Supplementary Figure 1 Identified major cell types, cell proportion analysis and CD112 and CD155 expression in NB tumor scRNA-seq analysis. (A) Violin plots showing the number of features, RNA counts and percent mitochondrial transcripts following quality control. (B) Violin plots showing the average expression of the genes for annotating the major cell types identified in NB tumors. (C) UMAP plots showing the distribution of cell types for each sample. (D) Bar plots showing the proportions of cell types for each sample. (E) UMAP plots showing the expression levels of CD112 and CD155.

Lymphatic malformation (LM) is a low-flow vascular malformation caused by the abnormal development of the lymphatic system. This study aimed to provide an update on pediatric LM by investigating its clinical characteristics and hospitalization burden in multiple medical centers across China. This study utilized data from the Futang Research Center of Pediatric Development (FRCPD). All hospitalization records from January 1, 2016, to December 31, 2023 (N = 10,974,595) were screened to identify children with LM. For these patients, a comprehensive analysis of clinical characteristics (e.g., demographics, lesion location, treatment, comorbidity/complication and ICU admissions) hospitalization burden (i.e., length of stay and total cost) was conducted. Our study comprised 24,207 inpatients with LM, predominantly from the eastern region. The typical hospitalized patient was a child aged 1–3 years or on trunk involvement. Strikingly, sclerotherapy was 1.57 times more common than surgical resection. The most frequent comorbidity/complication were infectious diseases. Treatment patterns also varied by anatomy: lesions in the trunk favored surgical resection, while other areas usually undergo sclerotherapy. Critically, hospitalization burden differed significantly based on lesion site, treatment received, and the presence of comorbidity and complication. By analyzing a national cohort of pediatric inpatients with LM, this study provides contemporary insights into the clinical characteristics and hospitalization burden of this condition in China. These findings underscore the substantial healthcare impact of LM and are critical for informing future healthcare strategies and resource allocation.

Supplementary Figure 3 The expression analysis of CD112 and CD155 in NB. (A) and (B) The expression of CD112 in NB cell lines and primary NB tumor tissues are identified using qPCR and Western blot. (C) and (D) The expression of CD112 in NB cell lines and primary NB tumor tissues are identified using FACS. (E) Soluble CD112 in serum is detected by ELISA (HC, n = 17, NB, n = 22) and is further analyzed between different groups divided according to INSS stage, risk and MYCN status. (F) and (G) The expression of CD155 in NB cell lines and primary NB tumor tissues are identified using qPCR and Western blot. (H) and (I) The expression of CD155 in NB cell lines and primary NB tumor tissues are identified using FACS. (J) Soluble CD155 in serum is detected by ELISA (HC, n = 21, NB, n = 35) and is further analyzed between different groups divided according to INSS stage, risk and MYCN status. The results are expressed as the means ± SEMs from at least three independent experiments. Significant differences between groups are represented by ns no significance, *** p < 0.001.

Supplementary Figure 8 Enriched pathways and functional characteristics of CD8 T and NK cells. (A) The enriched KEGG pathways in CD8 T cells from high-ratio and low-ratio groups are shown in dot plots. The dot size in KEGG enrichment analysis represents enriched gene numbers. (B) and (C) Cell-cycle score and GSVA score analysis of CD8 T cells. (D) Violin plots showing the expression levels of DNAM-1, TIGIT, CD96, PD-1, TIM-3, IFN-γ, granzyme B and perforin in CD8 T cells. (E) The enriched KEGG pathways in NK cells from high-ratio and low-ratio groups are shown in dot plots. The dot size in KEGG enrichment analysis represents enriched gene numbers. (F) and (G) Cell-cycle score and GSVA score analysis of NK cells. (D) and (H) Violin plots showing the expression levels of DNAM-1, TIGIT, CD96, PD-1, TIM-3, IFN-γ, granzyme B and perforin in NK cells.

Supplementary Figure 11 Low-dose doxorubicin affects the expression of CD112 and CD155 in NB cells. (A) The apoptosis of SH-SY5Y and SK-N-BE2 cells treated with low-dose doxorubicin (0.1 μM) is detected by FACS (left) and AV+ proportion is labeled in the representative FACS histograms. (B-F) CD112 and CD155 expression in SH-SY5Y and SK-N-BE2 cells or primary NB tumor cells treated with low-dose doxorubicin (0.1 μM) are detected by FACS and Western blot. MFI is labeled in the representative FACS histograms. (G) and (H) The cytotoxicity assays are performed to detected γδT-cell cytotoxicity against SH-SY5Y and SK-N-BE2 cells pretreated with 0.1 μM doxorubicin. The results are expressed as the means ± SEMs from at least three independent experiments. Significant differences between groups are represented by * p < 0.05 and *** p < 0.001.

Supplementary Figure 10 Identification of Tet-on system, ROC curves and survival curves of different groups for NB tumor. (A) and (B) The variation of CD112 and CD155 expression in SK-N-BE2 cells treated with different concentration of doxycycline (0, 2 nM, 20 nM, 200 nM, 2 μM, 20 μM) is detected by qPCR and Western blot. (C) ROC curves are generated for CD112, CD155, and CD112/CD155 ratio to predict NB patient death, tumor risk or MYCN amplification. The results are expressed as the means ± SEMs from at least three independent experiments. Significant differences between groups are represented by ns no significance, * p < 0.05, ** p < 0.01 and *** p < 0.001.

Supplementary Figure 2 Gating strategies of FACS analysis and the expression pattern of TIGIT in NB. (A) Gating strategies for the FACS analysis of circulating γδT cells. (B) Gating strategies for the FACS analysis of tumor infiltrating immune cells. (C) and (D) The expression analysis of CD96 and CD112R in peripheral blood (PB) γδT cells from healthy controls (HC, n = 10) and neuroblastoma patients (NB, n = 18). The proportions of γδT cells are shown in the left panel statistically and the representative FACS histograms are shown in the right panel with mean fluorescence intensity (MFI) labeled in corresponding histograms. (E) Using GSE49711 from GEO database and R2: Genomics Analysis and Visualization Platform, the expression pattern of TIGIT in NB tumors (n = 498) with different INSS stages, risks, MYCN status and death events is analyzed and illustrated by box plots. (F) The overall and eventfree survival curves are generated by grouping samples with the median of TIGIT expression. (G) The expression correlation between TIGIT and DNAM-1 are analyzed using GSE49711 dataset. The results are expressed as the means ± SEMs. Significant differences between groups are represented by ns no significance, ** p < 0.01 and **** p < 0.0001.

Supplementary Figure 7 Metabolic states of high-ratio and low-ratio tumor cells. (A) Enriched metabolic reactions in high-ratio and low-ratio tumor cells revealed by COMPASS analysis. (B) Changes in metabolic KEGG pathways revealed by spatial metabolome analysis. (C) The heatmaps of representing metabolites.

Supplementary Figure 13 The effects of CD155 on γδT-cell-cytotoxicity against neuroblastoma, and on proliferation and migration of NB cells. (A) and (B) α-TIGIT is used in the rhCD155 treatment of γδT cells. DNAM-1 is detected by FACS and Western blot. (C-F) rhCD155 is used to treat γδT cells for 24 h. TIGIT, CD96 and CD112R are detected by FACS (left) or Western blot and MFI is labeled in the representative FACS histograms (right). (G) γδT-cell numbers are counted and statistically analyzed during in vitro culture in presence of rhCD155. (H) The CFSE proliferation assays are performed during in vitro culture in presence of rhCD155. γδT cells are detected by FACS (right) and statistically analyzed (left). MFI is labeled in the representative FACS histograms. (I) γδT cells are pre-treated with rhCD155 for 24 h and subjected to calcium flux detection by Fluo-4 AM labeling and low-concentration PMA and ionomycin stimulation. The calcium flux results are used to benchmark the levels of activation. The results are recorded and illustrated by FACS (right) and statistically analyzed (left). (J) The phosphorylation of Akt and ERK1/2 in γδT cells is detected by Western blot after treatment using rhCD155 for 24 h. (K) and (L) The cytotoxicity assays are performed with γδT cells pre-treated with rhCD155 or α-TIGIT for 24 h and NB cell lines. (M) and (N) The cytotoxicity assays are performed to detected γδT-cell cytotoxicity against SH-SY5Y and CHLA-255 cells in presence of α-CD155. (O-Q) IFN-γ, perforin and granzyme B expression are detected by FACS upon PMA and ionomycin stimulation after rhCD155 pre-treatment (left) and MFI is labeled in the representative FACS histograms (right). (R-T) CCK-8, Transwell and wound-healing sassays are performed using SK-N-BE2-NC and SK-N-BE2-KoCD155 cells. Representative images of SK-N-BE2 cell migration obtained from the Transwell (magnification × 100) and wound-healing (magnification × 100) assays are shown (right). The cell numbers obtained from the Transwell assays are counted, and the relative migration rate obtained from the wound-healing assays is calculated by dividing the change in the distance between the scratch edges by the initial distance (left). (U) The phosphorylation of Akt and ERK1/2 in γδT cells is detected by Western blot after SK-N-BE2-NC and SK-N-BE2-KoCD155 stimulation. (V) TIGIT is detected by FACS after 24 h co-culture with SK-N-BE2-NC and SK-N-BE2-KoCD155 cells (left) and MFI is labeled in the representative FACS histograms (right). The results are expressed as the means ± SEMs from at least three independent experiments. Significant differences between groups are represented by ns no significance, * p < 0.05, ** p < 0.01 and *** p < 0.001.

Supplementary Figure 12 The effects of CD112 on γδT-cell-cytotoxicity against neuroblastoma, and on proliferation and migration of NB cells. (A-D) rhCD112 is used to treat γδT cells for 24 h. TIGIT, CD96 and CD112R are detected by FACS (left) or Western blot and MFI is labeled in the representative FACS histograms (right). (E) γδT-cell numbers are counted and statistically analyzed during in vitro culture in presence of rhCD112. (F) The CFSE proliferation assays are performed during in vitro culture in presence of rhCD112. γδT cells are detected by FACS (right) and statistically analyzed (left). MFI is labeled in the representative FACS histograms. (G) γδT cells are pre-treated with rhCD112 for 24 h and subjected to calcium flux detection by Fluo-4 AM labeling and low-concentration PMA and ionomycin stimulation. The calcium flux results are used to benchmark the levels of activation. The results are recorded and illustrated by FACS (right) and statistically analyzed (left). (H) The phosphorylation of Akt and ERK1/2 in γδT cells is detected by Western blot after treatment using rhCD112 with or without α-DNAM-1 for 24 h. (I) and (J) The cytotoxicity assays are performed with γδT cells pre-treated with rhCD112 for 24 h and NB cell lines. (K) and (L) The cytotoxicity assays are performed to detected γδT-cell cytotoxicity against SH-SY5Y and CHLA-255 cells in presence of α-CD112. (M-O) CCK-8, Transwell and wound-healing sassays are performed using CHLA-255-NC and CHLA-255-shCD112 cells. Representative images of CHLA-255 cell migration obtained from the Transwell (magnification × 100) and wound-healing (magnification × 100) assays are shown (right). The cell numbers obtained from the Transwell assays are counted, and the relative migration rate obtained from the wound-healing assays is calculated by dividing the change in the distance between the scratch edges by the initial distance (left). (P) CD112 is overexpressed in IMR-32 cells transiently by transfecting constructed CD112 vector and CD112 expression is detected by qPCR, FACS and Western blot. MFI is labeled in the representative FACS histograms. (Q) DNAM-1 reduction is detected by FACS after 24 h co-culture with IMR-32-vector or IMR-32-CD112 cells (left) and MFI is labeled in the representative FACS histograms (right). (R) The phosphorylation of Akt and ERK1/2 in γδT cells is detected by Western blot after CHLA-255-NC and CHLA-255-shCD112 stimulation. (S) The cytotoxicity assays are performed to detected γδT-cell cytotoxicity against IMR-32-vector and IMR-32-CD112 cells. The results are expressed as the means ± SEMs from at least three independent experiments. Significant differences between groups are represented by ns no significance, * p < 0.05, ** p < 0.01 and *** p < 0.001.

Supplementary Figure 4 Quality control and annotation genes for NB tumor spatial transcriptome analysis. (A) Violin plots and heatmaps showing the number of features, RNA counts and percent mitochondrial transcripts found in NB tumor sample following quality control. (B) UMAP plot showing the major cell types in spatial transcriptome analysis. Dots represent individual cells, and colors represent different cell populations. (C) UMAP plots and dot plots showing the expression of the genes for annotating the major cell types identified in NB tumor.

Lipoblastoma is a rare benign tumour, with a higher recurrence rate in the head and neck region owing to complex anatomy. The study summarizes the clinical features, surgical risks, and complications of paediatric lipoblastoma in various anatomical domains of the head and neck, aiming to improve diagnosis and management. A retrospective review was conducted on children who were treated for lipoblastoma of the head and neck between 2016 and 2023. Data assessed included location, demographics, clinical presentation, imaging, treatment, complications, recurrence, and follow-up. A total of 32 patients with lipoblastoma were retrospectively analyzed. These lipoblastomas located in the face (n = 2, 6.3

Supplementary Figure 14 The effects of soluble CD155 or CD155 on NB cells on DNAM-1, PD-1, TIM-3 and TIGIT expression levels in γδT cells during the interaction with NB microenvironment. (A-C) α-CD155 is used in the co-culture of γδT cells and SH-SY5Y cells. DNAM-1, PD-1, TIM-3 and TIGIT are detected by FACS after 24 h co-culture (left) and MFI is labeled in the representative FACS histograms (right). (D-G) α-CD155 is used in the co-culture of γδT cells and primary NB tumor cells. DNAM-1, PD-1, TIM-3 and TIGIT expression are detected by FACS after 24 h co-culture (left) and MFI is labeled in the representative FACS histograms (right). (H-K) DNAM-1, PD-1, TIM-3 and TIGIT expression are detected by FACS after 24 h NB TTCS treatment (left) and MFI is labeled in the representative FACS histograms (right). The results are expressed as the means ± SEMs from at least three independent experiments. Significant differences between groups are represented by ns no significance, * p < 0.05 and ** p < 0.01.

Supplementary Figure 6 The cell-cycle scores and expression of functional genes in tumor cells and γδT cells in spatial transcriptome analysis and scRNA sequencing analysis. (A) Cell-cycle score analysis of tumor cells in spatial transcriptome analysis. (B) Spatial distribution and expression of proliferating markers. (C) Cell-cycle score analysis of tumor cells in scRNA sequencing analysis. (D) Violin plots showing the expression levels of neuroblastoma related genes in tumor cells in scRNA sequencing analysis. (E) and (F) Cell-cycle score and GSVA score analysis of γδT cells in scRNA sequencing analysis. (G) Violin plots showing the expression levels of DNAM-1, TIGIT, CD96, PD-1, TIM-3, IFN-γ, granzyme B and perforin in γδT cells in scRNA sequencing analysis.

Lymphatic malformations (LMs) are rare pediatric conditions that present variable symptoms depending on lesion size and anatomical location, necessitating diverse treatment approaches. This study aims to characterize the clinical features and prognosis of LMs across different anatomical sites and to compare the therapeutic efficacy of various treatment modalities. A retrospective analysis was performed on 478 pediatric patients diagnosed with LMs at Beijing Children’s Hospital between January 2014 and December 2024. Data regarding patient demographics, clinical characteristics, treatment modalities, and therapeutic outcomes were extracted and analyzed. The head and neck region was the most involved site (45.0

Supplementary Figure 5 The expression of CD112 and CD155 in tumor and para-tumor tissue sections detected by immunohistochemistry.

Supplementary Tables and Supplementary Figure legends

Supplementary Figure 9 The characteristics of NB tumors and immune cells in CD112/CD155 high-ratio and low-ratio groups revealed by bulk RNA sequencing analysis. (A) A total of 3537 DEGs between CD112/CD155 high-ratio and low-ratio groups (grouping according to the ratio median in the violin plot) are identified by RNA sequencing analysis with 38 NB tumor samples and illustrated in volcano plot. (B) The expression of genes related with neuroblastoma in KEGG pathway transcriptional misregulation in cancer is illustrated in heatmap. (C) and (D) The enriched top 20 KEGG pathways in high-ratio or low-ratio tumors are shown in dot plots. (E) and (F) The enriched top 30 GO pathways (top 10 in BP, CC and MF respectively) in high-ratio or low-ratio tumors are shown in dot plots. (G) and (H) The representative GSEA pathways enriched in high-ratio or low-ratio tumors are labeled with NES and p values.

To compare real-world initial treatment strategies for pediatric head and neck lymphatic malformations using a multidimensional outcome framework that distinguishes complete radiological response, objective lesion response, progression control, and treatment burden, and to examine how lesion complexity influences treatment selection and outcomes. We conducted a retrospective cohort study of pediatric patients with head and neck lymphatic malformations treated at a tertiary referral center between January 2016 and December 2025. Patients were categorized into surgery alone, interventional therapy alone, medical therapy alone, or combined therapy according to the initial treatment strategy. Radiological outcomes were classified as complete response, partial response, stable disease, or progression/recurrence. Primary outcomes included complete response, objective response rate, progression/recurrence, and progression-free survival. Lesion complexity was classified using an exploratory, literature-informed framework based on De Serres stage, cystic subtype, number of involved anatomical regions, and critical structure involvement. Multivariable logistic regression, generalized propensity score-based overlap weighting, propensity score matching, event-density analysis, and follow-up sensitivity analyses were used to address confounding by indication and unequal follow-up. Among 210 patients, 40 received surgery alone, 110 interventional therapy alone, 23 medical therapy alone, and 37 combined therapy. Surgery alone achieved the highest complete response rate (75.0

This study aimed to retrospectively analyze the clinical characteristics, tumor staging, surgical outcomes, and prognosis of children with MEN2B who presented with MTC. By summarizing our single-center experience, we emphasize the importance of early recognition and genetic screening to provide clinical insights for improving prognosis. Clinical data of six pediatric patients diagnosed with MEN2B in BCH, between January 2019 and December 2023, were reviewed. Demographic characteristics, clinical presentation, laboratory and imaging findings, genetic testing, surgical details, and follow-up information were analyzed. Descriptive statistical methods were used for data analysis. The cohort included 6 patients (2 males, 4 females) with a median age of 9.2 years (range 8.1–14.4 years). All patients harbored the de novo p.M918T RET mutation. All patients exhibited the classic MEN2B phenotype (marfanoid habitus, ocular signs, multiple oral mucosal neuromas, vocal cord nodules, high-arched palate, etc.) accompanied by alacrima and gastrointestinal symptoms. All underwent total thyroidectomy and bilateral central compartment neck dissection, with five undergoing concurrent lateral neck dissection due to suspected metastasis. Postoperative pathology confirmed MTC in all cases, with cervical lymph node metastases present in 4 patients (66.7