Oral Lentinula edodes mycelia extract enhances the antitumor effect of radiotherapy via gut-associated activation of dendritic and cytotoxic T cells

Oral Lentinula edodes mycelia extract enhances the antitumor effect of radiotherapy via gut-associated activation of dendritic and cytotoxic T cells | 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 Article Oral Lentinula edodes mycelia extract enhances the antitumor effect of radiotherapy via gut-associated activation of dendritic and cytotoxic T cells Tsuguhide Takeshima, Yang Wang, Sumitaka Hasegawa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8096960/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Oral administration of Lentinula edodes mycelia extract (L.E.M.) has been shown to stimulate systemic T cell–mediated antitumor immunity and inhibit tumor growth in mice, suggesting its potential to modulate host immune responses. However, the route of this systemic antitumor effect remains unclear. This study focused on gut-associated immune mechanisms by analyzing mesenteric lymph nodes (MLNs), a major component of the gut-associated lymphoid tissue (GALT), and examined whether oral L.E.M. enhances the antitumor efficacy of radiation therapy (RT) in a B16F10-OVA melanoma model. L.E.M. administration upregulated MHC class II and CD86 expression on CD11c + dendritic cells (DCs) in MLNs and significantly increased the proportion of CD103 + subsets, indicating DC maturation within the GALT. In a radiation-induced tumor model, L.E.M. further enhanced DC maturation and increased CD8α + DCs in the spleen, accompanied by elevated effector and central memory fractions of CD8 + T cells in peripheral blood. Antigen-specific CD8 + T cells (OVA tetramer + ) were significantly enriched within tumors, and L.E.M. combined with RT achieved greater tumor growth inhibition than RT alone. These findings demonstrate that oral L.E.M. activates GALT-mediated DC and CD8 + T-cell responses, thereby augmenting the antitumor immune effects of RT. Biological sciences/Cancer Biological sciences/Immunology Health sciences/Oncology Lentinula edodes mycelia extract Gut-associated lymphoid tissue Dendritic cells CD8+ T cells Radiation Therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cancer immunotherapy attempts to control tumor growth by activating T cells that recognize tumor antigens. However, the tumor microenvironment contains regulatory T cells, myeloid-derived suppressor cells, and inhibitory cytokines that suppress antitumor immune responses [ 1 , 2 ]. Therefore, new treatment methods are needed to reduce immune suppression and to promote antigen presentation and T cell activation. A hot water extract of dried powder extracted from Lentinula edodes mycelia (L.E.M.), prepared from cultured biomass grown in a bagasse-rice bran medium and harvested prior to fruiting, has been reported to reduce regulatory T cells and induce CD8 + T cell–dependent antitumor effects following oral administration [ 3 – 10 ]. The gut-associated lymphoid tissue (GALT) has been suggested as the first site where L.E.M. acts in the body [ 8 ], indicating that L.E.M. may regulate immune activity through the intestinal immune system. The GALT and mesenteric lymph nodes (MLNs) are important lymphoid tissues that start immune responses to orally delivered antigens and help build systemic immunity [ 11 ]. Dendritic cells (DCs) in the intestine have several functional subsets. Among them, CD103 + DCs can move from the intestinal mucosa to the MLNs and activate T cells [ 12 – 14 ]. CD11c + MHC II + CD86 + cells are also common indicators of DC activation [ 15 , 16 ]. Radiation therapy (RT) not only eradicates tumor cells directly but also promotes the release of tumor antigens and danger-associated molecular patterns (DAMPs), which activate antigen-presenting cells and enhance T-cell–mediated antitumor immunity [ 17 – 19 ]. These immunogenic effects of RT provide a mechanistic rationale for combining RT with immune modulators that can prime systemic immunity. We hypothesized that oral administration of L.E.M. activates DCs within the intestinal immune system and that its combination with RT amplifies antitumor T-cell responses. This study investigated how L.E.M. modulates the intestinal immune pathway and evaluated the immunological and therapeutic effects of combining L.E.M. with RT in a tumor-bearing mouse model. Results Effects of oral L.E.M. administration on CD11c + DCs in mesenteric lymph nodes First, we examined how oral administration of L.E.M. affects DCs within the GALT, particularly in the MLNs, which serve as the principal draining lymphoid organs for intestinal immune responses. Mice were divided into two groups: Control (Cont.) and L.E.M.. Representative flow-cytometric plots illustrate the gating strategy used to identify CD11c + DCs in the MLNs after excluding doublets and dead cells (Fig. 1 A). After selecting the CD11c + population, we analyzed the expression of MHC class II (I-A/I-E) and CD86, which are well-established surface markers of DC maturation involved in antigen presentation and co-stimulation of T cells 15,16 . We also evaluated the proportion of CD103 + DCs, a subset representing intestinal-type migratory DCs that transport luminal antigens from the gut epithelium to the MLNs, thereby priming cytotoxic T lymphocytes (CTLs) [ 12 – 14 ]. Quantitative analyses (Figs. 1 B and 1 C) showed that the proportion of MHC II + CD86 + cells among CD11c + DCs in the MLNs was significantly higher in the L.E.M. group (32.5 ± 7.4%) than in the Cont. group (18.8 ± 4.3%; p = 0.0073). Likewise, the proportion of CD103 + DCs was markedly increased in the L.E.M. group (41.3 ± 1.6%) compared with the Cont. group (32.4 ± 3.0%; p = 0.0004). These effects were consistently observed across independent experiments, demonstrating a reproducible enhancement of DC activation following oral L.E.M. intake. All these findings demonstrate that oral administration of L.E.M. promotes DC maturation and selectively expands the CD103 + migratory DC subset in the MLNs. Because CD103 + DCs are key mediators of cross-presentation and CTL priming, these results suggest that L.E.M. enhances systemic antitumor immunity by strengthening GALT-mediated intestinal immune activation. Effects of oral L.E.M. administration and X-ray irradiation on CD11c DCs in the spleen Then, we investigated how oral administration of L.E.M. and X-ray irradiation influence systemic immune responses, focusing on the activation and subsets of DCs in the spleen. The spleen plays a central role in systemic antigen presentation and immune regulation, and splenic DC activation reflects systemic immune priming following local or mucosal stimulation. Representative flow-cytometric plots (Fig. 2 A) show the gating strategy used to identify CD11c + DCs in the spleen after excluding doublets and dead cells. Within the CD11c + population, we analyzed MHC class II and CD86 expression as indicators of DC maturation and co-stimulatory potential. We quantified CD8α + DCs—a subset specialized in cross-presentation and CTL priming. Quantitative analyses (Figs. 2 B and 2 C) demonstrated that the proportion of MHC II + CD86 + cells among splenic CD11c + DCs was 18.8 ± 4.2% in the Cont. group, 22.6 ± 3.4% in the L.E.M. group, 17.7 ± 1.8% in the X-ray group, and 26.1 ± 3.4% in the X + L.E.M. group. Although the increase in the L.E.M. group compared with the Cont. group was not statistically significant (p = 0.2920), the X + L.E.M. group showed a significant elevation relative to the X-ray group (p = 0.0051). The proportion of CD8α + DCs was 14.3 ± 3.2% in the Cont. group, 19.8 ± 1.4% in the L.E.M. group, 16.7 ± 2.7% in the X-ray group, and 21.2 ± 3.3% in the X + L.E.M. group. The L.E.M. group showed a significant increase compared with the Cont. group (p = 0.0276), and the X + L.E.M. group had values higher than did the X-ray group (p = 0.0831). All these results indicate that oral L.E.M. administration alone promotes DC maturation and increases the proportion of CD8α + DCs in the spleen, and that these effects are further enhanced when L.E.M. is combined with RT. They suggest that L.E.M. contributes not only to mucosal immune activation in the MLNs but also to systemic DC priming, thereby amplifying CTL-mediated antitumor immunity in combination with X-ray irradiation. (A) Representative gating strategy for splenic CD11c + DCs. Expression of MHC II, CD86, and CD8α was analyzed. (B) Percentage of MHC II + CD86 + cells within the CD11c + population. (C) Percentage of CD8α + cells within the CD11c + population. Each group, n = 5. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison test; p < 0.05 was considered statistically significant. The experiment was independently repeated twice with similar results. Effects of oral L.E.M. administration and X-ray irradiation on peripheral CD8 T-cell memory subsets We further examined whether L.E.M. and X-ray irradiation affected the differentiation of peripheral CD8 + T cells into memory subsets (Fig. 3 ). Memory-type CTLs are critical for sustaining long-term antitumor immunity and for rapid recall responses after antigen exposure. Representative flow-cytometric plots (Fig. 3 A) show the gating strategy for CD8 + T cells in peripheral blood. After selecting CD45 + CD8 + cells, effector-memory (EM; CD44 + CD62L − ) and central-memory (CM; CD44 + CD62L + ) subsets were defined according to CD44 and CD62L expression. EM cells represent effector-type memory T cells that rapidly respond in peripheral tissues, whereas CM cells maintain long-term immune memory within secondary lymphoid organs. Quantitative analyses demonstrated that L.E.M. alone did not significantly change either subset: the proportion of EM cells (Fig. 3 B) was 2.4 ± 0.8% in the Cont. group and 2.0 ± 0.8% in the L.E.M. group (p = 0.9880), while that of CM cells (Fig. 3 C) was 21.7 ± 2.0% in the Cont. group and 17.5 ± 5.9% in the L.E.M. group (p = 0.7230). Conversely, combination treatment with L.E.M. and X-ray irradiation significantly increased both subsets: EM cells (X-ray 2.4 ± 0.8%, X + L.E.M. 5.9 ± 3.4%, p = 0.0334) and CM cells (X-ray 14.6 ± 7.0%, X + L.E.M. 28.5 ± 8.7%, p = 0.0158). These findings indicate that oral L.E.M. alone did not markedly alter peripheral CTL memory differentiation, but when combined with RT, it promoted both effector- and central-memory CTL subsets. They suggest that L.E.M. enhances the generation of memory-type CTLs in response to radiation-induced tumor-antigen release, thereby contributing to durable systemic antitumor immunity. (C) Proportion of CM (CD44 + CD62L + ) CD8 + T cells. Each group, n = 5. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test; p < 0.05 was considered statistically significant. The experiment was independently repeated twice with similar results. Effects of oral L.E.M. administration and X-ray irradiation on intratumoral CD8 T-cell responses We next evaluated the impact of L.E.M. and X-ray irradiation on antigen-specific CTL infiltration and activation within the tumor (Fig. 4 ). Tumor-infiltrating CTLs are essential effector cells that directly eliminate tumor cells and play a central role in local immune control. Representative flow-cytometric plots (Fig. 4 A) show the gating strategy used for the analysis. Live (7-AAD − ) cells were gated, and within these, CD8 + T cells were identified and further analyzed for OVA tetramer binding to detect antigen-specific CTLs. Quantitative analyses (Fig. 4 B) demonstrated that the mean proportions of OVA-tetramer + cells among CD8 + T cells were 12.6 ± 4.5% in the Cont. group, 9.3 ± 1.7% in the L.E.M. group, 15.7 ± 2.9% in the X-ray group, and 25.6 ± 7.4% in the X + L.E.M. group. One-way ANOVA revealed a significant overall difference among the four groups (p = 0.002). Post hoc Tukey’s multiple-comparison test showed that the X + L.E.M. group exhibited significantly higher proportions of antigen-specific CTLs than the Cont. (p = 0.0091), L.E.M. (p = 0.0016), and X-ray (p = 0.0470) groups. However, no significant differences were observed among the other comparisons. These findings indicate that oral L.E.M. alone did not markedly enhance antigen-specific CTL infiltration into the tumor, but when combined with RT, it markedly augmented this response. They suggest that L.E.M. enhances radiation-induced, tumor-specific CTL immunity, thereby contributing to stronger local antitumor effects. Effects of oral L.E.M. administration and X-ray irradiation on tumor growth Finally, we evaluated how oral administration of L.E.M. and X-ray irradiation affected tumor growth in vivo (Fig. 5 ). Tumor volume was measured every few days, and the experimental endpoints corresponded to the time points when the maximum allowable tumor diameter (17 mm), defined by the animal ethics protocol, was reached. Representative tumor growth curves and quantitative analyses are shown in Figs. 5 A and 5 B, respectively. Oral L.E.M. alone significantly suppressed tumor progression. On day 10, the mean tumor volume in the L.E.M. group (670.9 ± 296.2 mm³) was significantly smaller than that in the Cont. group (1471.0 ± 692.6 mm 3 , p = 0.0449). In irradiated mice, tumor growth was delayed compared with controls, and this effect was further enhanced by combination treatment: on day 25, the mean tumor volume in the X + L.E.M. group (631.6 ± 272.7 mm 3 ) was significantly lower than in the X-ray group (1613.0 ± 892.6 mm 3 , p = 0.0466). These findings indicate that oral L.E.M. alone exerted a measurable antitumor effect, and that its combination with RT further enhanced tumor growth suppression, suggesting a synergistic interaction between intestinal immune modulation by L.E.M. and radiation-induced local tumor control. Discussion This study demonstrated that oral administration of L.E.M. activates DCs within the GALT and, when combined with RT, enhances CD8 + T-cell responses and tumor growth control. L.E.M. alone promoted DC maturation in the MLNs and spleen. However, its combination with X-ray irradiation further amplified CD8 + T-cell activation in the peripheral blood and tumor microenvironment. These results support our hypothesis that intestinal immune modulation by oral L.E.M. synergizes with local RT to augment systemic antitumor immunity. Although L.E.M. alone promoted DC maturation in MLNs and the spleen (Figs. 1 and 2 ), it caused no marked change in peripheral memory CD8 + T-cells (Fig. 3 ) and tumor-infiltrating OVA-specific CD8 + T-cells (Fig. 4 ), despite clear inhibition of tumor growth (Fig. 5 ). This apparent discrepancy may be attributable to two possible factors. First, the limited tumor-antigen release and weak damage-associated molecular pattern (DAMP) signaling under L.E.M. treatment alone likely restrict systemic CD8 + T-cell differentiation and tumor infiltration. Second, the CD8 + T-cell response induced by L.E.M. could have occurred transiently at a different time point from that observed after X-ray irradiation, and thus, the transient response was not captured in the present analysis. Nevertheless, even if such a temporal fluctuation was missed, the absence of a sustained CD8 + T-cell increase suggests that L.E.M. primarily modulates the quality rather than the quantity of T-cell responses. This interpretation is consistent with previous descriptions of intestinal DC subsets specialized in cross-presentation [ 14 , 20 , 21 ] and with reports that L.E.M. restores antigen-presenting function and T-cell priming by attenuating IL-6 production and myeloid-derived suppressor cell (MDSC) accumulation [ 10 ]. This mechanism is also consistent with earlier studies describing L.E.M. as an immune-priming modulator. Those studies showed that L.E.M. enhances IFN-γ production by CTLs upon ex vivo restimulation with tumor antigens or tumor cells, thereby evaluating post-stimulation reactivity rather than baseline immunity [ 7 , 9 , 10 ]. In contrast, the present study directly quantified CD8 + T-cells in vivo without antigenic restimulation, demonstrating that L.E.M. establishes an immunologically responsive environment that becomes functionally activated upon radiation-induced antigen release. When radiation provided the additional antigenic and danger signals, CTL responses became prominent within the L.E.M.-conditioned immune context, resulting in maximal tumor control. Thus, L.E.M. acts as an “immune-priming” phase that precedes and amplifies the “antigen-triggering” phase elicited by RT. RT promotes DC maturation and CTL activation through the release of tumor antigens and DAMPs (e.g., high mobility group box-1 (HMGB-1), ATP, calreticulin) and the activation of the cyclic GMP–AMP synthase–stimulator of interferon genes (cGAS–STING) pathway, which induces type I interferon (IFN) production and immunogenic cell death [ 18 , 22 ]. These mechanisms provide a rationale for combining oral immunomodulators with RT to enhance antigen presentation and CTL priming. Improved survival was reported when PSK (a b-glucan from Coriolus versicolor ) was combined with RT [ 23 ], and clinical data have shown maintenance or improvement of immune indices such as CD4/CD8 ratio and natural killer (NK) cell activity under such regimens [ 24 ]. Our findings that L.E.M. augments the immunogenicity of RT thus support a new concept of intestinal immune-priming–mediated radioimmunotherapy. The proposed intestinal mechanism of L.E.M. aligns with evidence from other mushroom-derived polysaccharides. The a-glucan YM-2A from Grifola frondosa (maitake) activates DCs and macrophages in Peyer’s patches, increases MHC class II and CD86 expression, and enhances IL-12 and TNF-α production, promoting systemic CTL responses [ 25 ]. Polysaccharides from Ganoderma lucidum (reishi) reshape gut microbiota, improve mucosal immunity, and suppress inflammation-associated tumorigenesis [ 26 ]. Likewise, Hericium erinaceus (lion’s mane) polysaccharides boost short-chain fatty acid production, strengthen intestinal barrier function, and restore immune balance [ 27 ]. In addition, orally administered β-glucans such as PSK have been clinically tested as adjuvant therapies to sustain host immunity and enhance CTL/NK-cell responses during RT or chemotherapy [ 28 ]. The enhanced DC maturation and expansion of CD103 + DCs observed in MLNs in our study fit well with this framework, suggesting that L.E.M. functions as an oral immunomodulator that activates gut immunity and, in turn, amplifies systemic CTL responses. This study has certain limitations. It was based on a single tumor model and limited time points, and the causal relationships among GALT-mediated DC activation, CD8⁺ T-cell responses, and radiation-induced immunity were not directly tested. Nevertheless, the present results strongly support the idea that oral L.E.M. enhances radiation-induced antitumor immunity via intestinal immune activation. Future studies using time-course and functional inhibition analyses will help further clarify this mechanism. In conclusion, our study demonstrates that oral L.E.M. activates DCs via GALT and synergistically enhances CTL-mediated antitumor immunity and tumor control when combined with RT. L.E.M. is orally administrable in outpatient settings, minimally invasive, and suitable for long-term immune modulation. Future studies should aim to (i) clarify the molecular basis of intestinal immune activation, (ii) comprehensively delineate L.E.M.-induced immunostimulatory pathways, (iii) investigate links between gut microbiota alterations and systemic immunity, (iv) optimize radiation dose and fractionation, and (v) evaluate combinations with immune-checkpoint inhibitors or cancer vaccines. Integrating these efforts may establish a novel therapeutic paradigm of “oral immune adjuvant + radiation therapy,” enabling safer and more durable induction of antitumor immunity. Materials and Methods Cell lines Mouse melanoma B16F10 cells were obtained from the American Type Culture Collection (ATCC). Cells were cultured in RPMI 1640 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal calf serum (FCS), 2 mmol/L L-glutamine, 0.05 mmol/L 2-mercaptoethanol, penicillin, and streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. B16F10-ovalbumin (OVA) cells were established by stable transfection with pAc-Neo-OVA (Addgene, Watertown, MA, USA). Animals and tumor models Female C57BL/6 mice (6–8 weeks old) were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and maintained under specific pathogen–free (SPF) conditions with free access to food and water. Mice of this age typically weighed approximately 18–22 g, consistent with supplier records for C57BL/6 females. All animal experiments were approved by the Institutional Animal Care and Use Committee of the National Institutes for Quantum Science and Technology (approval no. 18-1024). The study is reported in accordance with ARRIVE guidelines, and all procedures were performed in accordance with institutional regulations and relevant national guidelines. B16F10-OVA melanoma cells (7.5 × 10 5 cells/mouse) were injected intradermally into the right hind footpad. L.E.M. feeding (2% in diet) started one day after tumor inoculation and continued until the end of the experiment. All procedures, including irradiation and tissue collection, were performed under deep anesthesia with secobarbital sodium (intraperitoneal injection, 5 mg/ml; 0.5 ml/10 g body weight, overdose level) (Nichi-Iko Pharmaceutical Co., Ltd. Toyama, Japan) to minimize distress. For euthanasia, the same agent was administered at overdose concentration followed by cervical dislocation to ensure death. Oral administration of Lentinula edodes mycelia extract (L.E.M.) L.E.M. powder was kindly provided by Kobayashi Pharmaceutical Co., Ltd. (Osaka, Japan). L.E.M. is a dried hot-water extract obtained from Lentinula edodes mycelia cultured in a bagasse-rice bran medium and harvested prior to fruiting, as previously described [ 3 ]. For oral administration, standard powdered chow (CE-2; CLEA Japan, Tokyo, Japan) was mixed with 2% (w/w) L.E.M. Mice were allowed free access to this diet throughout the experiment. Control mice received the same chow without L.E.M. Local X-ray irradiation When tumors reached approximately 6–8 mm in diameter (day 7 after inoculation), local X-ray irradiation (8 Gy) was delivered using an X-ray generator with a tungsten target (TITAN, Shimadzu, Kyoto, Japan) operating at 200 kV and 20 mA. Aluminum (0.5 mm) and copper (0.5 mm) filters were applied, and the dose rate was 2.2 Gy/min. Mice were anesthetized with thiamylal sodium (5 mg/mL in PBS, 0.1 mL/10 g body weight) administered intraperitoneally 20 min before irradiation. For tumor growth monitoring, non-irradiated mice were euthanized when tumors reached the humane endpoint (~ 17 mm in diameter). For immunological analyses, a separate cohort—including non-irradiated mice—was sampled on day 11, when tumors remained below the endpoint (< 17 mm) owing to individual growth variation. Preparation of single-cell suspensions In the non-irradiated group (Fig. 1 ), MLNs were collected 18 days after tumor inoculation. In irradiated groups (Figs. 2 – 4 ), X-ray irradiation was performed 7 days after inoculation (defined as day 0), and MLNs, spleens, peripheral blood mononuclear cells (PBMCs), and tumors were harvested 11 days later. Collected tissues were digested at 37°C for 30 min with 125 U/mL collagenase IV (Worthington Biochemical, Lakewood, NJ) and 60 U/mL DNase I Type IV (Sigma-Aldrich, St. Louis, MO). PBMCs were isolated using OptiPrep lymphocyte separation solution (ρ = 1.0875 g/mL; Serumwerk Bernburg AG, Bernburg, Germany). After digestion and separation, samples were washed with PBS containing 2% FBS and filtered through a 70 µm cell strainer. Flow cytometry Fluorescence data were acquired using an SA3800 cell analyzer (Sony, Tokyo, Japan) and analyzed with Kaluza software (Beckman Coulter, Brea, CA, USA). Cells were first blocked with anti-CD16/32 antibody (clone 2.4G2; BioLegend, San Diego, CA, USA) and then stained with fluorochrome-conjugated anti-mouse antibodies. The following antibodies were purchased from BioLegend (San Diego, CA, USA): CD11c-Pacific Blue (clone N418), I-A/I-E-APC-Cy7 or -PerCP-Cy5.5 (clone M5/114.15.2), CD86-APC or -APC-Cy7 (clone GL-1), CD103-PE (clone 2E7), CD45-Alexa Fluor 700 (clone 30-F11), CD44-PE-Cy7 (clone IM7), and CD62L-APC (clone MEL-14). CD8α-FITC was obtained from MLB (Tokyo, Japan). OVA tetramer-PE (MLB, Tokyo, Japan) was used to detect antigen-specific CD8 + T cells, and 7-AAD (Beckman Coulter, Brea, CA, USA) was used for viability staining. Tumor growth curve Tumor size was measured every 3–4 days with a digital caliper, and tumor volume (V) was calculated using the formula: V = (π/6) × L × D × H, where L, D, and H represent length, width, and height, respectively. Measurements were performed in a blinded manner by an investigator independent of group allocation. In irradiation groups, local X-ray irradiation (8 Gy) was delivered when the tumor diameter reached 5–6 mm. Humane endpoints were defined as tumor diameter ≥ 17 mm, ulceration, > 20% necrosis, or > 20% body weight loss. Statistical analysis Statistical analyses were performed using R software version 4.5.1 (The R Foundation for Statistical Computing, Vienna, Austria). Data are presented as the mean ± standard deviation (SD). Differences between two groups were analyzed using an unpaired two-tailed Student’s t-test. For comparisons among more than four groups, one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was applied. p < 0.05 was considered statistically significant. Declarations Acknowledgements We thank Drs. Satoru Ishikawa and Yasunori Matsui from Kobayashi Pharmaceutical Co., Ltd. (Osaka, Japan) for kindly providing the Lentinula edodes mycelia (L.E.M.) powder used in this study. We also thank our laboratory members for their technical assistance and helpful discussions. Finally, we would like to thank Editage (www.editage.jp) for English language editing. Funding This work was supported by a research fund from the National Institutes for Quantum Science and Technology, Japan. Author information Authors and Affiliations Department of Charged Particle Therapy Research, National Institutes for Quantum Science and Technology, Chiba 263-8555, Japan T. Takeshima and S. Hasegawa Epilepsy Center, Xiamen Humanity Hospital, Fujian Medical University, Xiamen 361000, Fujian, China Y. Wang Author Contributions Conceptualization, T.T.; Data curation, Y.W. and T.T.; Formal analysis, Y.W. and T.T.; Funding acquisition, S.H.; Methodology, T.T.; Project administration, T.T. and S.H.; Supervision, T.T. and S.H.; Writing - original draft, T.T.; Writing - review & editing, T.T. and S.H. All authors have read and agreed to the published version of the manuscript. Corresponding Author Correspondence to Tsuguhide Takeshima. Data availability statement The data generated in this study are available upon request to the corresponding author. Ethics declarations Competing interests The authors declare no competing interests. References Gajewski, T. F., Schreiber, H. & Fu, Y. X. Innate and adaptive immune cells in the tumor microenvironment. Nat. Immunol. 14 , 1014–1022 (2013). Sharma, P. & Allison, J. P. The future of immune checkpoint therapy. Science 348 , 56–61 (2015). Kojima, H., Akaki, J., Nakajima, S., Kamei, K. & Tamesada, M. Structural analysis of glycogen-like polysaccharides having macrophage-activating activity in extracts of Lentinula edodes mycelia. J. Nat. Med. 64 , 16–23 (2010). Sugano, N., Hibino, Y., Choji, Y. & Maeda, H. Anticarcinogenic actions of water-soluble and alcohol-insoluble fractions from culture medium of Lentinus edodes mycelia. Cancer Lett. 17 , 109–114 (1982). Sugano, N., Choji, Y., Hibino, Y., Yasumura, S. & Maeda, H. Anticarcinogenic action of an alcohol-insoluble fraction (LAP1) from culture medium of Lentinus edodes mycelia. Cancer Lett. 27 , 1–6 (1985). Suzuki, H. et al. Structural characterization of the immunoactive and antiviral water-solubilized lignin in an extract of the culture medium of Lentinus edodes mycelia (L.E.M). Agric. Biol. Chem. 54 , 479–487 (1990). Tanaka, K. et al. Oral ingestion of Lentinula edodes mycelia extract inhibits B16 melanoma growth via mitigation of regulatory T cell-mediated immunosuppression. Cancer Sci. 102 , 516–521 (2011). Tanaka, K., Matsui, Y., Ishikawa, S., Kawanishi, T. & Harada, M. Oral ingestion of Lentinula edodes mycelia extract can restore the antitumor T cell response of mice inoculated with colon-26 cells into the subserosal space of the cecum. Oncol. Rep. 27 , 325–332 (2012). Tanaka, K. et al. Combining a peptide vaccine with oral ingestion of Lentinula edodes mycelia extract enhances antitumor activity in B16 melanoma-bearing mice. Cancer Immunol. Immunother . 61 , 2143–2152 (2012). Ishikawa, S. et al. Age-associated impairment of antitumor immunity in carcinoma-bearing mice and restoration by oral administration of Lentinula edodes mycelia extract. Cancer Immunol. Immunother . 65 , 961–972 (2016). Geem, D. et al. Contribution of mesenteric lymph nodes and GALT to the intestinal Foxp3 + regulatory T-cell compartment. Cell. Mol. Gastroenterol. Hepatol. 2 , 274–280 (2016). Scott, C. L., Aumeunier, A. M. & Mowat, A. M. Intestinal CD103(+) dendritic cells: master regulators of tolerance? Trends Immunol. 32 , 412–419 (2011). Cerovic, V. et al. Intestinal CD103(-) dendritic cells migrate in lymph and prime effector T cells. Mucosal Immunol. 6 , 104–113 (2013). Jaensson, E. et al. Small intestinal CD103 + dendritic cells display unique functional properties that are conserved between mice and humans. J. Exp. Med. 205 , 2139–2149 (2008). Villadangos, J. A. & Schnorrer, P. Intrinsic and cooperative antigen-presenting functions of dendritic-cell subsets in vivo. Nat. Rev. Immunol. 7 , 543–555 (2007). Kadowaki, T. et al. Reconsideration of macrophage and dendritic cell classification. Anticancer Res. 32 , 2257–2261 (2012). Takeshima, T. et al. Local Radiation Therapy Inhibits Tumor Growth through the Generation of Tumor-Specific CTL: Its Potentiation by Combination with Th1 Cell Therapy. Cancer Res. 70 , 2697–2706 (2010). Demaria, S. & Formenti, S. C. Radiotherapy effects on antitumor immunity: implications for cancer treatment. Front. Oncol. 3 , 128 (2013). Sharabi, A. B., Lim, M., DeWeese, T. L. & Drake, C. G. Radiation and checkpoint blockade immunotherapy: radiosensitisation and potential mechanisms of synergy. Lancet Oncol. 16 , e498–e509 (2015). Edelson, B. T. et al. Peripheral CD103 + dendritic cells form a unified subset developmentally related to CD8alpha + conventional dendritic cells. J. Exp. Med. 207 , 823–836 (2010). Hildner, K. et al. Batf3 deficiency reveals a critical role for CD8alpha + dendritic cells in cytotoxic T cell immunity. Science 322 , 1097–1100 (2008). Guo, S. et al. Radiation-induced tumor immune microenvironments and potential targets for combination therapy. Signal. Transduct. Target. Ther. 8 , 205 (2023). Hayakawa, K. et al. Effect of krestin (PSK) as adjuvant treatment on the prognosis after radical radiotherapy in patients with non-small cell lung cancer. Anticancer Res. 13 , 1815–1820 (1993). Fritz, H. et al. Polysaccharide K and Coriolus versicolor extracts for lung cancer: a systematic review. Integr. Cancer Ther. 14 , 201–211 (2015). Masuda, Y., Nakayama, Y., Tanaka, A., Naito, K. & Konishi, M. Antitumor activity of orally administered maitake alpha-glucan by stimulating antitumor immune response in murine tumor. PLOS One . 12 , e0173621 (2017). Guo, C. et al. Ganoderma lucidum polysaccharide modulates gut microbiota and immune cell function to inhibit inflammation and tumorigenesis in colon. Carbohydr. Polym. 267 , 118231 (2021). Tian, B. et al. Modulating effects of Hericium erinaceus polysaccharides on the immune response by regulating gut microbiota in cyclophosphamide-treated mice. J. Sci. Food Agric. 103 , 3050–3064 (2023). de Graaff, P., Govers, C., Wichers, H. J. & Debets, R. Consumption of beta-glucans to spice up T cell treatment of tumors: a review. Expert Opin. Biol. Ther. 18 , 1023–1040 (2018). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 16 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 31 Dec, 2025 Reviews received at journal 27 Dec, 2025 Reviews received at journal 22 Dec, 2025 Reviewers agreed at journal 17 Dec, 2025 Reviewers agreed at journal 14 Dec, 2025 Reviewers invited by journal 12 Dec, 2025 Editor assigned by journal 12 Dec, 2025 Editor invited by journal 10 Dec, 2025 Submission checks completed at journal 09 Dec, 2025 First submitted to journal 09 Dec, 2025 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. 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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-8096960","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":558382498,"identity":"ec581b71-0170-4f40-bccf-5223344d57e2","order_by":0,"name":"Tsuguhide Takeshima","email":"data:image/png;base64,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","orcid":"","institution":"National Institutes for Quantum Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Tsuguhide","middleName":"","lastName":"Takeshima","suffix":""},{"id":558382499,"identity":"680c70e1-98d7-4c9e-8cb9-5d60bcc67341","order_by":1,"name":"Yang Wang","email":"","orcid":"","institution":"Xiamen Humanity Hospital Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Wang","suffix":""},{"id":558382500,"identity":"23f1d3c3-09a2-48cb-af98-97e51eae8ed6","order_by":2,"name":"Sumitaka Hasegawa","email":"","orcid":"","institution":"National Institutes for Quantum Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Sumitaka","middleName":"","lastName":"Hasegawa","suffix":""}],"badges":[],"createdAt":"2025-11-12 13:38:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8096960/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8096960/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-35752-7","type":"published","date":"2026-01-16T16:28:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":98427150,"identity":"76bf3db2-272b-4c7f-abca-140e245e887f","added_by":"auto","created_at":"2025-12-17 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1","display":"","copyAsset":false,"role":"figure","size":95869,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOral administration of L.E.M. increases the activated phenotype (MHC II\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e CD86\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e) and the CD103\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e subset of DCs in mesenteric lymph nodes\u003c/strong\u003e. Mesenteric lymph nodes (MLNs) were collected 18 days after tumor inoculation from mice that received L.E.M. (2% in chow) starting the day after tumor inoculation. (A) Representative gating strategy for CD11c\u003csup\u003e+\u003c/sup\u003e DCs. Expression of MHC II, CD86, and CD103 was analyzed. (B) Percentage of MHCII\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e cells within the CD11c\u003csup\u003e+\u003c/sup\u003e population. (C) Percentage of CD103\u003csup\u003e+\u003c/sup\u003e cells within the CD11c\u003csup\u003e+\u003c/sup\u003e population. Each dot represents an individual mouse (n = 5 per group). Data are presented as mean ± SD. Statistical analysis was performed using an unpaired two-tailed Student’s t-test; p \u0026lt; 0.05 was considered statistically significant. Cont, control group; L.E.M., Lentinula edodes mycelia extract; MLN, mesenteric lymph node. The experiment was independently repeated twice with similar results.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8096960/v1/7efa201c669ac3f6a69c7562.png"},{"id":98043173,"identity":"7ef35d08-1510-4dfa-8be3-512159db0d9c","added_by":"auto","created_at":"2025-12-12 07:39:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":128501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOral administration of L.E.M. and X-ray irradiation increase the activated phenotype and the CD8α\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e subset of splenic DCs. \u003c/strong\u003eLocal X-ray irradiation (8 Gy) was performed 7 days after tumor inoculation (defined as day 0), and spleens were collected 11 days after irradiation for flow cytometric analysis.\u003c/p\u003e\n\u003cp\u003e(A) Representative gating strategy for splenic CD11c\u003csup\u003e+\u003c/sup\u003e DCs. Expression of MHC II, CD86, and CD8α was analyzed. (B) Percentage of MHC II\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e cells within the CD11c\u003csup\u003e+\u003c/sup\u003e population. (C) Percentage of CD8α\u003csup\u003e+\u003c/sup\u003e cells within the CD11c\u003csup\u003e+\u003c/sup\u003e population. Each group, n = 5. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison test; p \u0026lt; 0.05 was considered statistically significant. The experiment was independently repeated twice with similar results.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8096960/v1/ea7a843406a67c7c6ee5dd5e.png"},{"id":98425661,"identity":"f570f1fd-b159-4cb4-bc35-26d936ad587f","added_by":"auto","created_at":"2025-12-17 16:35:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":117844,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOral administration of L.E.M. and X-ray irradiation increase effector-memory (EM) and central-memory (CM) subsets of peripheral CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells.\u003c/strong\u003e Local X-ray irradiation (8 Gy) was performed 7 days after tumor inoculation (defined as day 0), and peripheral blood was collected 11 days after irradiation for flow-cytometric analysis. (A) Representative gating strategy for peripheral CD8\u003csup\u003e+\u003c/sup\u003e T cells. Expression of CD44 and CD62L was analyzed to identify EM (CD44\u003csup\u003e+\u003c/sup\u003eCD62L⁻) and CM (CD44\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003e) subsets. (B) Proportion of EM (CD44\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e-\u003c/sup\u003e) CD8\u003csup\u003e+\u003c/sup\u003e T cells.\u003c/p\u003e\n\u003cp\u003e(C) Proportion of CM (CD44\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003e) CD8\u003csup\u003e+\u003c/sup\u003e T cells. Each group, n = 5. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test; p \u0026lt; 0.05 was considered statistically significant. The experiment was independently repeated twice with similar results.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8096960/v1/3cb8fac5876cbba60b5d5fc3.png"},{"id":98043174,"identity":"3345d2a8-1ed4-4c98-8fd7-67d325c575f3","added_by":"auto","created_at":"2025-12-12 07:39:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":104473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOral administration of L.E.M. and X-ray irradiation enhance tumor-infiltrating antigen-specific CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T-cell responses.\u003c/strong\u003e Local X-ray irradiation (8 Gy) was performed 7 days after tumor inoculation (defined as day 0), and tumors were collected 11 days after irradiation for flow-cytometric analysis. (A) Representative gating strategy for tumor-infiltrating CD8\u003csup\u003e+\u003c/sup\u003e T cells. Live (7-AAD⁻) cells were gated, CD8\u003csup\u003e+\u003c/sup\u003e T cells were identified, and OVA-tetramer\u003csup\u003e+\u003c/sup\u003e CTLs were quantified to evaluate antigen-specific responses within the tumor. (B) Quantitative analysis of the proportion of OVA-tetramer\u003csup\u003e+\u003c/sup\u003e CTLs among CD8\u003csup\u003e+\u003c/sup\u003e T cells. Each group, n = 4. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison test; p \u0026lt; 0.05 was considered statistically significant. The experiment was independently repeated twice with similar results.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8096960/v1/60c7b4eabf4320b2663f9edd.png"},{"id":98425869,"identity":"7c1506ca-4bd2-47ba-8fd8-b59eedd03795","added_by":"auto","created_at":"2025-12-17 16:35:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":80199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOral administration of L.E.M. and X-ray irradiation suppress tumor growth in vivo.\u003c/strong\u003e C57BL/6 mice were subcutaneously inoculated with B16F10-OVA cells and randomly assigned to each group. L.E.M. feeding (2% in chow) began the day after inoculation, and local X-ray irradiation (8 Gy) was performed on day 7 after tumor inoculation, when tumors reached 5–7 mm in diameter. (A) Tumor growth curves. Mice in the Cont. and L.E.M. groups were sacrificed on day 10, and those in the X-ray and X + L.E.M. groups on day 25. (B) Final tumor volume on day 10 (Cont. vs L.E.M.). (C) Final tumor volume on day 25 (X-ray vs X + L.E.M.). Each group, n = 5. Data are presented as mean ± SD. Statistical analysis was performed using an unpaired two-tailed Student’s t-test; p \u0026lt; 0.05 was considered statistically significant. The experiment was independently repeated twice with similar results.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8096960/v1/d2f47456b93a4a0a929763a9.png"},{"id":100614314,"identity":"c5e7769b-5dda-4167-9809-0645a2a6a1e4","added_by":"auto","created_at":"2026-01-19 17:18:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1504433,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8096960/v1/08015bf1-1cab-4470-aa74-1efd5e737aa7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oral Lentinula edodes mycelia extract enhances the antitumor effect of radiotherapy via gut-associated activation of dendritic and cytotoxic T cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCancer immunotherapy attempts to control tumor growth by activating T cells that recognize tumor antigens. However, the tumor microenvironment contains regulatory T cells, myeloid-derived suppressor cells, and inhibitory cytokines that suppress antitumor immune responses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Therefore, new treatment methods are needed to reduce immune suppression and to promote antigen presentation and T cell activation.\u003c/p\u003e \u003cp\u003eA hot water extract of dried powder extracted from \u003cem\u003eLentinula edodes\u003c/em\u003e mycelia (L.E.M.), prepared from cultured biomass grown in a bagasse-rice bran medium and harvested prior to fruiting, has been reported to reduce regulatory T cells and induce CD8\u003csup\u003e+\u003c/sup\u003e T cell\u0026ndash;dependent antitumor effects following oral administration [\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7 CR8 CR9\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The gut-associated lymphoid tissue (GALT) has been suggested as the first site where L.E.M. acts in the body [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], indicating that L.E.M. may regulate immune activity through the intestinal immune system.\u003c/p\u003e \u003cp\u003eThe GALT and mesenteric lymph nodes (MLNs) are important lymphoid tissues that start immune responses to orally delivered antigens and help build systemic immunity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Dendritic cells (DCs) in the intestine have several functional subsets. Among them, CD103\u003csup\u003e+\u003c/sup\u003e DCs can move from the intestinal mucosa to the MLNs and activate T cells [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. CD11c\u003csup\u003e+\u003c/sup\u003eMHC II\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e cells are also common indicators of DC activation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRadiation therapy (RT) not only eradicates tumor cells directly but also promotes the release of tumor antigens and danger-associated molecular patterns (DAMPs), which activate antigen-presenting cells and enhance T-cell\u0026ndash;mediated antitumor immunity [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These immunogenic effects of RT provide a mechanistic rationale for combining RT with immune modulators that can prime systemic immunity.\u003c/p\u003e \u003cp\u003eWe hypothesized that oral administration of L.E.M. activates DCs within the intestinal immune system and that its combination with RT amplifies antitumor T-cell responses. This study investigated how L.E.M. modulates the intestinal immune pathway and evaluated the immunological and therapeutic effects of combining L.E.M. with RT in a tumor-bearing mouse model.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEffects of oral L.E.M. administration on CD11c\u003csup\u003e+\u003c/sup\u003e DCs in mesenteric lymph nodes\u003c/h2\u003e \u003cp\u003eFirst, we examined how oral administration of L.E.M. affects DCs within the GALT, particularly in the MLNs, which serve as the principal draining lymphoid organs for intestinal immune responses. Mice were divided into two groups: Control (Cont.) and L.E.M..\u003c/p\u003e \u003cp\u003eRepresentative flow-cytometric plots illustrate the gating strategy used to identify CD11c\u003csup\u003e+\u003c/sup\u003e DCs in the MLNs after excluding doublets and dead cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). After selecting the CD11c\u003csup\u003e+\u003c/sup\u003e population, we analyzed the expression of MHC class II (I-A/I-E) and CD86, which are well-established surface markers of DC maturation involved in antigen presentation and co-stimulation of T cells \u003csup\u003e15,16\u003c/sup\u003e. We also evaluated the proportion of CD103\u003csup\u003e+\u003c/sup\u003e DCs, a subset representing intestinal-type migratory DCs that transport luminal antigens from the gut epithelium to the MLNs, thereby priming cytotoxic T lymphocytes (CTLs) [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eQuantitative analyses (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) showed that the proportion of MHC II\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e cells among CD11c\u003csup\u003e+\u003c/sup\u003e DCs in the MLNs was significantly higher in the L.E.M. group (32.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4%) than in the Cont. group (18.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3%; p\u0026thinsp;=\u0026thinsp;0.0073). Likewise, the proportion of CD103\u003csup\u003e+\u003c/sup\u003e DCs was markedly increased in the L.E.M. group (41.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6%) compared with the Cont. group (32.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0%; p\u0026thinsp;=\u0026thinsp;0.0004). These effects were consistently observed across independent experiments, demonstrating a reproducible enhancement of DC activation following oral L.E.M. intake.\u003c/p\u003e \u003cp\u003eAll these findings demonstrate that oral administration of L.E.M. promotes DC maturation and selectively expands the CD103\u003csup\u003e+\u003c/sup\u003e migratory DC subset in the MLNs. Because CD103\u003csup\u003e+\u003c/sup\u003e DCs are key mediators of cross-presentation and CTL priming, these results suggest that L.E.M. enhances systemic antitumor immunity by strengthening GALT-mediated intestinal immune activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEffects of oral L.E.M. administration and X-ray irradiation on CD11c DCs in the spleen\u003c/h3\u003e\n\u003cp\u003eThen, we investigated how oral administration of L.E.M. and X-ray irradiation influence systemic immune responses, focusing on the activation and subsets of DCs in the spleen. The spleen plays a central role in systemic antigen presentation and immune regulation, and splenic DC activation reflects systemic immune priming following local or mucosal stimulation.\u003c/p\u003e \u003cp\u003eRepresentative flow-cytometric plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) show the gating strategy used to identify CD11c\u003csup\u003e+\u003c/sup\u003e DCs in the spleen after excluding doublets and dead cells. Within the CD11c\u003csup\u003e+\u003c/sup\u003e population, we analyzed MHC class II and CD86 expression as indicators of DC maturation and co-stimulatory potential. We quantified CD8α\u003csup\u003e+\u003c/sup\u003e DCs\u0026mdash;a subset specialized in cross-presentation and CTL priming.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eQuantitative analyses (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) demonstrated that the proportion of MHC II\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e cells among splenic CD11c\u003csup\u003e+\u003c/sup\u003e DCs was 18.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2% in the Cont. group, 22.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4% in the L.E.M. group, 17.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8% in the X-ray group, and 26.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4% in the X\u0026thinsp;+\u0026thinsp;L.E.M. group. Although the increase in the L.E.M. group compared with the Cont. group was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.2920), the X\u0026thinsp;+\u0026thinsp;L.E.M. group showed a significant elevation relative to the X-ray group (p\u0026thinsp;=\u0026thinsp;0.0051). The proportion of CD8α\u003csup\u003e+\u003c/sup\u003e DCs was 14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2% in the Cont. group, 19.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4% in the L.E.M. group, 16.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7% in the X-ray group, and 21.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3% in the X\u0026thinsp;+\u0026thinsp;L.E.M. group. The L.E.M. group showed a significant increase compared with the Cont. group (p\u0026thinsp;=\u0026thinsp;0.0276), and the X\u0026thinsp;+\u0026thinsp;L.E.M. group had values higher than did the X-ray group (p\u0026thinsp;=\u0026thinsp;0.0831).\u003c/p\u003e \u003cp\u003eAll these results indicate that oral L.E.M. administration alone promotes DC maturation and increases the proportion of CD8α\u003csup\u003e+\u003c/sup\u003e DCs in the spleen, and that these effects are further enhanced when L.E.M. is combined with RT. They suggest that L.E.M. contributes not only to mucosal immune activation in the MLNs but also to systemic DC priming, thereby amplifying CTL-mediated antitumor immunity in combination with X-ray irradiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) Representative gating strategy for splenic CD11c\u003csup\u003e+\u003c/sup\u003e DCs. Expression of MHC II, CD86, and CD8α was analyzed. (B) Percentage of MHC II\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e cells within the CD11c\u003csup\u003e+\u003c/sup\u003e population. (C) Percentage of CD8α\u003csup\u003e+\u003c/sup\u003e cells within the CD11c\u003csup\u003e+\u003c/sup\u003e population. Each group, n\u0026thinsp;=\u0026thinsp;5. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical analysis was performed using one-way ANOVA followed by Tukey\u0026rsquo;s multiple-comparison test; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. The experiment was independently repeated twice with similar results.\u003c/p\u003e\n\u003ch3\u003eEffects of oral L.E.M. administration and X-ray irradiation on peripheral CD8 T-cell memory subsets\u003c/h3\u003e\n\u003cp\u003eWe further examined whether L.E.M. and X-ray irradiation affected the differentiation of peripheral CD8\u003csup\u003e+\u003c/sup\u003e T cells into memory subsets (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Memory-type CTLs are critical for sustaining long-term antitumor immunity and for rapid recall responses after antigen exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRepresentative flow-cytometric plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) show the gating strategy for CD8\u003csup\u003e+\u003c/sup\u003e T cells in peripheral blood. After selecting CD45\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e cells, effector-memory (EM; CD44\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e\u0026minus;\u003c/sup\u003e) and central-memory (CM; CD44\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003e) subsets were defined according to CD44 and CD62L expression. EM cells represent effector-type memory T cells that rapidly respond in peripheral tissues, whereas CM cells maintain long-term immune memory within secondary lymphoid organs.\u003c/p\u003e \u003cp\u003eQuantitative analyses demonstrated that L.E.M. alone did not significantly change either subset: the proportion of EM cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) was 2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8% in the Cont. group and 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8% in the L.E.M. group (p\u0026thinsp;=\u0026thinsp;0.9880), while that of CM cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) was 21.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0% in the Cont. group and 17.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9% in the L.E.M. group (p\u0026thinsp;=\u0026thinsp;0.7230). Conversely, combination treatment with L.E.M. and X-ray irradiation significantly increased both subsets: EM cells (X-ray 2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8%, X\u0026thinsp;+\u0026thinsp;L.E.M. 5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4%, p\u0026thinsp;=\u0026thinsp;0.0334) and CM cells (X-ray 14.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.0%, X\u0026thinsp;+\u0026thinsp;L.E.M. 28.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.7%, p\u0026thinsp;=\u0026thinsp;0.0158).\u003c/p\u003e \u003cp\u003eThese findings indicate that oral L.E.M. alone did not markedly alter peripheral CTL memory differentiation, but when combined with RT, it promoted both effector- and central-memory CTL subsets. They suggest that L.E.M. enhances the generation of memory-type CTLs in response to radiation-induced tumor-antigen release, thereby contributing to durable systemic antitumor immunity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(C) Proportion of CM (CD44\u003csup\u003e+\u003c/sup\u003eCD62L\u003csup\u003e+\u003c/sup\u003e) CD8\u003csup\u003e+\u003c/sup\u003e T cells. Each group, n\u0026thinsp;=\u0026thinsp;5. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical analysis was performed using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. The experiment was independently repeated twice with similar results.\u003c/p\u003e\n\u003ch3\u003eEffects of oral L.E.M. administration and X-ray irradiation on intratumoral CD8 T-cell responses\u003c/h3\u003e\n\u003cp\u003eWe next evaluated the impact of L.E.M. and X-ray irradiation on antigen-specific CTL infiltration and activation within the tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Tumor-infiltrating CTLs are essential effector cells that directly eliminate tumor cells and play a central role in local immune control.\u003c/p\u003e \u003cp\u003eRepresentative flow-cytometric plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) show the gating strategy used for the analysis. Live (7-AAD\u003csup\u003e\u0026minus;\u003c/sup\u003e) cells were gated, and within these, CD8\u003csup\u003e+\u003c/sup\u003e T cells were identified and further analyzed for OVA tetramer binding to detect antigen-specific CTLs.\u003c/p\u003e \u003cp\u003eQuantitative analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) demonstrated that the mean proportions of OVA-tetramer\u003csup\u003e+\u003c/sup\u003e cells among CD8\u003csup\u003e+\u003c/sup\u003e T cells were 12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5% in the Cont. group, 9.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7% in the L.E.M. group, 15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9% in the X-ray group, and 25.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4% in the X\u0026thinsp;+\u0026thinsp;L.E.M. group. One-way ANOVA revealed a significant overall difference among the four groups (p\u0026thinsp;=\u0026thinsp;0.002). Post hoc Tukey\u0026rsquo;s multiple-comparison test showed that the X\u0026thinsp;+\u0026thinsp;L.E.M. group exhibited significantly higher proportions of antigen-specific CTLs than the Cont. (p\u0026thinsp;=\u0026thinsp;0.0091), L.E.M. (p\u0026thinsp;=\u0026thinsp;0.0016), and X-ray (p\u0026thinsp;=\u0026thinsp;0.0470) groups. However, no significant differences were observed among the other comparisons.\u003c/p\u003e \u003cp\u003eThese findings indicate that oral L.E.M. alone did not markedly enhance antigen-specific CTL infiltration into the tumor, but when combined with RT, it markedly augmented this response. They suggest that L.E.M. enhances radiation-induced, tumor-specific CTL immunity, thereby contributing to stronger local antitumor effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEffects of oral L.E.M. administration and X-ray irradiation on tumor growth\u003c/h3\u003e\n\u003cp\u003eFinally, we evaluated how oral administration of L.E.M. and X-ray irradiation affected tumor growth in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Tumor volume was measured every few days, and the experimental endpoints corresponded to the time points when the maximum allowable tumor diameter (17 mm), defined by the animal ethics protocol, was reached.\u003c/p\u003e \u003cp\u003eRepresentative tumor growth curves and quantitative analyses are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, respectively. Oral L.E.M. alone significantly suppressed tumor progression. On day 10, the mean tumor volume in the L.E.M. group (670.9\u0026thinsp;\u0026plusmn;\u0026thinsp;296.2 mm\u0026sup3;) was significantly smaller than that in the Cont. group (1471.0\u0026thinsp;\u0026plusmn;\u0026thinsp;692.6 mm\u003csup\u003e3\u003c/sup\u003e, p\u0026thinsp;=\u0026thinsp;0.0449). In irradiated mice, tumor growth was delayed compared with controls, and this effect was further enhanced by combination treatment: on day 25, the mean tumor volume in the X\u0026thinsp;+\u0026thinsp;L.E.M. group (631.6\u0026thinsp;\u0026plusmn;\u0026thinsp;272.7 mm\u003csup\u003e3\u003c/sup\u003e) was significantly lower than in the X-ray group (1613.0\u0026thinsp;\u0026plusmn;\u0026thinsp;892.6 mm\u003csup\u003e3\u003c/sup\u003e, p\u0026thinsp;=\u0026thinsp;0.0466).\u003c/p\u003e \u003cp\u003eThese findings indicate that oral L.E.M. alone exerted a measurable antitumor effect, and that its combination with RT further enhanced tumor growth suppression, suggesting a synergistic interaction between intestinal immune modulation by L.E.M. and radiation-induced local tumor control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrated that oral administration of L.E.M. activates DCs within the GALT and, when combined with RT, enhances CD8\u003csup\u003e+\u003c/sup\u003e T-cell responses and tumor growth control. L.E.M. alone promoted DC maturation in the MLNs and spleen. However, its combination with X-ray irradiation further amplified CD8\u003csup\u003e+\u003c/sup\u003e T-cell activation in the peripheral blood and tumor microenvironment. These results support our hypothesis that intestinal immune modulation by oral L.E.M. synergizes with local RT to augment systemic antitumor immunity.\u003c/p\u003e \u003cp\u003eAlthough L.E.M. alone promoted DC maturation in MLNs and the spleen (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e), it caused no marked change in peripheral memory CD8\u003csup\u003e+\u003c/sup\u003e T-cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and tumor-infiltrating OVA-specific CD8\u003csup\u003e+\u003c/sup\u003e T-cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e), despite clear inhibition of tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This apparent discrepancy may be attributable to two possible factors. First, the limited tumor-antigen release and weak damage-associated molecular pattern (DAMP) signaling under L.E.M. treatment alone likely restrict systemic CD8\u003csup\u003e+\u003c/sup\u003e T-cell differentiation and tumor infiltration. Second, the CD8\u003csup\u003e+\u003c/sup\u003e T-cell response induced by L.E.M. could have occurred transiently at a different time point from that observed after X-ray irradiation, and thus, the transient response was not captured in the present analysis. Nevertheless, even if such a temporal fluctuation was missed, the absence of a sustained CD8\u003csup\u003e+\u003c/sup\u003e T-cell increase suggests that L.E.M. primarily modulates the quality rather than the quantity of T-cell responses.\u003c/p\u003e \u003cp\u003eThis interpretation is consistent with previous descriptions of intestinal DC subsets specialized in cross-presentation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and with reports that L.E.M. restores antigen-presenting function and T-cell priming by attenuating IL-6 production and myeloid-derived suppressor cell (MDSC) accumulation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This mechanism is also consistent with earlier studies describing L.E.M. as an immune-priming modulator. Those studies showed that L.E.M. enhances IFN-γ production by CTLs upon ex vivo restimulation with tumor antigens or tumor cells, thereby evaluating post-stimulation reactivity rather than baseline immunity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In contrast, the present study directly quantified CD8\u003csup\u003e+\u003c/sup\u003e T-cells in vivo without antigenic restimulation, demonstrating that L.E.M. establishes an immunologically responsive environment that becomes functionally activated upon radiation-induced antigen release. When radiation provided the additional antigenic and danger signals, CTL responses became prominent within the L.E.M.-conditioned immune context, resulting in maximal tumor control. Thus, L.E.M. acts as an \u0026ldquo;immune-priming\u0026rdquo; phase that precedes and amplifies the \u0026ldquo;antigen-triggering\u0026rdquo; phase elicited by RT.\u003c/p\u003e \u003cp\u003eRT promotes DC maturation and CTL activation through the release of tumor antigens and DAMPs (e.g., high mobility group box-1 (HMGB-1), ATP, calreticulin) and the activation of the cyclic GMP\u0026ndash;AMP synthase\u0026ndash;stimulator of interferon genes (cGAS\u0026ndash;STING) pathway, which induces type I interferon (IFN) production and immunogenic cell death [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These mechanisms provide a rationale for combining oral immunomodulators with RT to enhance antigen presentation and CTL priming. Improved survival was reported when PSK (a b-glucan from \u003cem\u003eCoriolus versicolor\u003c/em\u003e) was combined with RT [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and clinical data have shown maintenance or improvement of immune indices such as CD4/CD8 ratio and natural killer (NK) cell activity under such regimens [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Our findings that L.E.M. augments the immunogenicity of RT thus support a new concept of intestinal immune-priming\u0026ndash;mediated radioimmunotherapy.\u003c/p\u003e \u003cp\u003eThe proposed intestinal mechanism of L.E.M. aligns with evidence from other mushroom-derived polysaccharides. The a-glucan YM-2A from \u003cem\u003eGrifola frondosa\u003c/em\u003e (maitake) activates DCs and macrophages in Peyer\u0026rsquo;s patches, increases MHC class II and CD86 expression, and enhances IL-12 and TNF-α production, promoting systemic CTL responses [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Polysaccharides from \u003cem\u003eGanoderma lucidum\u003c/em\u003e (reishi) reshape gut microbiota, improve mucosal immunity, and suppress inflammation-associated tumorigenesis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Likewise, \u003cem\u003eHericium erinaceus\u003c/em\u003e (lion\u0026rsquo;s mane) polysaccharides boost short-chain fatty acid production, strengthen intestinal barrier function, and restore immune balance [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In addition, orally administered β-glucans such as PSK have been clinically tested as adjuvant therapies to sustain host immunity and enhance CTL/NK-cell responses during RT or chemotherapy [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The enhanced DC maturation and expansion of CD103\u003csup\u003e+\u003c/sup\u003e DCs observed in MLNs in our study fit well with this framework, suggesting that L.E.M. functions as an oral immunomodulator that activates gut immunity and, in turn, amplifies systemic CTL responses.\u003c/p\u003e \u003cp\u003eThis study has certain limitations. It was based on a single tumor model and limited time points, and the causal relationships among GALT-mediated DC activation, CD8⁺ T-cell responses, and radiation-induced immunity were not directly tested. Nevertheless, the present results strongly support the idea that oral L.E.M. enhances radiation-induced antitumor immunity via intestinal immune activation. Future studies using time-course and functional inhibition analyses will help further clarify this mechanism.\u003c/p\u003e \u003cp\u003eIn conclusion, our study demonstrates that oral L.E.M. activates DCs via GALT and synergistically enhances CTL-mediated antitumor immunity and tumor control when combined with RT. L.E.M. is orally administrable in outpatient settings, minimally invasive, and suitable for long-term immune modulation.\u003c/p\u003e \u003cp\u003eFuture studies should aim to (i) clarify the molecular basis of intestinal immune activation, (ii) comprehensively delineate L.E.M.-induced immunostimulatory pathways, (iii) investigate links between gut microbiota alterations and systemic immunity, (iv) optimize radiation dose and fractionation, and (v) evaluate combinations with immune-checkpoint inhibitors or cancer vaccines. Integrating these efforts may establish a novel therapeutic paradigm of \u0026ldquo;oral immune adjuvant\u0026thinsp;+\u0026thinsp;radiation therapy,\u0026rdquo; enabling safer and more durable induction of antitumor immunity.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell lines\u003c/h2\u003e \u003cp\u003eMouse melanoma B16F10 cells were obtained from the American Type Culture Collection (ATCC). Cells were cultured in RPMI 1640 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal calf serum (FCS), 2 mmol/L L-glutamine, 0.05 mmol/L 2-mercaptoethanol, penicillin, and streptomycin at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂. B16F10-ovalbumin (OVA) cells were established by stable transfection with pAc-Neo-OVA (Addgene, Watertown, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and tumor models\u003c/h2\u003e \u003cp\u003eFemale C57BL/6 mice (6\u0026ndash;8 weeks old) were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and maintained under specific pathogen\u0026ndash;free (SPF) conditions with free access to food and water. Mice of this age typically weighed approximately 18\u0026ndash;22 g, consistent with supplier records for C57BL/6 females. All animal experiments were approved by the Institutional Animal Care and Use Committee of the National Institutes for Quantum Science and Technology (approval no. 18-1024). The study is reported in accordance with ARRIVE guidelines, and all procedures were performed in accordance with institutional regulations and relevant national guidelines. B16F10-OVA melanoma cells (7.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mouse) were injected intradermally into the right hind footpad. L.E.M. feeding (2% in diet) started one day after tumor inoculation and continued until the end of the experiment. All procedures, including irradiation and tissue collection, were performed under deep anesthesia with secobarbital sodium (intraperitoneal injection, 5 mg/ml; 0.5 ml/10 g body weight, overdose level) (Nichi-Iko Pharmaceutical Co., Ltd. Toyama, Japan) to minimize distress. For euthanasia, the same agent was administered at overdose concentration followed by cervical dislocation to ensure death.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOral administration of Lentinula edodes mycelia extract (L.E.M.)\u003c/h2\u003e \u003cp\u003eL.E.M. powder was kindly provided by Kobayashi Pharmaceutical Co., Ltd. (Osaka, Japan). L.E.M. is a dried hot-water extract obtained from \u003cem\u003eLentinula edodes\u003c/em\u003e mycelia cultured in a bagasse-rice bran medium and harvested prior to fruiting, as previously described [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. For oral administration, standard powdered chow (CE-2; CLEA Japan, Tokyo, Japan) was mixed with 2% (w/w) L.E.M. Mice were allowed free access to this diet throughout the experiment. Control mice received the same chow without L.E.M.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLocal X-ray irradiation\u003c/h2\u003e \u003cp\u003eWhen tumors reached approximately 6\u0026ndash;8 mm in diameter (day 7 after inoculation), local X-ray irradiation (8 Gy) was delivered using an X-ray generator with a tungsten target (TITAN, Shimadzu, Kyoto, Japan) operating at 200 kV and 20 mA. Aluminum (0.5 mm) and copper (0.5 mm) filters were applied, and the dose rate was 2.2 Gy/min. Mice were anesthetized with thiamylal sodium (5 mg/mL in PBS, 0.1 mL/10 g body weight) administered intraperitoneally 20 min before irradiation. For tumor growth monitoring, non-irradiated mice were euthanized when tumors reached the humane endpoint (~\u0026thinsp;17 mm in diameter). For immunological analyses, a separate cohort\u0026mdash;including non-irradiated mice\u0026mdash;was sampled on day 11, when tumors remained below the endpoint (\u0026lt;\u0026thinsp;17 mm) owing to individual growth variation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of single-cell suspensions\u003c/h2\u003e \u003cp\u003eIn the non-irradiated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), MLNs were collected 18 days after tumor inoculation. In irradiated groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e), X-ray irradiation was performed 7 days after inoculation (defined as day 0), and MLNs, spleens, peripheral blood mononuclear cells (PBMCs), and tumors were harvested 11 days later. Collected tissues were digested at 37\u0026deg;C for 30 min with 125 U/mL collagenase IV (Worthington Biochemical, Lakewood, NJ) and 60 U/mL DNase I Type IV (Sigma-Aldrich, St. Louis, MO). PBMCs were isolated using OptiPrep lymphocyte separation solution (ρ\u0026thinsp;=\u0026thinsp;1.0875 g/mL; Serumwerk Bernburg AG, Bernburg, Germany). After digestion and separation, samples were washed with PBS containing 2% FBS and filtered through a 70 \u0026micro;m cell strainer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eFluorescence data were acquired using an SA3800 cell analyzer (Sony, Tokyo, Japan) and analyzed with Kaluza software (Beckman Coulter, Brea, CA, USA). Cells were first blocked with anti-CD16/32 antibody (clone 2.4G2; BioLegend, San Diego, CA, USA) and then stained with fluorochrome-conjugated anti-mouse antibodies. The following antibodies were purchased from BioLegend (San Diego, CA, USA): CD11c-Pacific Blue (clone N418), I-A/I-E-APC-Cy7 or -PerCP-Cy5.5 (clone M5/114.15.2), CD86-APC or -APC-Cy7 (clone GL-1), CD103-PE (clone 2E7), CD45-Alexa Fluor 700 (clone 30-F11), CD44-PE-Cy7 (clone IM7), and CD62L-APC (clone MEL-14). CD8α-FITC was obtained from MLB (Tokyo, Japan). OVA tetramer-PE (MLB, Tokyo, Japan) was used to detect antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells, and 7-AAD (Beckman Coulter, Brea, CA, USA) was used for viability staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTumor growth curve\u003c/h2\u003e \u003cp\u003eTumor size was measured every 3\u0026ndash;4 days with a digital caliper, and tumor volume (V) was calculated using the formula: V = (π/6) \u0026times; L \u0026times; D \u0026times; H, where L, D, and H represent length, width, and height, respectively. Measurements were performed in a blinded manner by an investigator independent of group allocation. In irradiation groups, local X-ray irradiation (8 Gy) was delivered when the tumor diameter reached 5\u0026ndash;6 mm. Humane endpoints were defined as tumor diameter\u0026thinsp;\u0026ge;\u0026thinsp;17 mm, ulceration, \u0026gt; 20% necrosis, or \u0026gt;\u0026thinsp;20% body weight loss.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using R software version 4.5.1 (The R Foundation for Statistical Computing, Vienna, Austria). Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences between two groups were analyzed using an unpaired two-tailed Student\u0026rsquo;s t-test. For comparisons among more than four groups, one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s multiple comparison test was applied. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Drs. Satoru Ishikawa and Yasunori Matsui from Kobayashi Pharmaceutical Co., Ltd. (Osaka, Japan) for kindly providing the \u003cem\u003eLentinula edodes\u0026nbsp;\u003c/em\u003emycelia (L.E.M.) powder used in this study. We also thank our laboratory members for their technical assistance and helpful discussions. Finally, we would like to thank Editage (www.editage.jp) for English language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a research fund from the National Institutes for Quantum Science and Technology, Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors and Affiliations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Charged Particle Therapy Research, National Institutes for Quantum Science and Technology, Chiba 263-8555, Japan\u003c/p\u003e\n\u003cp\u003eT. Takeshima and S. Hasegawa\u003c/p\u003e\n\u003cp\u003eEpilepsy Center, Xiamen Humanity Hospital, Fujian Medical University, Xiamen 361000, Fujian, China\u003c/p\u003e\n\u003cp\u003eY. Wang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, T.T.; Data curation, Y.W. and T.T.; Formal analysis, Y.W. and T.T.; Funding acquisition, S.H.; Methodology, T.T.; Project administration, T.T. and S.H.; Supervision, T.T. and S.H.; Writing - original draft, T.T.; Writing - review \u0026amp; editing, T.T. and S.H. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCorresponding Author\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Tsuguhide Takeshima.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated in this study are available upon request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGajewski, T. F., Schreiber, H. \u0026amp; Fu, Y. X. Innate and adaptive immune cells in the tumor microenvironment. \u003cem\u003eNat. Immunol.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 1014\u0026ndash;1022 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma, P. \u0026amp; Allison, J. P. The future of immune checkpoint therapy. \u003cem\u003eScience\u003c/em\u003e \u003cb\u003e348\u003c/b\u003e, 56\u0026ndash;61 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKojima, H., Akaki, J., Nakajima, S., Kamei, K. \u0026amp; Tamesada, M. Structural analysis of glycogen-like polysaccharides having macrophage-activating activity in extracts of Lentinula edodes mycelia. \u003cem\u003eJ. Nat. Med.\u003c/em\u003e \u003cb\u003e64\u003c/b\u003e, 16\u0026ndash;23 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSugano, N., Hibino, Y., Choji, Y. \u0026amp; Maeda, H. Anticarcinogenic actions of water-soluble and alcohol-insoluble fractions from culture medium of Lentinus edodes mycelia. \u003cem\u003eCancer Lett.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 109\u0026ndash;114 (1982).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSugano, N., Choji, Y., Hibino, Y., Yasumura, S. \u0026amp; Maeda, H. Anticarcinogenic action of an alcohol-insoluble fraction (LAP1) from culture medium of Lentinus edodes mycelia. \u003cem\u003eCancer Lett.\u003c/em\u003e \u003cb\u003e27\u003c/b\u003e, 1\u0026ndash;6 (1985).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuzuki, H. et al. Structural characterization of the immunoactive and antiviral water-solubilized lignin in an extract of the culture medium of Lentinus edodes mycelia (L.E.M). \u003cem\u003eAgric. Biol. Chem.\u003c/em\u003e \u003cb\u003e54\u003c/b\u003e, 479\u0026ndash;487 (1990).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanaka, K. et al. Oral ingestion of Lentinula edodes mycelia extract inhibits B16 melanoma growth via mitigation of regulatory T cell-mediated immunosuppression. \u003cem\u003eCancer Sci.\u003c/em\u003e \u003cb\u003e102\u003c/b\u003e, 516\u0026ndash;521 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanaka, K., Matsui, Y., Ishikawa, S., Kawanishi, T. \u0026amp; Harada, M. Oral ingestion of Lentinula edodes mycelia extract can restore the antitumor T cell response of mice inoculated with colon-26 cells into the subserosal space of the cecum. \u003cem\u003eOncol. Rep.\u003c/em\u003e \u003cb\u003e27\u003c/b\u003e, 325\u0026ndash;332 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanaka, K. et al. Combining a peptide vaccine with oral ingestion of Lentinula edodes mycelia extract enhances antitumor activity in B16 melanoma-bearing mice. \u003cem\u003eCancer Immunol. Immunother\u003c/em\u003e. \u003cb\u003e61\u003c/b\u003e, 2143\u0026ndash;2152 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIshikawa, S. et al. Age-associated impairment of antitumor immunity in carcinoma-bearing mice and restoration by oral administration of Lentinula edodes mycelia extract. \u003cem\u003eCancer Immunol. Immunother\u003c/em\u003e. \u003cb\u003e65\u003c/b\u003e, 961\u0026ndash;972 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeem, D. et al. Contribution of mesenteric lymph nodes and GALT to the intestinal Foxp3\u0026thinsp;+\u0026thinsp;regulatory T-cell compartment. \u003cem\u003eCell. Mol. Gastroenterol. Hepatol.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, 274\u0026ndash;280 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScott, C. L., Aumeunier, A. M. \u0026amp; Mowat, A. M. Intestinal CD103(+) dendritic cells: master regulators of tolerance? \u003cem\u003eTrends Immunol.\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e, 412\u0026ndash;419 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCerovic, V. et al. Intestinal CD103(-) dendritic cells migrate in lymph and prime effector T cells. \u003cem\u003eMucosal Immunol.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 104\u0026ndash;113 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJaensson, E. et al. Small intestinal CD103\u0026thinsp;+\u0026thinsp;dendritic cells display unique functional properties that are conserved between mice and humans. \u003cem\u003eJ. Exp. Med.\u003c/em\u003e \u003cb\u003e205\u003c/b\u003e, 2139\u0026ndash;2149 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVilladangos, J. A. \u0026amp; Schnorrer, P. Intrinsic and cooperative antigen-presenting functions of dendritic-cell subsets in vivo. \u003cem\u003eNat. Rev. Immunol.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 543\u0026ndash;555 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKadowaki, T. et al. Reconsideration of macrophage and dendritic cell classification. \u003cem\u003eAnticancer Res.\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e, 2257\u0026ndash;2261 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakeshima, T. et al. Local Radiation Therapy Inhibits Tumor Growth through the Generation of Tumor-Specific CTL: Its Potentiation by Combination with Th1 Cell Therapy. \u003cem\u003eCancer Res.\u003c/em\u003e \u003cb\u003e70\u003c/b\u003e, 2697\u0026ndash;2706 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDemaria, S. \u0026amp; Formenti, S. C. Radiotherapy effects on antitumor immunity: implications for cancer treatment. \u003cem\u003eFront. Oncol.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e, 128 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharabi, A. B., Lim, M., DeWeese, T. L. \u0026amp; Drake, C. G. Radiation and checkpoint blockade immunotherapy: radiosensitisation and potential mechanisms of synergy. \u003cem\u003eLancet Oncol.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e, e498\u0026ndash;e509 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdelson, B. T. et al. Peripheral CD103\u0026thinsp;+\u0026thinsp;dendritic cells form a unified subset developmentally related to CD8alpha\u0026thinsp;+\u0026thinsp;conventional dendritic cells. \u003cem\u003eJ. Exp. Med.\u003c/em\u003e \u003cb\u003e207\u003c/b\u003e, 823\u0026ndash;836 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHildner, K. et al. Batf3 deficiency reveals a critical role for CD8alpha\u0026thinsp;+\u0026thinsp;dendritic cells in cytotoxic T cell immunity. \u003cem\u003eScience\u003c/em\u003e \u003cb\u003e322\u003c/b\u003e, 1097\u0026ndash;1100 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo, S. et al. Radiation-induced tumor immune microenvironments and potential targets for combination therapy. \u003cem\u003eSignal. Transduct. Target. Ther.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 205 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayakawa, K. et al. Effect of krestin (PSK) as adjuvant treatment on the prognosis after radical radiotherapy in patients with non-small cell lung cancer. \u003cem\u003eAnticancer Res.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 1815\u0026ndash;1820 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFritz, H. et al. Polysaccharide K and Coriolus versicolor extracts for lung cancer: a systematic review. \u003cem\u003eIntegr. Cancer Ther.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 201\u0026ndash;211 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasuda, Y., Nakayama, Y., Tanaka, A., Naito, K. \u0026amp; Konishi, M. Antitumor activity of orally administered maitake alpha-glucan by stimulating antitumor immune response in murine tumor. \u003cem\u003ePLOS One\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e, e0173621 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo, C. et al. Ganoderma lucidum polysaccharide modulates gut microbiota and immune cell function to inhibit inflammation and tumorigenesis in colon. \u003cem\u003eCarbohydr. Polym.\u003c/em\u003e \u003cb\u003e267\u003c/b\u003e, 118231 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian, B. et al. Modulating effects of Hericium erinaceus polysaccharides on the immune response by regulating gut microbiota in cyclophosphamide-treated mice. \u003cem\u003eJ. Sci. Food Agric.\u003c/em\u003e \u003cb\u003e103\u003c/b\u003e, 3050\u0026ndash;3064 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Graaff, P., Govers, C., Wichers, H. J. \u0026amp; Debets, R. Consumption of beta-glucans to spice up T cell treatment of tumors: a review. \u003cem\u003eExpert Opin. Biol. Ther.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e, 1023\u0026ndash;1040 (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lentinula edodes mycelia extract, Gut-associated lymphoid tissue, Dendritic cells, CD8+ T cells, Radiation Therapy","lastPublishedDoi":"10.21203/rs.3.rs-8096960/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8096960/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOral administration of \u003cem\u003eLentinula edodes\u003c/em\u003e mycelia extract (L.E.M.) has been shown to stimulate systemic T cell\u0026ndash;mediated antitumor immunity and inhibit tumor growth in mice, suggesting its potential to modulate host immune responses. However, the route of this systemic antitumor effect remains unclear. This study focused on gut-associated immune mechanisms by analyzing mesenteric lymph nodes (MLNs), a major component of the gut-associated lymphoid tissue (GALT), and examined whether oral L.E.M. enhances the antitumor efficacy of radiation therapy (RT) in a B16F10-OVA melanoma model. L.E.M. administration upregulated MHC class II and CD86 expression on CD11c\u003csup\u003e+\u003c/sup\u003e dendritic cells (DCs) in MLNs and significantly increased the proportion of CD103\u003csup\u003e+\u003c/sup\u003e subsets, indicating DC maturation within the GALT. In a radiation-induced tumor model, L.E.M. further enhanced DC maturation and increased CD8α\u003csup\u003e+\u003c/sup\u003e DCs in the spleen, accompanied by elevated effector and central memory fractions of CD8\u003csup\u003e+\u003c/sup\u003e T cells in peripheral blood. Antigen-specific CD8\u003csup\u003e+\u003c/sup\u003e T cells (OVA tetramer\u003csup\u003e+\u003c/sup\u003e) were significantly enriched within tumors, and L.E.M. combined with RT achieved greater tumor growth inhibition than RT alone. These findings demonstrate that oral L.E.M. activates GALT-mediated DC and CD8\u003csup\u003e+\u003c/sup\u003e T-cell responses, thereby augmenting the antitumor immune effects of RT.\u003c/p\u003e","manuscriptTitle":"Oral Lentinula edodes mycelia extract enhances the antitumor effect of radiotherapy via gut-associated activation of dendritic and cytotoxic T cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-12 07:39:47","doi":"10.21203/rs.3.rs-8096960/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-31T21:00:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-27T21:07:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-23T03:46:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"270398609354667630829807344917393158003","date":"2025-12-17T06:40:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223347962275508233452266738924098435500","date":"2025-12-14T08:31:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-12T14:40:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-12T14:39:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-10T13:51:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-09T13:00:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-09T12:49:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"83406b75-f9c7-43ed-83a5-f963528606ec","owner":[],"postedDate":"December 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":59439603,"name":"Biological sciences/Cancer"},{"id":59439604,"name":"Biological sciences/Immunology"},{"id":59439605,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-01-19T16:44:21+00:00","versionOfRecord":{"articleIdentity":"rs-8096960","link":"https://doi.org/10.1038/s41598-026-35752-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-16 16:28:40","publishedOnDateReadable":"January 16th, 2026"},"versionCreatedAt":"2025-12-12 07:39:47","video":"","vorDoi":"10.1038/s41598-026-35752-7","vorDoiUrl":"https://doi.org/10.1038/s41598-026-35752-7","workflowStages":[]},"version":"v1","identity":"rs-8096960","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8096960","identity":"rs-8096960","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}