Search Results (244)

Tumor-draining lymph nodes (TDLNs) are important immune organs linking primary tumors with systemic immune responses. They support tumor antigen presentation, T-cell priming, and effector immune responses, but under sustained tumor influence they can also be remodeled into microenvironments that promote immune escape and metastatic colonization. Conventional methods, including flow cytometry, bulk RNA sequencing, and routine immunohistochemistry, have advanced our understanding of TDLN immunity but cannot simultaneously preserve tissue architecture, cell identity, and spatial cell–cell relationships. Recent advances in spatial transcriptomics, spatial proteomics, and multiplexed imaging enable in situ analysis of immune cells, stromal cells, vascular structures, and their interactions within TDLNs. Emerging evidence suggests that TDLNs are not immunologically homogeneous organs, but gradually develop spatially distinct immune-activation and immunosuppressive regions during tumor progression. Activation regions are associated with HEV-mediated lymphocyte entry, DC–T-cell priming, B-cell follicles, and germinal-center reactions, whereas suppressive regions are enriched in Treg cells, exhausted T cells, suppressive myeloid cells, tumor-reprogrammed FRCs, and myeloid–CAF niches. This review summarizes spatial multi-omics studies of TDLN functional compartmentalization and discusses the potential value of TDLN spatial immune states in predicting immunotherapy response, assessing metastatic risk, and guiding precision treatment, thereby providing a reference for future spatial multi-omics studies of TDLNs. Full article

Immune checkpoint inhibitors (ICIs) are now being introduced into perioperative treatment for several solid tumors. This strategy is usually explained by tumor reduction before surgery or by the elimination of minimal residual disease (MRD) after surgery. However, these explanations may not be sufficient to understand why the timing of ICI treatment, especially before lymph node (LN) removal, is important. In this review, we discuss tumor-draining lymph nodes (tdLNs) from two different aspects. tdLNs are anatomical routes for regional and distant metastasis, but they are also sites where tumor antigens are presented and tumor-specific T cell responses are generated. In particular, preclinical and translational studies suggest that tdLNs may maintain stem-like or progenitor-exhausted T cells (TPEX) that can respond to PD-1 blockade and supply more differentiated exhausted T cells to tumor sites. However, current clinical trials of perioperative ICIs demonstrate therapeutic benefit in specific diseases and regimens, but do not directly establish tdLN preservation or tdLN-resident TPEX maintenance as the decisive mechanism of efficacy. We therefore present the tdLN-reservoir model as a hypothesis-generating framework rather than as a clinically validated basis for modifying lymph node management. We also discuss the possible roles of neoadjuvant and adjuvant ICI in relation to antigen flow, minimal residual disease, metastatic-site draining LNs, postoperative lymphatic dysfunction, and future immune-guided clinical trials. Importantly, current evidence does not support altering standard lymph node surgery or radiotherapy solely to preserve putative tdLN immune-reservoir function. Full article

Introduction: Hairy cell leukemia is a rare, indolent chronic B cell lymphoproliferative disorder characterized by cytopenia, splenomegaly and profound immune dysfunction, predisposing affected individuals to opportunistic infections including non-tuberculous mycobacteria. Concurrent presentation with disseminated mycobacterial infection is un-common and may pose significant diagnostic challenges. Case Presentation: We report the case of a 68-year-old Caucasian man with a history of splenectomy who presented with fever, lymphadenopathy, leukopenia and a generalized rash. Initial investigations including infectious, hematological and vasculitis workups were inconclusive. Lymph node histology demonstrated necrotizing lymphadenitis, which in the context of an otherwise negative investigations was initially suggestive of Kikuchi–Fujimoto disease. Subsequently, cultures from lymph node tissue and blood yielded Mycobacterium avium complex, establishing a diagnosis of disseminated infection. Further bone marrow evaluation with flow cytometry ultimately confirmed underlying hairy cell leukemia. Conclusions: This case highlights how an impaired immune milieu may obscure classic clinical and histopathological features, contributing to diagnostic delay and potentially inappropriate immunosuppressive treatment. Clinicians should maintain a high index of suspicion for underlying hematological malignancy in patients presenting with unexplained cytopenia in association with atypical infections. Early consideration of bone marrow evaluation can be crucial. Full article

Background/Objectives: Kimura disease is a rare, chronic immune-mediated disorder that classically produces painless head-and-neck masses, regional lymphadenopathy, blood eosinophilia, and a raised serum immunoglobulin E (IgE). Almost all reported patients are young East Asian men, and the condition is seldom encountered in the Middle East, where it is easily mistaken for lymphoma or another eosinophilic disorder. We describe a histologically confirmed case in a Saudi adolescent and review the literature to compare treatment strategies and outcomes across populations. Methods: We documented the clinical course, laboratory profile, histopathological findings, and 15-month outcome of the patient, and searched PubMed for reported cases of Kimura disease, with emphasis on pediatric and non-Asian series. Results: A 16-year-old boy presented with a one-year history of painless right cervical swelling, constitutional symptoms and striking eosinophilia (36%; absolute eosinophil count 6.5 × 10⁹/L). Hematological malignancy was excluded through bone-marrow examination, flow cytometry and molecular studies. Excisional lymph node biopsy revealed the diagnostic triad—reactive follicular hyperplasia, a dense eosinophilic infiltrate with microabscesses, and vascular proliferation—together with IgE-positive immunostaining. Complete remission followed surgical excision alone and was maintained at 15 months without systemic corticosteroids. Conclusions: Kimura disease occurs well beyond its traditional geographic boundaries and belongs in the differential diagnosis of eosinophilia accompanied by lymphadenopathy. Although corticosteroids remain the mainstay for extensive disease, relapse on tapering is common, whereas complete excision can secure lasting remission in localized pediatric disease while sparing patients from steroid-related toxicity. Full article

Periodontitis is a chronic inflammatory disease characterized by alveolar bone loss driven by dysregulated immune responses. We previously showed that extracellular vesicles derived from retinoic acid-induced regulatory T lymphocytes (RA-Treg EVs) suppress pathogenic CD4⁺ T-lymphocyte responses and reduce alveolar bone loss during periodontitis. Herein, we investigated whether RA-Treg EVs also modulate macrophage and CD8⁺ T-lymphocyte responses during experimental periodontitis. Ligature-induced periodontitis was generated in mice, followed by local administration of RA-Treg EVs. Alveolar bone loss was analyzed by micro-computed tomography, and periodontal tissues and cervical lymph nodes were analyzed by flow cytometry to quantify antigen-presenting cells, macrophages, macrophage subsets, and CD8⁺ T lymphocytes. The direct effects of RA-Treg EVs on macrophage phenotype and CD8⁺ T-cell proliferation and activation were assessed in vitro. RA-Treg EV treatment attenuated alveolar bone loss and preserved trabecular microarchitecture. This effect was associated with reduced macrophage infiltration into periodontal tissues, modulation of macrophage polarization, and restoration of CD8⁺ T-cell abundance in periodontal tissues and draining cervical lymph nodes, without major changes in CD8⁺IFN-γ⁺ or CD8⁺RANKL⁺ cells. In vitro, RA-Treg EVs induced heterogeneous macrophage phenotypes distinct from the classical M1/M2 polarization states while markedly enhancing CD8⁺ T-cell proliferation and activation. These findings indicate that RA-Treg EVs preserve alveolar bone during experimental periodontitis while selectively modulating macrophage and CD8⁺ T-lymphocyte responses. Full article

Lymph nodes are central hubs of immune regulation and coordination, serving as primary sites for antigen presentation, lymphocyte activation, and the orchestration of adaptive immune responses. The composition and activation state of lymph node-resident immune cells critically shape both local and systemic immunity. Comprehensive immunophenotyping of these populations is therefore essential for understanding immune organization and functional heterogeneity. Here, we present an optimized protocol for the characterization of mouse lymph node-associated immune populations using 14-color multiparametric flow cytometry. The method combines lymph node isolation based on anatomical landmarks with mechanical dissociation and enzymatic digestion to generate high-quality single-cell suspensions suitable for downstream analysis. Furthermore, the described flow cytometry panel and gating strategy enable reliable identification and quantification of major lymphoid subsets, including helper CD4⁺ and cytotoxic CD8⁺ T cells with their differentiation states, as well as natural killer (NK) cells across distinct maturation stages. Although optimized for assessing lymphocyte maturation after lipopolysaccharide (LPS) challenge, the protocol serves as a reproducible platform for broad immunophenotyping of T and NK cell subsets in mouse lymphoid tissues under experimental conditions. Full article

Background/Objectives: Preclinical studies of head and neck squamous cell carcinoma (HNSCC) commonly use subcutaneous heterotopic (flank) tumor models for simplicity; however, orthotopic models may better reflect the native tumor environment. Direct comparisons of the tumor immune microenvironments (TIME) and tumor-draining lymph nodes (tdLNs) between these models remain limited. Better understanding of site-specific immune differences could improve model selection and interpretation of translational HNSCC studies. Methods: ROC1 tumors were established in murine heterotopic and orthotopic sites, followed by assessment of tumor growth kinetics, survival, and the tumor microenvironment. Immune composition of tumors, blood, tdLNs, and spleen was evaluated at three tumor progression timepoints using multiparameter spectral flow cytometry. Results: Heterotopic and orthotopic tumor models showed similar growth kinetics and survival. Immune profiling revealed increased infiltration of CD3⁺ T-cells, natural killer (NK) cells, and myeloid populations in both models. Heterotopic tumors were enriched in dendritic cells (DCs), plasmacytoid DCs, and monocytic myeloid-derived suppressor cells (M-MDSCs), whereas orthotopic tumors showed increased macrophages, granulocytic MDSCs, and M-MDSCs. Despite temporal variation, both TIMEs were dominated by macrophages, DCs, and CD3⁺ T-cells. Late-stage heterotopic tumors contained more CD4⁺ T-cells. Reduced T-cell cytotoxicity (PD-1, CD107a) and increased immune checkpoint expression across myeloid cells indicated an immunosuppressive TIME. Systemically, effector cells were preserved despite suppressive cell trafficking, and tdLNs in both models exhibited immunosuppressive PD-L1 expression. Conclusions: Heterotopic and orthotopic ROC1 tumors share key immune features, but site-specific differences in the TIME and tdLNs reveal tissue-dependent regulation. These local effects align with systemic changes, supporting global tumor-associated immunosuppression. Full article

Background: Lymph node biopsy is an important means of etiologic diagnosis for patients with fever of unknown origin (FUO) and lymphadenopathy. However, it is an invasive procedure and may yield negative results. It is worth exploring which kinds of patients could benefit most from lymph node biopsy. Methods: FUO patients (n = 242) who had lymphadenopathy and underwent lymph node biopsy were enrolled into this retrospective single-center study. Clinical manifestations were documented and risk factors suggestive of an underlying positive lymph node biopsy (providing diagnostic clues) and lymphoma were analyzed. Results: The etiologies were as follows: infectious disease in 10 (4.1%) cases, connective tissue diseases in 51 (21.1%) cases, neoplastic diseases in 57 (23.6%) cases, other diseases in 28 (11.6%) cases, and unknown diagnosis in 96 (39.7%) cases. A total of 88 patients (36.4%) were diagnosed through lymph node biopsy. The following four independent risk factors were found to be related to positive lymph node biopsy: male gender (OR 2.471; 95% CI 1.158–5.270; p = 0.019), no rash (OR 3.531; 95% CI 1.595–7.816; p = 0.002), shape index of the lymph node (OR 8.566; 95% CI 1.035–70.915; p = 0.046), and hypoalbuminemia (OR 3.370; 95% CI 1.470–7.728; p = 0.004). Later, 146 patients with a confirmed diagnosis (including 57 cases of lymphoma and 89 cases of non-lymphoma) were included in the analysis of lymphoma-related factors. Age older than 45 years (OR 8.663; 95% CI 3.045–24.647; p < 0.001), no rash (OR 4.946; 95% CI 1.646–14.859; p = 0.004), serositis (OR 3.588; 95% CI 1.137–11.318; p = 0.029), abnormal blood flow of the lymph node (OR 3.025; 95% CI 1.034–8.848; p = 0.043), abnormal central lymph nodes (OR 6.546; 95% CI 1.721–24.898; p = 0.006), focal lesions in the spleen (OR 13.386; 95% CI 2.067–86.706; p = 0.006), and serum lactate dehydrogenase (LDH) > 250 U/L (OR 3.885; 95% CI 1.111–13.584; p = 0.034) were independent risk factors for lymphoma. Conclusions: Lymph node biopsy is a valuable diagnostic procedure for patients with FUO and lymphadenopathy. For male patients without rash, more rounded lymph nodes, and hypoalbuminemia, we strongly recommend lymph node biopsy. Age older than 45 years, no rash, serositis, abnormal blood flow of the lymph node, abnormal central lymph nodes, focal lesions in the spleen, and serum LDH > 250 U/L are risk factors suggesting an underlying lymphoma, and multi-site biopsy should be considered if necessary. Full article

Background/Objectives: Follicular helper T-cell (TFH)-type lymphomas, including angioimmunoblastic T-cell lymphoma (AITL) and TFH lymphoma, not otherwise specified (TFH-NOS), are characterized by marked immune dysregulation in the tumor microenvironment. We investigated whether TFH-type lymphomas are enriched in immunosuppressive cells and explored immunologic changes associated with denileukin diftitox (DD) treatment. Methods: FOXP3-positive mononuclear cells were quantified by immunohistochemistry in lymph node specimens from 10 patients with TFH-type lymphoma and six with non-TFH-type T-cell lymphoma. Paired skin biopsy specimens obtained before and after DD treatment from two patients with AITL were evaluated for CD4, CD8, CD68, CD163, and FOXP3 expression. Longitudinal flow cytometric T-cell receptor Vβ repertoire analysis of CD8-positive T-cell subsets was performed in three patients with AITL treated with DD. Clinical responses were retrospectively assessed in seven patients with relapsed or refractory TFH-type lymphoma treated with DD. Results: TFH-type lymphomas showed significantly higher intra-tumoral FOXP3-positive cell densities than non-TFH-type lymphomas (p = 0.0024). In paired skin biopsies, DD treatment was associated with reductions in CD68- and CD163-positive macrophage-rich infiltrates and a suggested decrease in FOXP3-positive cells. Among seven patients treated with DD, the overall response rate was 86%, including one complete response and five partial responses. Responders showed selective Vβ over-representation patterns in effector and/or memory CD8-positive T-cell subsets, whereas such findings were not convincing in the non-responder. Conclusions: TFH-type lymphomas were associated with higher FOXP3-positive cell density than selected non-TFH-type comparators. DD treatment may be associated with changes in tissue immune infiltrates and peripheral CD8/TCR Vβ skewing in responding cases, although these findings remain exploratory and do not establish causality. Full article

Lung adenocarcinoma (LUAD) is the most common subtype of non-small cell lung cancer and remains a leading cause of cancer-related mortality worldwide. MicroRNAs (miRNAs) are critical regulators of tumor progression; however, the biological role and molecular mechanisms of miR-589-3p in LUAD remain unclear. In this study, the expression levels of miR-589-3p and WWC2 were analyzed using The Cancer Genome Atlas lung adenocarcinoma (TCGA-LUAD) datasets via the UALCAN platform. Flow cytometric apoptosis analysis and functional assays including CCK-8, colony formation, AO/EB staining, and Transwell invasion assays were performed in LUAD cell lines. The interaction between miR-589-3p and WWC2 was validated using dual-luciferase reporter assays, Western blotting, and rescue experiments. miR-589-3p expression was significantly elevated in LUAD tissues compared with normal lung tissues (p < 0.05) and was positively associated with an advanced tumor stage and lymph node metastasis (p < 0.05). Inhibition of miR-589-3p significantly suppressed proliferation and colony formation (p < 0.05), reduced invasive capacity (p < 0.05), and markedly increased apoptosis (p < 0.01) in LUAD cells. Dual-luciferase reporter assays confirmed WWC2 as a direct target of miR-589-3p, with miR-589-3p mimics significantly reducing WWC2 wild-type reporter activity (p < 0.05). WWC2 expression was significantly downregulated in LUAD tissues (p < 0.05), and WWC2 knockdown reversed the anti-proliferative, pro-apoptotic, and anti-invasive effects induced by miR-589-3p inhibition (p < 0.01). These findings demonstrate that miR-589-3p promotes lung adenocarcinoma progression by directly suppressing WWC2. The miR-589-3p/WWC2 axis represents a novel molecular mechanism contributing to LUAD malignancy and may provide a foundation for future mechanistic and translational studies. Full article

Attention-Deficit/Hyperactivity Disorder (ADHD), affecting 5–10% of children globally, faces treatment limitations due to adverse effects and uncertain long-term risks of current pharmacotherapies. This study investigated the therapeutic potential of sepia ink (SI), a marine-derived natural complex from cuttlefish, in a scopolamine-induced ADHD-like mouse model. The chemical constituents of SI were characterized via Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS). The behavioral assessments, histopathological examinations, flow cytometry, and complete blood counts were utilized to evaluate its effects on ADHD-like phenotypes, neuroinflammation, and immune function. Integrated transcriptomic, plasma metabolomic, and 16S rRNA sequencing were used to explore the underlying mechanisms. SI significantly alleviated hyperactivity and improved spatial learning and memory deficits. It reduced hippocampal neuronal damage, attenuated neuroinflammation, and reversed scopolamine-induced immunosuppression in spleen and thymus. SI also restored the balance of immune cell subsets in both mesenteric lymph nodes and spleen, and the peripheral blood cell counts. Multi-omics analyses suggested that the beneficial effects of SI were associated with reduced neuroinflammation, rebalanced systemic immune responses, partial correction of lipid metabolic disturbances, and restoration of gut microbiota homeostasis. Collectively, our findings indicate that SI effectively mitigates the in vivo ADHD-like impairments by coordinating immune, metabolic, and gut microbiota-related processes, thereby supporting its potential as a marine-derived therapeutic candidate for further ADHD treatment. Full article

Background: Occult lymph node metastasis (LNM) occurs in 30–80% of patients with clinically node-negative papillary thyroid carcinoma (cN0-PTC), partly owing to the limited sensitivity of current preoperative nodal assessment, and may contribute to postoperative recurrence. Conventional sentinel lymph node (SLN) biopsy, typically performed with a single tracer, has limited reliability for detecting occult metastatic nodes, which can result in either overtreatment or undertreatment with lymph node dissection. We aimed to develop a highly accurate multimodal prediction framework to accurately identify second-echelon lymph node metastasis (SeLNM) and non-sentinel lymph node metastasis (NsLNM). Methods: We prospectively enrolled 301 patients with cN0-PTC between April and October 2024, of whom 131 met the inclusion criteria. Intraoperatively, a dual-tracer technique combining carbon nanoparticles and indocyanine green was applied, and near-infrared imaging was used to record the entire SLN visualization process in real time. For each case, a 3 min video clip (150 frames) was captured. Two senior surgeons delineated regions of interest to generate 19,650 mask images. A total of 2048 spatial features and 20 temporal features were extracted, combined with 32 clinical variables, including demographics, ultrasound characteristics, and gene mutation status. Nine deep learning models were developed and evaluated using 10-fold cross-validation. Model performance was quantified using receiver operating characteristic curves, decision curve analysis curves, calibration curves, precision–recall curves, learning curves, and 12 metrics. Statistical comparisons were performed using the DeLong test, and models were further evaluated using a probability-based ranking approach. Shapley Additive Explanations (SHAP) analysis was applied to interpret key predictive features. The primary outcomes were SeLNM and NsLNM, defined based on postoperative histopathology. Results: The Long Short-Term Memory (LSTM) + Transformer model showed the best performance for both prediction tasks, with stable AUCs across training and testing (SeLNM: 0.980/0.982; NsLNM: 0.986/0.983). In the testing set, the model reached the same accuracy for both outcomes (94.7%) and showed strong sensitivity/specificity for SeLNM (94.7%/94.6%) and NsLNM (96.4%/91.5%). SHAP analysis indicated that time-series fluorescence flow features were the most influential predictors, followed by spatial structural features and SLN status. Conclusions: Dual-tracer SLN mapping with deep learning demonstrated encouraging intraoperative prediction of lymph node metastasis with interpretable features in this single-center cohort. Independent multicenter validation and prospective outcome studies are needed before considering clinical adoption. Full article

Pancreatic ductal adenocarcinoma (PDAC) is characterized by immune cell dysfunction and poor prognosis. CD44, a cell surface glycoprotein with multiple splice variants, has been implicated in tumor progression, but its compartment-specific roles in PDAC remain unclear. CD44 standard and variant isoform expression was analyzed in patient-derived lymph nodes (LNs) by quantitative PCR. Immune cell-specific CD44 expression was assessed by flow cytometry in LNs and peripheral blood. Soluble and exosome-associated CD44 (exo-CD44) were measured in plasma. Clinical associations and survival analyses were performed. Transcriptomic, immune infiltration, immune checkpoint, and drug sensitivity analyses were conducted using TCGA-PAAD and pharmacogenomic datasets. CD44 standard isoform expression was unchanged in PDAC LNs, whereas multiple CD44 variant isoforms (v4–v10) were significantly reduced and associated with metastatic disease and poor survival, particularly CD44v5, v6, v7, and v10. CD44 expression was enriched in CD45⁺ immune cells, with highest levels in CD4⁺ T cells in both LNs and blood. Soluble CD44 levels showed no clinical associations. In contrast, exo-CD44 levels were reduced overall in PDAC but increased in patients with distant metastasis, positive resection margins, systemic inflammation, and reduced survival. High CD44 expression was associated with advanced disease, immune cell infiltration, immune checkpoint gene expression, reduced sensitivity to gemcitabine, paclitaxel, rapamycin, and FMK, and distinct CTLA4/PD-L1 checkpoint profiles. CD44 exhibits compartment-specific regulation in PDAC, linking immune remodeling, exosome signaling, and therapeutic resistance to adverse clinical outcome. Full article

Background/Objectives: Antigen presenting cells (APCs) and immune cells have unique properties to drive or suppress immune responses. They are therefore key targets for the expression of vaccine antigens or transgene proteins. To better determine the utility of different molecular therapies to modify these cells, mRNA and DNA-based molecular therapy vectors were compared for their ability to genetically modify immune cells after intradermal injections in mice. DNA-based vectors included naked plasmid DNA, plasmid packaged in lipid nanoparticles (LNPs), and replication-defective adenovirus (Ad) vectors. mRNA delivery was mediated by packaging into LNPs like those used in COVID-19 vaccines. Methods: Each vector was used to deliver Cre recombinase into Cre reporter mice whose cells were activated to express green fluorescent protein (GFP) and firefly luciferase after Cre recombination. The mice were injected intradermally (ID) near the base of their tail at a site that drains into the inguinal lymph node. Luciferase activity was imaged in the living mice 1 or 4 days after vector injection. The animals were then euthanized, and luciferase activity was imaged in the draining inguinal lymph node. Cells were prepared from the intradermal injection site and from the draining lymph node to determine which immune cells were genetically modified by phenotyping CD45, CD3, and CD11b GFP-positive cells by flow cytometry. Given that the skin uniquely contains Langerhans dendritic cells, these CD207⁺ cells were also phenotyped in skin samples and in the draining lymph node. Results: In both the skin and in the draining lymph node, the rank order of luciferase and GFP activation by the vectors were: (1) Ad; (2) mRNA-LNP; (3) DNA-LNP; and (4) naked DNA. Only mRNA-LNP and Ad vectors mediated obvious luciferase activity in the living animals and in the draining lymph nodes by imaging. Notably, both vectors appeared to leak from the ID injection site and not only modify the draining lymph node but also strongly modify the livers of the mice. Naked DNA and DNA-LNP mediated detectable GFP activation in the skin and draining lymph node in some mice, but this activity was low and did not reach statistical significance when compared to PBS-treated animals. mRNA-LNPs and Ad both mediated significant Cre delivery in CD45⁺, CD3⁺, CD11b⁺, and CD207⁺ immune cells in the skin and in the lymph node, with adenovirus mediating consistently higher levels of expression in all of the tested cells. Conclusions: These data indicate that mRNA-LNP and Ad vectors mediate stronger modification of skin and lymph node immune cells after intradermal injections. Naked DNA and DNA-LNPs were markedly less potent at this activity than the other vectors. These data are consistent with the higher vaccine potency of mRNA-LNP and Ad vectors and suggest that approaches that increase targeting of immune cell subsets may have utility to increase efficacy while also reducing off-target modification of tissues like the liver. Full article

Fluorescence emission-guided blood flow and lymph node location detection are important observation methods in cancer removal surgery, where near-infrared LED illumination is used to induce fluorescence emission. However, conventional LED light sources have narrow beam widths, resulting in a limited excitation area and a restricted field of view (FOV). In this study, we propose a balanced optical illumination module that combines a beam-focusing condenser lens and a beam-diffusing lens to expand the beam width while efficiently redistributing optical energy. When only the LED was used, the beam diameter and central irradiance were 4.0 cm and 1.43 mW/cm², respectively. With the condenser lens, the beam diameter remained nearly unchanged (3.98 cm), while the central irradiance decreased to 0.91 mW/cm². When the condenser was combined with the proposed diffuser structure, the beam diameter increased to 14.1 cm, corresponding to an approximately 3.5-fold expansion, while the central irradiance was measured at 0.72 mW/cm², reflecting the redistribution of optical energy from an initially Gaussian-like irradiance distribution into a wider and more uniform illumination area. This irradiance level exceeds the minimum threshold of 0.6 mW/cm² required to induce fluorescence emission, as defined for the experimental working distance of 30 cm and LED power of 200 mW. By integrating the irradiance distributions of both the bare LED and the proposed structure over their respective illuminated surfaces, the measured total power is physically consistent with energy conservation, showing an expected transmission loss of 18.8% due to optical absorption and scattering. These results demonstrate that the proposed beam diffusion-concentration approach provides an effective and practical solution for wide-field fluorescence-guided lesion observation during cancer removal surgery. Full article