/p\u003e\n\u003cp\u003ePDT: photodynamic therapy\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was partly supported by the Grant-in-Aid for Scientific Research (Grant no. 22K16465). This study was also funded by Taiho Pharmaceutical Co., Ltd. (Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated in the present study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTY, SO, KY, MN, TT and MS conceived and designed the study. TY, SO, HK and YW performed the experiments and collected the data. TY, SO, TNa, TNo and CT analyzed and interpreted the data. TY and SO wrote the paper. TT, TNa, TNo and MN reviewed the data and information and edited the manuscript. KY and MS revised the paper critically for important intellectual content. KY, TY and TT confirmed the authenticity of all the raw data. All authors agreed to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was conducted in compliance with the Division for Animal Research Resources, Institute of Health Biosciences, University of Tokushima, Japan. The experiments and procedures were approved by the Animal Care and Use Committee of the University of Tokushima, Japan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Patient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHatakeyama T, Murayama Y, Komatsu S, et al. Efficacy of 5-aminolevulinic acid-mediated photodynamic therapy using light-emitting diodes in human colon cancer cells. Oncology reports. 2013;29(3):911-6.\u003c/li\u003e\n\u003cli\u003eHodgkinson N, Kruger CA, Abrahamse H. Targeted photodynamic therapy as potential treatment modality for the eradication of colon cancer and colon cancer stem cells. Tumor Biology. 2017;39(10):1010428317734691.\u003c/li\u003e\n\u003cli\u003eOh P-S, Hwang H, Jeong H-S, et al. Blue light emitting diode induces apoptosis in lymphoid cells by stimulating autophagy. The international journal of biochemistry \u0026amp; cell biology. 2016;70:13-22.\u003c/li\u003e\n\u003cli\u003edel Olmo-Aguado S, N\u0026uacute;\u0026ntilde;ez-\u0026Aacute;lvarez C, Osborne NN. Blue light action on mitochondria leads to cell death by necroptosis. Neurochemical research. 2016;41(9):2324-35.\u003c/li\u003e\n\u003cli\u003eOh P-S, Na KS, Hwang H, et al. Effect of blue light emitting diodes on melanoma cells: involvement of apoptotic signaling. Journal of Photochemistry and Photobiology B: Biology. 2015;142:197-203.\u003c/li\u003e\n\u003cli\u003eSparsa A, Faucher K, Sol V, et al. Blue light is phototoxic for B16F10 murine melanoma and bovine endothelial cell lines by direct cytocidal effect. Anticancer research. 2010;30(1):143-7.\u003c/li\u003e\n\u003cli\u003eYan G, Zhang L, Feng C, et al. Blue light emitting diodes irradiation causes cell death in colorectal cancer by inducing ROS production and DNA damage. The international journal of biochemistry \u0026amp; cell biology. 2018;103:81-8.\u003c/li\u003e\n\u003cli\u003eYoshimoto T, Morine Y, Takasu C, et al. Blue light‐emitting diodes induce autophagy in colon cancer cells by Opsin 3. Annals of gastroenterological surgery. 2018;2(2):154-61.\u003c/li\u003e\n\u003cli\u003eKoyanagi M, Terakita A. Diversity of animal opsin-based pigments and their optogenetic potential. Biochimica et Biophysica Acta (BBA)-Bioenergetics. 2014;1837(5):710-6.\u003c/li\u003e\n\u003cli\u003eJiao J, Hong S, Zhang J, et al. Opsin3 sensitizes hepatocellular carcinoma cells to 5-fluorouracil treatment by regulating the apoptotic pathway. Cancer letters. 2012;320(1):96-103.\u003c/li\u003e\n\u003cli\u003eKoyanagi M, Takada E, Nagata T, et al. Homologs of vertebrate Opn3 potentially serve as a light sensor in nonphotoreceptive tissue. Proceedings of the National Academy of Sciences. 2013;110(13):4998-5003.\u003c/li\u003e\n\u003cli\u003eTerakita A, Nagata T. Functional properties of opsins and their contribution to light-sensing physiology. Zoological science. 2014;31(10):653-9.\u003c/li\u003e\n\u003cli\u003ede Assis L, Moraes M, da Silveira Cruz-Machado S, et al. The effect of white light on normal and malignant murine melanocytes: a link between opsins, clock genes, and melanogenesis. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 2016;1863(6):1119-33.\u003c/li\u003e\n\u003cli\u003eYoshimoto T, Shimada M, Tokunaga T, et al. Blue light irradiation inhibits the growth of colon cancer and activation of cancer associated fibroblasts. Oncology Reports. 2022 May 1;47(5):1-9.\u003c/li\u003e\n\u003cli\u003eLivak KJ and Schmittgen TD: Analysis of relative gene expression data using real‑time quantitative PCR and the 2(‑Delta Delta C(T)) method. Methods 25: 402‑408, 2001.\u003c/li\u003e\n\u003cli\u003eOkikawa S, Morine Y, Saito Y, et al. Inhibition of the VEGF signaling pathway attenuates tumor‑associated macrophage activity in liver cancer. Oncology reports. 2022 Apr 1;47(4):1-1. \u003c/li\u003e\n\u003cli\u003eGharibi, T., Babaloo, Z., Hosseini, A., et al. Targeting STAT3 in cancer and autoimmune diseases. European journal of pharmacology, 2020, 878: 173107.\u003c/li\u003e\n\u003cli\u003eDe Beule, N., De Veirman, K., Maes, K., et al. Tumour‐associated macrophage‐mediated survival of myeloma cells through STAT3 activation. The Journal of Pathology, 2017, 241.4: 534-546.\u003c/li\u003e\n\u003cli\u003eSoyama, T., Sakuragi, A., Oishi, D., et al. Photodynamic therapy exploiting the anti-tumor activity of mannose-conjugated chlorin e6 reduced M2-like tumor-associated macrophages. Translational oncology, 2021, 14.2: 101005.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Blue light, colorectal cancer, light-emitting diode, Opsin3, tumor-associated macrophage","lastPublishedDoi":"10.21203/rs.3.rs-3951809/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3951809/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRecent studies have shown that blue light-emitting diode (LED) light has anti-tumor effects, suggesting the possibility of using visible light in cancer therapy. However, the effects of blue light irradiation on cells in the tumor microenvironment, including tumor-associated macrophages (TAMs), are unknown. Here, THP-1 cells were cultured in the conditioned medium (CM) of HCT-116 cells to prepare TAMs. TAMs were divided into LED-irradiated and control groups. Then, the effects of blue LED irradiation on TAM activation were examined. Expression levels of M2 macrophage markers CD163 and CD206 expression were significantly decreased in LED-irradiated TAMs compared with the control group. While control TAM-CM could induce HCT-116 cell migration, these effects were not observed in cells cultured in TAM-CM with LED irradiation. Vascular endothelial growth factor (VEGF) secretion was significantly suppressed in LED-exposed TAMs. PD-L1 expression was upregulated in HCT-116 cells cultured with TAM-CM but attenuated in cells cultured with LED-irradiated TAM-CM. In an \u003cem\u003ein vivo\u003c/em\u003e model, protein expression levels of F4/80 and CD163, which are TAM markers, were reduced in the LED-exposed group. These results indicate that blue LED light may have an inhibitory effect on TAMs, as well as anti-tumor effects on colon cancer cells.\u003c/p\u003e","manuscriptTitle":"Blue light irradiation inhibits the activation of cancer-associated macrophages in colon cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-15 17:47:11","doi":"10.21203/rs.3.rs-3951809/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-30T07:34:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-29T00:29:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"e99684bc-9b97-4983-9a46-6665c8331517","date":"2024-02-28T00:07:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-27T19:43:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"155ea8bb-92e4-4041-a700-479281edce3b","date":"2024-02-27T14:10:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-23T06:54:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-02-15T12:23:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-14T04:35:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-14T04:35:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2024-02-12T19:31:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"60d18bc5-1657-47ad-8032-6953f607fd9f","owner":[],"postedDate":"February 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-28T08:30:33+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-15 17:47:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3951809","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3951809","identity":"rs-3951809","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}