Light‐emitting diode irradiation targets aerobic glycolysis in cancer‐associated fibroblasts to inhibit metabolic coupling with colon cancer cells

Abstract

Background

The concepts examined in tumor metabolism research have been moving away from cancer cells themselves and the tumor microenvironment has become a focus of investigation. Epigenetic changes affecting glucose metabolism in cancer‐associated fibroblasts (CAFs) in the tumor microenvironment regulate the malignancy of cancer cells. Light‐emitting diode (LED) have several functional effects, including anti‐tumor effects, through a variety of mechanisms. We previously reported that blue LED irradiation had an anti‐tumor effect via Opsin 3 that suppress CAF function in vivo and in vitro. However, the detailed mechanism by which LED affect CAF regulation remained unclear.

Methods

CAFs were induced from human intestinal fibroblasts co‐cultured with colon cancer cells. Using lactate assays and reverse transcription‐quantitative PCR, the effects of LED irradiation on glycolysis in CAFs were investigated. The effects of LED irradiation for metabolic coupling were evaluated in vitro.

Results

Reverse transcription‐quantitative PCR showed higher expression of CAF marker genes in induced CAFs. Blue LED irradiation of induced CAFs suppressed their ability to promote characteristics of tumor malignancy of colon cancer cell line. LED treatment decreased expression of glycolysis markers, including phosphofructokinase and monocarboxylate transporter 4, indicating inhibition of glycolytic activity in CAFs. Extracellular secretion of lactate from CAFs was decreased by LED irradiation.

Conclusions

Blue LED irradiation targeted metabolic coupling between CAFs and colon cancer cells, thereby reducing the promotion of tumor progression by CAFs. Blue LED targeting glucose metabolism in CAFs is a promising anti‐cancer treatment option.

Blue LED irradiation targeted metabolic coupling between CAFs and colon cancer cells.

1. INTRODUCTION

Colorectal cancer (CRC) is one of the most common and aggressive gastrointestinal malignancies. ¹ , ² Advances in surgery, radiotherapy, chemotherapy, and immunotherapy have greatly improved patient survival. However, CRC still causes many cancer deaths worldwide. ¹ , ²

Focusing solely on cancer cells is usually insufficient to eradicate a malignancy and does not usually cure patients. This is because many types of stromal cell in the tumor microenvironment (TME), such as cancer‐associated fibroblasts (CAFs), tumor‐associated macrophages, dendritic cells, neutrophils, lymphocytes, and other immune cells, promote tumor malignancy. Among these, CAFs have a crucial role in malignant behavior. ³

Altered glucose metabolism in cancer cells is well known and is called the “Warburg effect.” ⁴ , ⁵ A recent trend in tumor metabolism research has been a shift away from the focus on cancer cells themselves and toward the TME. ⁶ , ⁷ , ⁸ , ⁹ , ¹⁰ CAFs in TME undergo metabolic change, and metabolites from CAFs, such as lactate, glutamine, and lipids, are taken up by cancer cells and used to promote tumor progression. This phenomenon is called metabolic coupling. We previously reported that flavonoid regulate the metabolic coupling between cancer cells and CAFs. ⁶ , ⁷

Light‐emitting diode (LED) with specific wavelengths have several types of effects on organisms. In the field of cancer treatment, blue LED irradiation has phototoxic and anti‐proliferative effects on cancer cells by inhibiting growth, inducing apoptosis, autophagy, and cell cycle arrest, and causing DNA damage. ¹¹ , ¹² , ¹³ , ¹⁴ , ¹⁵ We previously showed that blue LED irradiation at a wavelength of 465 nm inhibits colon cancer cell proliferation via the photoreceptor, Opsin 3 (OPN3). ¹⁶ We recently proposed that LED can attenuate the cancer‐promoting ability of CAFs. However, the detailed mechanism of this effect was unclear. ¹⁷

Here, we propose and investigate the hypothesis that LED can regulate the metabolic coupling between CAFs and colon cancer cells by inhibiting aerobic glycolytic activity in CAFs to suppress characteristics of tumor malignancy in vitro. Our findings indicate that blue LED irradiation can target the glucose metabolism of CAFs and disrupt the metabolic coupling between colon cancer cells and CAFs.

2. MATERIALS AND METHODS

2.1. Cell culture and reagents

The human colon cancer cell line, HCT‐116 (CCL‐247), was purchased from ATCC (Manassas, VA, USA) and cultured in McCoy's 5A (Thermo Fisher Scientific, Inc. Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) and a 1% penicillin–streptomycin solution (Thermo Fisher Scientific, Inc.). Human intestinal fibroblasts (HIFs) were purchased from ScienCell Research Laboratories (Cat. 2920, Carlsbad, CA, USA), and were cultured in complete fibroblast medium (Cat. 2301, ScienCell Research Laboratories). Cells were cultured under mycoplasma‐free conditions at 37°C in a 5% CO₂ and normal oxygen atmosphere. Cells in the logarithmic growth phase were selected for experiments.

2.2. LED irradiation

Methods of LED irradiation have been reported previously. ¹⁶ , ¹⁷ A 465‐nm blue LED (NCSB119) was used (NICHIA Corporation, Tokushima, Japan). An LED irradiation device with an irradiation platform for the light source was made by the Department of Electrical and Electronic Engineering, Faculty of Engineering, The University of Tokushima, Tokushima, Japan. The LED was used in continuous mode. The distance from the light source to the cells was 15 cm for each irradiation. The irradiance was set to 30 mW/cm² (1.0 A) for 30 min.

2.3. Cell co‐culture and conditioned medium (CM) collection

To induce CAFs, HIFs were co‐cultured with HCT‐116 colon cancer cells. The cancer cells were then removed from the co‐culture system and the monocultures of induced CAFs incubated in fresh serum‐free Dulbecco's modified Eagle's medium. Supernatant was collected, centrifuged (500× g) at room temperature for 20 min and filtered using a 0.2‐μm filter to remove cell debris. To generate HIF‐CM, HIFs were cultured using the same procedure as for CAFs, except that the HCT‐116 cells were no longer present. All conditioned media were stored at −80°C and repeated freeze–thaw cycles were avoided. Conditioned media were added to fresh medium at a ratio of 1:2 for the experimental treatment of cancer cells.

2.4. Cell proliferation assay

HCT‐116 cells were seeded at a density of 1 × 10⁴ cells/well into 96‐well plates and allowed to adhere. After washing with PBS, indicated CMs were added to each group and cells were cultured for 48 h. A 10% volume of cell counting kit‐8 (CCK‐8) solution (Dojindo Molecular Technologies, Inc., Tokyo, Japan) was added to the medium. Two hours later, cell proliferation was analyzed by measuring absorbance at 450 nm with a SpectraMax i3 microplate reader (Molecular Devices, LLC).

2.5. Migration assay

A 24‐well Transwell system with 8.0‐μm pores (Corning, Inc.) was used for migration assays. A 100‐μL suspension of 7 × 10⁴ serum‐starved cancer cells/well were seeded into the upper chamber. After cell attachment, the culture medium was changed to the indicated CM for each group. The final fetal bovine serum concentration was 5% in the upper chamber and 10% in the lower chamber. After a period of incubation, the upper chambers were fixed, and stained and non‐migrated cells were removed. Pictures were then taken, and the number of remaining cells counted.

2.6. Quantitative real‐time reverse transcription PCR (qRT‐PCR)

Total RNA was extracted using an RNeasy Mini kit (Qiagen GmbH, Hilden, Germany) and RNA concentration was measured using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Subsequently, 2.5 μg RNA was reverse transcribed into cDNA using a High‐Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). Then, a StepOnePlus Real‐Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to perform TaqMan qPCR. The following TaqMan gene expression assays were used: ACTA2 (assay ID, Hs00426835_g1), FAP (assay ID, Hs00990791_m1), OPN3 (assay ID, Hs00173892_m1), PFKP (assay ID, Hs00737347_m1), and SLC16A3 (MCT4) (assay ID, Hs00358829_m1). GAPDH (assay ID, 4326317E; Thermo Fisher Scientific, Inc.) was used as the internal control to normalize the raw data. A 2^−∆∆Cq method was used for data analysis, and the results are presented as the fold changes of the relative mRNA expression for each experimental group compared with that in the control group.

2.7. Lactate assay

A lactate assay kit (MAK064, Sigma‐Aldrich, MO, USA) was used to detect the concentration of lactate in supernatant. In brief, supernatants and standard samples were mixed with lactate assay buffer at dilutions indicated in the manufacturer's instructions and added to a 96‐well plate. After incubation with 50 μL master reaction mix for 30 min at room temperature, the absorbance values at 570 nm were measured with a microplate reader, and lactate concentrations of samples were calculated using a standard curve constructed from standard sample values. The relative secretion of lactate was normalized to the cell number.

2.8. Western blot analysis

Freshly prepared RIPA lysis buffer (Thermo Fisher Scientific, Inc.) containing 1% protease inhibitor cocktail (P8849, Sigma‐Aldrich) and 1% Phosphatase Inhibitor Cocktail 3 (P0044, Sigma‐Aldrich) was used to extract total protein. Protein concentration was measured using a BCA kit (Thermo Fisher Scientific, Inc.). Equal amounts of protein (10 μg) were separated by 10% SDS‐PAGE and transferred onto PVDF membranes with 0.45‐μm pores (Sigma‐Aldrich). The blots were blocked with 5% skimmed milk for 1.5 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: anti‐MCT4 (1:2000; cat. no. 22787‐1‐AP; ProteinTech Group, Inc.) and anti‐β‐actin (1:2000; cat. no. 4970; Cell Signaling Technology, Inc.). The next day, anti‐rabbit IgG, HRP‐linked (1:3000; cat. no. 7074; Cell Signaling Technology, Inc.) was used as a secondary antibody, and proteins were detected using ECL Prime Western Blotting Detection Reagents (RPN2232, Cytiva, Sheffield, UK).

2.9. Statistical analysis

All statistical analyses were performed using JMP 8.0.1 (SAS, Cary, NC, USA). The Mann–Whitney U test was used for comparisons between two groups. All experiments were performed for at least three biological replicates and p < 0.05 (two‐sided) was considered to indicate statistical significance.

3. RESULTS

HIFs were co‐cultured with colon cancer HCT‐116 cells to induce CAFs (Figure 1). We previously reported that glycolytic activity and lactate secretion are enhanced in induced CAFs. ⁶ In this study, induced CAFs had higher gene expression of α‐smooth muscle actin (ACTA2) and fibroblast activation protein (FAP) and exhibited a CAF phenotype (Figure 2A,B).

FIGURE 1.

Experimental design. HIFs were co‐cultured with colon cancer HCT‐116 cells to induce CAFs. Induced CAFs were irradiated with blue LED light (465 nm, 30 mW/cm², 30 min) and the supernatant was collected. These supernatants were designated CAF‐conditioned medium (CAF‐CM). Cancer cells were incubated with CAF‐CM. LED, light‐emitting diode; CAF, cancer‐associated fibroblast; CM, conditioned medium; HIFs, human intestinal fibroblasts.

FIGURE 2.

HIFs co‐cultured with tumor cells exhibit a CAF phenotype. After co‐culture with HCT‐116 colon cancer cells, the gene expression levels of the CAF markers ACTA2 and FAP in HIFs were determined by RT‐qPCR (A, B). ACTA2; alpha‐smooth muscle Actin, FAP; fibroblast activation protein, CAF; cancer‐associated fibroblast, RT‐qPCR, reverse transcription‐quantitative PCR. *p < 0.05.

Next, we investigated the effects of blue LED on tumor malignancy promotion by CAFs. Induced CAFs were irradiated with blue LED light (465 nm, 30 mW/cm², 30 min) and the supernatant was collected. The supernatant from CAFs was designated CAF‐conditioned medium (CAF‐CM) (Figure 1). We previously showed that LED decreased ACTA2 and FAP expression and the CAF phenotype. ¹⁷ As shown in Figure 3A, CAF‐CM from LED‐irradiated CAFs suppressed the proliferation and migration of HCT‐116 cells, indicating that the tumor‐promoting activity of CAFs was also suppressed by LED irradiation. Transwell migration assays indicated that CAF‐CM from LED‐irradiated CAFs suppressed promotion of HCT‐116 cell migration (Figure 3B,C). These findings indicate that blue LED irradiation suppressed the promotion of tumor malignancy characteristics by CAFs.

FIGURE 3.

LED irradiation of CAFs suppresses cancer cell migration. Cell counting kit‐8 results demonstrated that LED irradiation of CAFs suppressed CAF‐CM promotion of cancer cell proliferation (A). Transwell migration assays indicated that LED irradiation of CAFs suppressed CAF‐CM promotion of HCT‐116 cell migration (B, C). CAF, cancer‐associated fibroblast; CM, conditioned medium; LED, light‐emitting diode. *p < 0.05.

LED irradiation decreased the production of lactate by CAFs (Figure 4) and reduced the gene expression of Phosphofructokinase (PFK) and Monocarboxylate transporter 4 (MCT4) (Figure 5), which are the key enzymes for glycolytic activity. Decreased lactate secretion into the TME inhibits tumor capability. ⁶ OPN3 expression in CAFs was significantly upregulated compared to HIFs, and LED decreased OPN3 expression of CAFs (Figure 5). Taken together, blue LED treatment inhibited the aerobic glycolytic metabolism of CAFs and disrupted the metabolic coupling between cancer cells and CAFs (Figure 6).

FIGURE 4.

Extracellular lactate secretions by HIFs and CAFs. Lactate concentration in CMs was evaluated by lactate assays. HIF, human intestinal fibroblast; CAF, cancer‐associated fibroblast; CM, conditioned medium; *p < 0.05.

FIGURE 5.

Gene expression levels of PFK (A) and MCT4 (B) in LED‐irradiated CAFs determined by RT‐qPCR analysis. Protein levels of MCT4 were evaluated by western bloating (C, D). Gene expression levels of OPN3 in HIF, CAF and LED‐irradiated CAFs determined by RT‐qPCR analysis (E). HIF, human intestinal fibroblast; CAF, cancer‐associated fibroblast; CM, conditioned medium; OPN3, Opsin 3; RT‐qPCR, reverse transcription‐quantitative PCR; PFK, Phosphofructokinase; MCT4, Monocarboxylate transporter 4; LED, light‐emitting diode; *p < 0.05.

FIGURE 6.

Proposed mechanism. LED treatment inhibits aerobic glycolytic metabolism in CAFs to suppress tumor‐stroma metabolic coupling. Schematic representation of LED hindrance of the metabolic coupling between colon cancer cells and CAFs by targeting aerobic glycolysis in CAFs. HIF, human intestinal fibroblast; LED, light‐emitting diode; CAF, cancer‐associated fibroblast; CM, conditioned medium; F‐6‐P, fructose l‐6‐phosphate; F‐1,6‐BP, fructose l‐1,6‐bisphosphate; PFK, Phosphofructokinase; MCT4, Monocarboxylate transporter 4; TCA cycle, tricarboxylic acid cycle.

4. DISCUSSION

In this study, we demonstrated that blue LED irradiation can reduce the aerobic glycolysis activity of CAFs, thereby inhibiting the metabolic coupling between colon cancer cells and CAFs, which can reduce tumor malignancy. Therapeutic application of blue LED to target the glucose metabolism of CAFs in the TME is a promising alternative anti‐cancer strategy.

In the TME, CAFs have various crucial effects on cancer progression, including proliferation, migration, invasion, epithelial mesenchymal transition, and angiogenesis. ³ , ¹⁸ These multimodal functions lead to tumor promotion. Furthermore, CAFs can constantly supply nutritional components to cancer cells for tumor survival and progression. Recent research on metabolism in the TME has shifted from focusing on cancer cells themselves to the reprogramming of tumor‐stroma metabolic coupling. ⁶ , ⁷ , ⁸ , ⁹ , ¹⁰ Metabolic coupling is the uptake and utilization of CAF‐produced energy metabolites by cancer cells. ⁶ , ⁷ , ⁸ , ⁹ , ¹⁰ If glucose metabolism is increased in CAFs, glucose is metabolized into pyruvate and lactate. The lactate is released extracellularly via the lactate transporter, MCT4, and then taken up by cancer cells via the lactate transporter, MCT1, to be used as an energy source. This induces VEGF and mTOR signaling and histone lactylation, which contribute to tumor malignancy. ¹⁹ , ²⁰ , ²¹ This new concept is called the “reverse Warburg effect.” The lactate from CAFs is a potential target for cancer treatment. ⁶ , ⁷ , ¹⁹ , ²⁰ , ²¹ Blocking this tumor‐stroma metabolic coupling between CAFs and cancer cells may provide a new anti‐tumor strategy.

LED are routinely used for lighting and displays in daily life. LED have several advantages, such as cost effectiveness and a variety of wavelengths. Furthermore, LED can be readily adapted for phototherapy, cancer treatment, regenerative medicine, and plant cultivation. The direct anti‐tumor mechanisms of LED in cancer therapy have been studied previously. ¹¹ , ¹² , ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ We previously showed that blue LED irradiation induces autophagy and has an anti‐tumor effect via the cell surface photoreceptor, OPN3, in the colon cancer. ¹⁶ Furthermore, blue LED irradiation also inhibited CAF activation, ¹⁷ however, the detailed mechanisms of CAF suppression were unclear. Here, LED irradiation inhibited glycolytic enzyme activity and reduced the aerobic glycolytic capacity of CAFs. Specifically, PFK and MCT4 were down‐regulated, and the efflux of lactic acid was reduced by LED irradiation. Interestingly, LED‐treated CAFs had reduced capacity to promote characteristics of colon cancer malignancy. Some reports have shown that phototherapy can significantly inhibit the glucose metabolic activity in cancer cells. ²² In addition, blue LED have an anti‐fibrotic effect via modulation of glycolytic capability. ²³

In our experiments, OPN3 expression in CAFs was significantly upregulated compared to HIFs, and LED decreased OPN3 expression in CAFs. HCT‐116 cell proliferation was more suppressed in LED‐treated CAFs CM than HIFs CM. LED irradiation might induce strong suppression of CAF activity. Regarding irradiation condition, the irradiation time and dose for cancer cells and HIF have been reported previously. ¹⁵ We did not change the LED irradiation time and dose in the current experiment, and the best irradiation time and dose for CAF are unclear. We have previously confirmed the inhibition of cell viability at 48 hours after LED irradiation (in vitro), ¹⁶ and the inhibition of tumor growth at 1 week after LED irradiation in vivo. ¹⁷ The change of cancer cell proliferation was observed from day 4, which seems reasonable given the previous results.

Regarding the signal pathway, adenylyl cyclase and cAMP are down stream of OPN3—G protein‐coupled receptor pathway. cAMP activates protein kinase A (PKA) and increase synthesis of fructose‐2,6‐bisphosphate (F‐2,6‐BP). F‐2,6‐BP activates PFK. ²⁴ Furthermore, cAMP may regulate MCT4. When cAMP is increased, cAMP/PKA pathway increases MCT4 expression via transcription factors (such as HIF‐1α). ²⁵ , ²⁶ Taken together, LED might regulate the expression of PFK and MCT4 through OPN3. We hypothesized that blue LED inhibit tumor‐stroma metabolic coupling (Figure 6) and can be safely used as an anti‐cancer adjuvant targeting the TME.

Although we have presented some groundbreaking results, further research is needed. First, the detailed mechanism by which blue LED irradiation reduces glycolytic enzymes is not yet fully understood. We think inhibition of PFK or MCT4 reverses metabolic coupling. LED irradiation reduces lactate excretion by CAFs and inhibits their tumor‐promoting potential. Metabolism involves a complex interplay of mechanisms, including feedback; when lactate production is reduced, feedback may cause PFK and MCT4 expression to increase. On the other hand, we hypothesized that reduced PFK/MCT4 expression could result in decreased lactate. Second, other CAF metabolites, such as proteins and lipids, have not been evaluated. Third, as reported previously, blue LED irradiation inhibited tumor progression in vivo, but glucose metabolism in vivo has not been evaluated. Fourth, we chose only the HCT‐116 cell line from among the many types of colorectal cancer cell lines. In our previous report, we performed experiments using CAFs generated from HT‐29 as well as HCT‐116. ⁶ , ⁷ In these previous experiments, there was no difference in experimental results between CAFs made with HCT‐116 and those made with HT‐29. In the current study, we followed a previous paper ¹⁷ that used HCT‐116.

Tumor microenvironment is composed of a variety of cells, including CAFs, tumor‐associated macrophages (TAMs), and so on. We have confirmed that LEDs inhibit not only CAFs ¹⁷ but also TAMs, ²⁷ indicating that LEDs have a broad inhibitory effect not only on tumor cells but also on cells constituting the TME. As a future perspective for clinical application, synergistic effects with anticancer agents and combination therapy with immune checkpoint inhibitor for advanced cancer are expected. LEDs may also be able to replace the role of radiotherapy because of being less disruptive to normal tissues, ¹⁵ compared to radiotherapy.

In conclusion, our research reveals the metabolic coupling between cancer cells and CAFs in colon cancer, and that this relationship supports tumor progression. We also further revealed the anti‐tumor mechanism of blue LED irradiation, which targets the glycolysis of CAFs in the TME. Our findings further clarify tumor–stroma metabolic coupling and will inform the development of new therapeutic strategies.

AUTHOR CONTRIBUTIONS

Masaaki Nishi: Conceptualization; funding acquisition; methodology; project administration; writing – original draft; writing – review and editing. Chiharu Nakasu: Data curation; formal analysis; investigation; methodology. Toshiaki Yoshimoto: Data curation; investigation; methodology. Takuya Tokunaga: Data curation; formal analysis. T. Hideya Kashihara: Formal analysis; investigation. Chie Takasu: Data curation; formal analysis; visualization; writing – review and editing. Daichi Ishikawa: Investigation; methodology. Yuma Wada: Data curation; visualization. Mitsuo Shimada: Funding acquisition; supervision; writing – review and editing.

FUNDING INFORMATION

This work was supported by Taiho Pharma Co Ltd. The funding source had no role in the design, practice, or analysis of this study.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest for this article.

ETHICS STATEMENT

Approval of the research protocol: The experiments and procedures were approved by the Animal Care and Use Committee of the University of Tokushima, Japan.

Informed consent: N/A.

Registry and the Registration No. of the study/trial: N/A.

Nishi M, Nakasu C, Yoshimoto T, Tokunaga T, Kashihara TH, Takasu C, et al. Light‐emitting diode irradiation targets aerobic glycolysis in cancer‐associated fibroblasts to inhibit metabolic coupling with colon cancer cells. Ann Gastroenterol Surg. 2025;9:761–768. 10.1002/ags3.70004