Mechanism Of Lymph Node Metastasis Research Articles

Unraveling the molecular mechanisms of lymph node metastasis in ovarian cancer: focus on MEOX1

BackgroundLymph node metastasis (LNM) is a major factor contributing to the high mortality rate of ovarian cancer, making the treatment of this disease challenging. However, the molecular mechanism underlying LNM in ovarian cancer is still not well understood, posing a significant obstacle to overcome.ResultsThrough data mining from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, we have identified MEOX1 as a specific gene associated with LNM in ovarian cancer. The expression of MEOX1 was found to be relatively high in serous ovarian adenocarcinoma, and its higher expression were associated with increased tumor grade and poorer clinical prognosis for ovarian cancer patients. Bioinformatics analysis revealed that MEOX1 exhibited the highest mRNA levels among all cancer types in ovarian cancer tissues and cell lines. Furthermore, gene set enrichment analysis (GSEA) and pathway analysis demonstrated that MEOX1 was involved in various LNM-related biological activities, such as lymphangiogenesis, lymphatic vessel formation during metastasis, epithelial-mesenchymal transition (EMT), G2/M checkpoint, degradation of extracellular matrix, and collagen formation. Additionally, the expression of MEOX1 was positively correlated with the expression of numerous prolymphangiogenic factors in ovarian cancer. To validate our findings, we conducted experiments using clinical tissue specimens and cell lines, which confirmed that MEOX1 was highly expressed in high-grade serous ovarian cancer (HGSOC) tissues and various ovarian cancer cell lines (A2780, SKOV3, HO8910, and OVCAR5) compared to normal ovarian tissues and normal ovarian epithelial cell line IOSE-80, respectively. Notably, we observed a higher protein level of MEOX1 in tumor tissues of LNM-positive HGSOC compared to LNM-negative HGSOC. Moreover, our fundamental experiments demonstrated that suppression of MEOX1 led to inhibitory effects on ovarian cancer cell proliferation and EMT, while overexpression of MEOX1 enhanced the proliferation and EMT capacities of ovarian cancer cells.ConclusionsThe results of our study indicate that MEOX1 plays a role in the lymph node metastasis of ovarian cancer by regulating multiple biological activities, including the proliferation and EMT of ovarian cancer, lymphangiogenesis, and ECM remodeling. Our findings suggest that MEOX1 could serve as a potential biomarker for the diagnosis and treatment of ovarian cancer with LNM.

The microprotein encoded by exosomal lncAKR1C2 promotes gastric cancer lymph node metastasis by regulating fatty acid metabolism

Lymph node metastasis (LNM) is the prominent route of gastric cancer dissemination, and usually leads to tumor progression and a dismal prognosis of gastric cancer. Although exosomal lncRNAs have been reported to be involved in tumor development, whether secreted lncRNAs can encode peptides in recipient cells remains unknown. Here, we identified an exosomal lncRNA (lncAKR1C2) that was clinically correlated with lymph node metastasis in gastric cancer in a VEGFC-independent manner. Exo-lncAKR1C2 secreted from gastric cancer cells was demonstrated to enhance tube formation and migration of lymphatic endothelial cells, and facilitate lymphangiogenesis and lymphatic metastasis in vivo. By comparing the metabolic characteristics of LN metastases and primary focuses, we found that LN metastases of gastric cancer displayed higher lipid metabolic activity. Moreover, exo-lncAKR1C2 encodes a microprotein (pep-AKR1C2) in lymphatic endothelial cells and promotes CPT1A expression by regulating YAP phosphorylation, leading to enhanced fatty acid oxidation (FAO) and ATP production. These findings highlight a novel mechanism of LNM and suggest that the microprotein encoded by exosomal lncAKR1C2 serves as a therapeutic target for advanced gastric cancer.

WDR4 promotes the progression and lymphatic metastasis of bladder cancer via transcriptional down-regulation of ARRB2

Lymph node (LN) metastasis is one of the key prognostic factors in bladder cancer, but its underlying mechanisms remain unclear. Here, we found that elevated expression of WD repeat domain 4 (WDR4) in bladder cancer correlated with worse prognosis. WDR4 can promote the LN metastasis and proliferation of bladder cancer cells. Mechanistic studies showed that WDR4 can promote the nuclear localization of DEAD-box helicase 20 (DDX20) and act as an adaptor to bind DDX20 and Early growth response 1 (Egr1), thereby inhibiting Egr1-promoted transcriptional expression of arrestin beta 2 (ARRB2) and ultimately contributing to the progression of bladder cancer. Immunohistochemical analysis confirmed that WDR4 expression is also an independent predictor of LN metastasis in bladder cancer. Our results reveal a novel mechanism of LN metastasis and progression in bladder cancer and identify WDR4 as a potential therapeutic target for metastatic bladder cancer.

Lymph node metastasis in oral squamous cell carcinoma: Where we are and where we are going

AbstractBackgroundOral cancer is a common malignant tumour in the head and neck region, with over 90% of cases being oral squamous cell carcinoma (OSCC). Lymph node metastasis (LNM) is a significant adverse prognostic factor in OSCC; however, the mechanism of LNM in OSCC remains unclear, while the strategies for managing cervical lymph nodes (CLNs) in OSCC continue to evolve. At present, neck dissection is necessary for the vast majority of OSCC patients, but complications of surgery can significantly impair patients’ postoperative quality of life. A recent clinical trial discovery indicates that immunotherapy can activate anti‐tumour T cells in nearby lymph nodes, leading to vibrant discussions among clinicians about the importance of preserving lymph nodes during surgical treatment.AimThis review aims to provide an overview of where we are: the current knowledge of OSCC LNM patterns and management strategies, and where we are going: prospects for clinical and basic research on the issue of OSCC LNM in the era of cancer immunotherapy.ResultWe summarize the general patterns and management strategies of OSCC LNM. The process of LNM in OSCC encompasses four stages: Preparation, Unleash, Migration, and Planting (‘PUMP’ principle). We propose adopting the ‘PRECISE’ model, an overall workflow that includes seven elements—prevention, radiology, preoperative evaluation, chemotherapy, immunotherapy, surgery, and postoperative evaluation—for neck management in OSCC, promoting personalized precision medicine augmented by neoadjuvant therapy. We further discussed recent advances in clinical and basic research on OSCC LNM and provided an outlook on future research directions.ConclusionOver the past two centuries, our understanding and management strategies of LNM in OSCC have evolved, seeking more precise and personalized approaches to reduce patient burden and enhance survival and quality of life. Further research should explore lymph nodes’ role in cancer immunotherapy and OSCC interactions, leading to better, less invasive treatments and improved outcomes.

ScRNA-seq revealed an immunosuppression state and tumor microenvironment heterogeneity related to lymph node metastasis in prostate cancer

BackgroundMetastasis is a crucial aspect of disease progression leading to death in patients with prostate cancer (PCa). However, its mechanism remains unclear. We aimed to explore the mechanism of lymph node metastasis (LNM) by analyzing the heterogeneity of tumor microenvironment (TME) in PCa using scRNA-seq.MethodsA total of 32,766 cells were obtained from four PCa tissue samples for scRNA-seq, annotated, and grouped. InferCNV, GSVA, DEG functional enrichment analysis, trajectory analysis, intercellular network evaluation, and transcription factor analysis were carried out for each cell subgroup. Furthermore, validation experiments targeting luminal cell subgroups and CXCR4 + fibroblast subgroup were performed.ResultsThe results showed that only EEF2 + and FOLH1 + luminal subgroups were present in LNM, and they appeared at the initial stage of luminal cell differentiation, which were comfirmed by verification experiments. The MYC pathway was enriched in the EEF2 + and FOLH1 + luminal subgroups, and MYC was associated with PCa LNM. Moreover, MYC did not only promote the progression of PCa, but also led to immunosuppression in TME by regulating PDL1 and CD47. The proportion of CD8 + T cells in TME and among NK cells and monocytes was lower in LNM than in the primary lesion, while the opposite was true for Th and Treg cells. Furthermore, these immune cells in TME underwent transcriptional reprogramming, including CD8 + T subgroups of CCR7 + and IL7R+, as well as M2-like monocyte subgroups expressing tumor-associated signature genes, like CCR7, SGKI, and RPL31. Furthermore, STEAP4+, ADGRF5 + and CXCR4+, and SRGNC + fibroblast subgroups were closely related to tumor progression, tumor metabolism, and immunosuppression, indicating their contributions in PCa metastasis. Meanwhile, The presence of CXCR4 + Fibroblasts in PCa was confirmed by polychromatic immunofluorescence.ConclusionsThe significant heterogeneity of luminal, immune, and interstitial cells in PCa LNM may not only directly contribute to tumor progression, but also indirectly result in TME immunosuppression, which may be the cause of metastasis in PCa and in which MYC played an role.

A NOTCH1 Mutation Found in a Newly Established Ovarian Cancer Cell Line (FDOVL) Promotes Lymph Node Metastasis in Ovarian Cancer

Peritoneal implantation and lymph node metastasis have different driving mechanisms in ovarian cancer. Elucidating the underlying mechanism of lymph node metastasis is important for treatment outcomes. A new cell line, FDOVL, was established from a metastatic lymph node of a patient with primary platinum-resistant ovarian cancer and was then characterized. The effect of NOTCH1-p.C702fs mutation and NOTCH1 inhibitor on migration was evaluated in vitro and in vivo. Ten paired primary sites and metastatic lymph nodes were analyzed by RNA sequencing. The FDOVL cell line with serious karyotype abnormalities could be stably passaged and could be used to generated xenografts. NOTCH1-p.C702fs mutation was found exclusively in the FDOVL cell line and the metastatic lymph node. The mutation promoted migration and invasion in cell and animal models, and these effects were markedly repressed by the NOTCH inhibitor LY3039478. RNA sequencing confirmed CSF3 as the downstream effector of NOTCH1 mutation. Furthermore, the mutation was significantly more common in metastatic lymph nodes than in other peritoneal metastases in 10 paired samples (60% vs. 20%). The study revealed that NOTCH1 mutation is probably a driver of lymph node metastasis in ovarian cancer, which offers new ideas for the treatment of ovarian cancer lymph node metastasis with NOTCH inhibitors.

CircVPRBP inhibits nodal metastasis of cervical cancer by impeding RACK1 O-GlcNAcylation and stability

Lymph node (LN) metastasis is one of the most malignant clinical features in patients with cervical cancer (CCa). Understanding the mechanism of lymph node metastasis will provide treatment strategies for patients with CCa. Circular RNAs (circRNA) play a critical role in the development of human cancers. However, the role and mechanism of circRNAs in lymph node metastasis remain largely unknown. Here, it is reported that loss expression of circRNA circVPRBP was closely associated with LN metastasis and poor survival of CCa patients. In vitro and in vivo assays showed that circVPRBP overexpression notably inhibited lymphangiogenesis and LN metastasis, whereas RfxCas13d mediated silencing of circVPRBP promoted lymphangiogenesis and the ability of the cervical cancer cells to metastasize to the LNs. Mechanistically, circVPRBP could bind to RACK1 and shield the S122 O-GlcNAcylation site to promote RACK1 degradation, resulting in inhibition of Galectin-1 mediated lymphangiogenesis and LN metastasis in CCa. Taken together, the results demonstrate that circVPRBP is a potential prognostic biomarker and a novel therapeutic target for LN metastasis in CCa patients.

Integrated single-cell transcriptome analysis of the tumor ecosystems underlying cervical cancer metastasis.

Cervical cancer (CC) is one of the most frequent female malignancies worldwide. However, the molecular mechanism of lymph node metastasis in CC remains unclear. In this study, we investigated the transcriptome profile of 51,507 single cells from primary tumors, positive lymph nodes (P-LN), and negative lymph nodes (N-LN) using single-cell sequencing. Validation experiments were performed using bulk transcriptomic datasets and immunohistochemical assays. Our results indicated that epithelial cells in metastatic LN were associated with cell- cycle-related signaling pathways, such as E2F targets, and mitotic spindle, and immune response-related signaling pathways, such as allograft rejection, IL2_STAT5_signaling, and inflammatory response. However, epithelial cells in primary tumors exhibited high enrichment of epithelial-mesenchymal translation (EMT), oxidative phosphorylation, and interferon alpha response. Our analysis then indicated that metastasis LN exhibited an early activated tumor microenvironment (TME) characterized by the decrease of naive T cells and an increase of cytotoxicity CD8 T cells, NK cells, FOXP3+ Treg cells compared with normal LN. By comparing the differently expressed gene of macrophages between tumor and metastatic LN, we discovered that C1QA+ MRC1low macrophages were enriched in a tumor, whereas C1QA+ MRC1high macrophages were enriched in metastatic LN. Finally, we demonstrated that cancer-associated fibroblasts (CAFs) in P-LN were associated with immune regulation, while CAFs in tumor underwent EMT. Our findings offered novel insights into the mechanisms of research, diagnosis, and therapy of CC metastasis.

Comprehensive analysis of tissue proteomics in patients with papillary thyroid microcarcinoma uncovers the underlying mechanism of lymph node metastasis and its significant sex disparities.

BackgroundLymph node metastasis (LNM) in papillary thyroid microcarcinoma (PTMC) is associated with an increased risk of recurrence and poor prognosis. Sex has been regarded as a critical risk factor for LNM. The present study aimed to investigate the molecular mechanisms underlying LNM and its significant sex disparities in PTMC development.MethodsA direct data-independent acquisition (DIA) proteomics approach was used to identify differentially expressed proteins (DEPs) in PTMC tumorous tissues with or without LNM and from male and female patients with LNM. The functional annotation of DEPs was performed using bioinformatics methods. Furthermore, The Cancer Genome Atlas Thyroid Carcinoma (TCGA-THCA) dataset and immunohistochemistry (IHC) were used to validate selected DEPs.ResultsThe proteomics profile in PTMC with LNM differed from that of PTMC without LNM. The metastasis-related DEPs were primarily enriched in categories associated with mitochondrial dysfunction and may promote tumor progression by activating oxidative phosphorylation and PI3K/AKT signaling pathways. Comparative analyses of these DEPs revealed downregulated expression of specific proteins with well-established links to tumor metastasis, such as SLC25A15, DIRAS2, PLA2R1, and MTARC1. Additionally, the proteomics profiles of male and female PTMC patients with LNM were dramatically distinguishable. An elevated level of ECM-associated proteins might be related to more LNM in male PTMC than in female PTMC patients. The upregulated expression levels of MMRN2 and NID2 correlated with sex disparities and showed a positive relationship with unfavorable variables, such as LNMs and poor prognosis.ConclusionsThe proteomics profiles of PTMC show significant differences associated with LNM and its sex disparities, which further expands our understanding of the functional networks and signaling pathways related to PTMC with LNM.

Molecular Features in Lymphatic Metastases Reflect the Metastasis Mechanism of Lymph Nodes With Non-Small-Cell Lung Cancers.

Lymphatic metastasis influences clinical treatment and prognosis of patients with non-small-cell lung cancer (NSCLC). There is an urgency to understand the molecular features and mechanisms of lymph node metastasis. We analyzed the molecular features on pairs of the primary tumor and lymphatic metastasis tissue samples from 15 NSCLC patients using targeted next-generation sequencing. The potential metastasis-related genes were screened from our cohort based on cancer cell fraction. After filtering with gene functions, candidate metastasis-related events were validated in the MSK cohort with Fisher’s exact test. The molecular signature and tumor mutational burden were similar in paired samples, and the average mutational concordance was 42.0% ± 28.9%. Its metastatic mechanism is potentially a linear progression based on the metastatic seeding theory. Furthermore, mutated ataxia telangiectasia mutated and Rad3-related (ATR) and tet methylcytosine dioxygenase 2 (TET2) genes were significantly enriched in lymphatic metastases (p ≤ 0.05). Alterations in these two genes could be considered metastasis-related driving events. Mutated ATR and TET2 might play an active role in the metastasis of lymph nodes with NSCLC. More case enrollment and long-term follow-up will further verify the clinical significance of these two genes.

Single-Cell Profiling Reveals Heterogeneity of Primary and Lymph Node Metastatic Tumors and Immune Cell Populations and Discovers Important Prognostic Significance of CCDC43 in Oral Squamous Cell Carcinoma.

Although substantial progress has been made in biological research and clinical treatment in recent years, the clinical prognosis of oral squamous cell carcinoma (OSCC) is still not satisfactory. Tumor immune microenvironment (TIME) is a potential target, which plays an essential role in the response of anti-tumor immunity and immunotherapy. In this study, we used scRNA-seq data, revealing the heterogeneity of TIME between metastatic and primary site. We found that in the metastatic site, the content of cytotoxic T cells and classical activated macrophages (M1 macrophages) increases significantly, while alternately activated macrophages (M2 macrophages) and inflammatory cancer-associated fibroblasts (iCAFs) decrease, which may be due to the increased immunogenicity of OSCC cells in the metastatic site and the changes in some signal pathways. We also found that iCAFs may recruit alternately activated macrophages (M2 macrophages) by secreting CXCL12. Then, we described a regulatory network for communication between various TIME cells centered on OSCC cells, which can help to clarify the possible mechanism of lymph node metastasis in OSCC cells. By performing pseudotime trajectory analysis, we found that the expression CCDC43 is upregulated in more advanced OSCC cells and is an independent prognostic factor for poor living conditions. Other than this, the high expression of CCDC43 may impair the antitumor immunity of the human body and promote the metastasis of OSCC cells. Our research provides a profound insight into the immunological study of OSCC and an essential resource for future drug discovery.

Siglec-15 Is an Immune Suppressor and Potential Target for Immunotherapy in the Pre-Metastatic Lymph Node of Colorectal Cancer.

Lymph node metastasis indicates a poor prognosis in colorectal cancer. To better understand the underlying mechanisms of lymph node metastasis, we analyzed transcriptome characteristics of the pre-metastatic lymph node, a putative microenvironment favorable for the seeding and proliferation of cancer cells. Thus, we tried to compare and elucidate the transcriptional and immune characteristics of sentinel lymph nodes (SNs) with matched non-sentinel lymph nodes (NSNs) in colorectal cancer patients. In this study, a total of 38 pairs of SNs and NSNs were collected, in which 26 pairs of non-metastatic lymph nodes were subjected to RNA-seq and bioinformatics analysis for the gene expression profiles. There were 16 differentially expressed genes between SNs and NSNs being identified, including 9 upregulated and 7 downregulated genes in SN. Gene Ontology (GO) classification analysis revealed that the differentially expressed genes were mainly involved in leukocyte differentiation, chemokine secretion, and immune system regulation. In the meantime, gene set enrichment analysis (GSEA) showed that immune-related signaling pathways, such as transforming growth factor beta (TGF-β) signaling and tumor necrosis factor alpha (TNF-α)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, were enriched in NSN, while cell proliferation–related signaling pathways were enriched in SN, including MYC signaling and G2M checkpoint signaling. We further identified SIGLEC15 as a top upregulated gene in SN. However, RNAscope assay showed that SIGLEC15 was not largely co-expressed with M2 macrophage marker CD163. We then selected eight pairs of lymph nodes for further cytological studies. Flow cytometry analysis revealed that Siglec-15 was expressed on all myeloid cell subsets. The relative expression of SEGLEC15 (SN/NSN) was correlated with the microsatellite instability (MSI) status in colorectal cancer patients. Further studies found that small interfering ribonucleic acid (siRNA)-mediated silencing of SLGLEC15 can enhance the anti-tumor function of T cells, as indicated by cytokine release analysis. In conclusion, we presented here a first report on the gene expression profiling of the pre-metastatic lymph node in colorectal cancer. The findings in this study suggest that SIGLEC15 plays an important role in SN immunosuppression. SEGLEC15 silencing could be a therapeutic strategy for restoring T cell function in tumor SNs.

Long Noncoding RNA VESTAR Regulates Lymphangiogenesis and Lymph Node Metastasis of Esophageal Squamous Cell Carcinoma by Enhancing VEGFC mRNA Stability.

Lymph node metastasis is one of the most malignant clinical features in patients with esophageal squamous cell carcinoma (ESCC). Understanding the mechanism of lymph node metastasis will provide treatment strategies for patients with ESCC. Long noncoding RNAs (lncRNA) play a critical role in the development and progression of human cancers. However, the role and mechanism of lncRNAs in lymph node metastasis remain largely unknown. Here we show that VEGFC mRNA stability-associated long noncoding RNA (VESTAR) is involved in lymph node metastasis of ESCC. VESTAR was overexpressed in ESCC tissues and was predictive of poor prognosis in patients with ESCC. In ESCC, NXF1 and SRSF3 facilitated nuclear export of VESTAR to the cytoplasm, which was associated with lymph node metastasis. Depletion of VESTAR inhibited ESCC-associated lymphangiogenesis and lymphatic metastasis. Mechanistically, VESTAR directly bound and stabilized VEGFC mRNA. VESTAR also interacted with HuR, a positive regulator of VEGFC mRNA stability, and increased HuR binding to VEGFC mRNA. Our study reveals a novel lncRNA-guided mechanism of lymph node metastasis in ESCC and may provide a potential target for treatment of ESCC lymphatic metastasis. SIGNIFICANCE: These findings illustrate the lncRNA-guided regulation of VEGFC mRNA stability via direct RNA-RNA interactions, highlighting a therapeutic target for patients with ESCC with lymphatic metastasis.

Lymph node metastasis-derived gastric cancer cells educate bone marrow-derived mesenchymal stem cells via YAP signaling activation by exosomal Wnt5a

Lymph node metastasis (LNM), a common metastatic gastric-cancer (GC) route, is closely related to poor prognosis in GC patients. Bone marrow-derived mesenchymal stem cells (BM-MSCs) preferentially engraft at metastatic lesions. Whether BM-MSCs are specifically reprogrammed by LNM-derived GC cells (LNM-GCs) and incorporated into metastatic LN microenvironment to prompt GC malignant progression remains unknown. Herein, we found that LNM-GCs specifically educated BM-MSCs via secretory exosomes. Exosomal Wnt5a was identified as key protein mediating LNM-GCs education of BM-MSCs, which was verified by analysis of serum exosomes collected from GC patients with LNM. Wnt5a-enriched exosomes induced YAP dephosphorylation in BM-MSCs, whereas Wnt5a-deficient exosomes exerted the opposite effect. Inhibition of YAP signaling by verteporfin blocked LNM-GC exosome- and serum exosome-mediated reprogramming in BM-MSCs. Analysis of MSC-like cells obtained from metastatic LN tissues of GC patients (GLN-MSCs) confirmed that BM-MSCs incorporated into metastatic LN microenvironment, and that YAP activation participated in maintaining their tumor-promoting phenotype and function. Collectively, our results show that LNM-GCs specifically educated BM-MSCs via exosomal Wnt5a-elicited activation of YAP signaling. This study provides new insights into the mechanisms of LNM in GC and BM-MSC reprogramming, and will provide potential therapeutic targets and detection indicators for GC patients with LNM.

Study of the Relationship Between Serum Amino Acid Metabolism and Lymph Node Metastasis in Patients with Colorectal Cancer.

PurposeLymph node metastasis is one of the important prognostic factors of colorectal cancer, and an important index of individualized treatment. The purpose of this study is to use metabonomics to identify potential molecular markers of lymph node metastasis in colorectal cancer (CRC).Patients and MethodsPeripheral blood samples of 223 CRC patients were collected. The metabolic levels of amino acids and carnitine in peripheral blood of CRC patients, with and without lymph node metastasis, were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).ResultsThe results show that there were significant differences in the levels of serum amino acids and carnitine between lymph node metastatic patients and lymph node non-metastatic patients. The diagnostic model that was constructed by 9 types of serum metabolites has a high diagnostic ability.ConclusionLC-MS/MS is a detection method that has a broad application in predicting CRC prognosis, individualized treatment, and in studying the mechanism of lymph node metastasis.

Development and validation of hub genes for lymph node metastasis in patients with prostate cancer.

Lymph node metastasis is one of the most important independent risk factors that can negatively affect the prognosis of prostate cancer (PCa); however, the exact mechanisms have not been well studied. This study aims to better understand the underlying mechanism of lymph node metastasis in PCa by bioinformatics analysis. We analysed a total of 367 PCa cases from the cancer genome atlas database and performed weighted gene co‐expression network analysis to explore some modules related to lymph node metastasis. Gene Ontology analysis and pathway enrichment analysis were conducted for functional annotation, and a protein‐protein interaction network was built. Samples from the International Cancer Genomics Consortium database were used as a validation set. The turquoise module showed the most relevance with lymph node metastasis. Functional annotation showed that biological processes and pathways were mainly related to activation of the processes of cell cycle and mitosis. Four hub genes were selected: CKAP2L, CDCA8, ERCC6L and ARPC1A. Further validation showed that the four hub genes well‐distinguished tumour and normal tissues, and they were good biomarkers for lymph node metastasis of PCa. In conclusion, the identified hub genes facilitate our knowledge of the underlying molecular mechanism for lymph node metastasis of PCa.

Distribution of Regulatory T-Cells and Other Phenotypes of T-Cells in Tumors and Regional Lymph Nodes of Colorectal Cancer Patients.

Tumor microenvironments consist of many types of immune cells, in which regulatory T-cells (Tregs) are supposed to play important roles to suppress anti-tumor immunity. Regional lymph nodes are essential for antitumor immunity in colorectal cancer (CRC). In this study, we compared the diversity of phenotypes of T-cells in normal tissue and regional lymph nodes in order to determine the immunosuppressive mechanism of lymph node metastasis of CRC. Fifty patients were enrolled in this study, and paired samples (tumor tissue, normal tissue, and three regional lymph node samples and as well as non-regional lymph node samples) were obtained from each patient. In each paired-sample set, the proportions of different immune cell types and T-cells expressing immune checkpoint molecules were compared using flow cytometry. Higher proportions of Tregs [7.58% (4.94%-13.87%) vs. 1.79% (0.03%-5.36%), p<0.001] and lower proportions of INFγ-producing CD4-positive T (iCD4+) cells [21.49% (12.08%-27.35%) vs. 26.55% (15.65%-37.63%), p<0.001] were observed in tumor tissue than in normal mucosa. Parts of regional lymph nodes nearest the tumor had a greater proportion of Tregs [5.86% (4.18%-7.69%)] and lower proportions of iCD4+ [5.94% (3.51%-9.04%)] and INFγ-producing CD8-positive T (iCD8+) cells [21.93% (14.92%-35.90%)] than distant parts of regional lymph nodes and non-regional lymph nodes. Both immune-suppressing molecules (CTLA-4 and PD-1) and immune-promoting molecules (OX-40 and ICOS) tended to be highly expressed in tumor tissue and local lymph nodes. In patients with CRC, regional lymph nodes, especially the parts nearest the tumor, had a higher proportion of Tregs and other suppressive immunophenotypes of T-cells than those located more distantly.

Integrated bioinformatics analysis to screen hub genes in the lymph node metastasis of thyroid cancer

Thyroid cancer (TC) is one of the most common types of malignancy of the endocrine-system. At present, there is a lack of effective methods to predict neck lymph node metastasis (LNM) in TC. The present study compared the expression profiles from The Cancer Genome Atlas between N1M0 and N0M0 subgroups in each T1-4 stages TC in order to identify the four groups of TC LNM-associated differentially expressed genes (DEGs). Subsequently, DEGs were combined to obtain a total of 493 integrated DEGs by using the method of Robust Rank Aggregation. Furthermore, the underlying mechanisms of LNM were investigated. The results from Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses demonstrated that the identified DEGs may promote LNM via numerous pathways, including extracellular matrix-receptor interaction, PI3K-AKT signaling pathway and focal adhesion. Following construction of a protein-protein interaction network, the significance score for each gene was calculated and seven hub genes were screened, including interleukin 6, actinin α2, collagen type I α 1 chain, actin α1, calbindin 2, thrombospondin 1 and parathyroid hormone. These genes were predicted to serve crucial roles in TC with LNM. The results from the present study could therefore improve the understanding of LNM in TC. In addition, the seven DEGs identified may be considered as potential novel targets for the development of biomarkers that could be used in the diagnosis and therapy of TC.

Genomic mutation signatures in primary breast cancer and their axillary metastatic lymph nodes

Abstract Breast cancer is one of the most common malignant tumors in women all over the world. Metastasis represents a major adverse progression of breast cancer, resulting in poor survival duration. Axillary lymph node metastasis is often the first step of systemic metastasis process of breast cancer. However, the mechanism of lymph node metastasis and the genomic signatures of primary breast tumors and lymph node metastasis are still under exploration. Whole exome sequencing was applied to primary breast cancer, axillary metastatic lymph nodes, and white blood cells from 10 Chinese women patients in our study. Single nucleotide variants (SNVs) and copy-number variants (CNVs) were compared between primary tumors and lymph nodes for individual patients. There are somatic SNVs (average 5.58 ± 2.56 per megabase) in primary breast cancers and somatic SNVs (average 5.46 ± 2.66 per megabase) in axillary metastatic lymph nodes were identified, which is corresponding to a semblable mutation burden in two malignant sites (P = 0.81). No difference was found in CNVs (P = 0.33). In primary breast cancer, somatic SNVs (48.12 ± 13.80%) and CNVs (61.72 ± 35.00%) were overlapping with somatic SNVs (49.43 ± 12.30%) and CNVs (72.01 ± 24.31%) in axillary metastatic lymph nodes. Nine genes were screened for significant specific mutations in primary tumors, and 15 genes were significantly mutated in metastatic lymph nodes. Using MutSigCV screening, it was found that HRNR and AHNAK2 are lymph node metastasis-specific genes. In our study, primary breast tumors are directly related to axillary lymph node metastases together and there are most SNVs and CNVs which were overlapping in primary and metastatic sites. These variants which are overlapping is closely related to the metastatic process of tumor invasion with early genetic variability. This is the first time to prove the concept of polyclonal metastatic model and in this model more than one clone migrates establish the metastases to axillary lymph nodes. This study was approved by the institutional review board (IRB) of the Cancer Hospital, Chinese Academy of Medical Sciences, and Peking Union Medical College, China (approval No. NCC2016G-030) on March 3, 2016.

Abstract P6-09-11: NFATC2 suppresses the metastatic cascade in breast cancer patients

Abstract For the patients with breast cancer, the pathological status of axillary lymph nodes is one of the most important predictor of prognosis. In recent years, the application of sentinel lymph node biopsy has provided us with an opportunity for the further study on the mechanism of lymph node metastasis in breast cancer. Through detailed pathological examination of sentinel lymph nodes, we can easily find patients who are in the early stage of lymph node metastasis - that is, the tumor cells from the primary tumor, invading the lymph nodes and successfully colonized, but not yet break through the lymph node microenvironment defense to the subsequent lymph node. It is the most important stage during lymph node metastasis. In the present study, we selected the pairing samples from breast cancer patients with early stage lymph node metastasis to carry out differential genomics research. A total of 182 genes with significant function and involved in significant signal transduction pathways were obtained. Bioinformatics method was used to screen out the mRNA, microRNA and transcription factor in the process of lymph node metastasis of breast cancer. We investigated the role of activated T cells c2 (NFATC2) in the core transcription factors as a target for further clinical and basic research. The expression of NFATC2 was detected in the tissue microarray prepared from 200 patients with primary breast cancer. After the median follow-up period of 89.5 months, it showed that the prognosis of patients with NFATC2 overexpression was significantly better than that of the control group (p = 0.022). We also measured the expression of NFATC2 in 50 pairs of breast cancer patients with lymph node metastasis. The results showed that the expression of NFATC2 in the primary tumor was significantly higher than that in the matched lymph node, suggesting that in patients with breast cancer, the down-regulate or deficiency of NFATC2 expression may be related with lymph node metastasis. We also attempted to add the expression of NFATC2 as a new parameter in the prediction model of lymph node metastasis based on preoperative clinical and pathological parameters. The area under the ROC curve obtained in the newly established model was 0.767, the expression state of NFATC2 had impact on the performance of the prediction model. Furthermore, when we removed the NFATC2 parameter, it could reduce the area under the ROC curve in the validation group, suggesting that the expression of NFATC2 in the primary tumor may be used as a predictor of the pathological state of axillary lymph nodes. In vitro and in vivo experiments, we demonstrated that NFATC2 has a significant suppression effect on the proliferation, migration and invasion of breast cancer cells. The potential NFATC2-target genes were determined by RNAseq and Chipseq. Nine differentially expressed genes were found to be regulated by NFATC2 in the Chip-seq analysis and bound to the promoter region of the gene, whereas GRAMD3 and SRGAP2 in the subsequent validation showed a positive correlation with the expression of NFATC2, suggesting that the two genes are likely to have a positive regulatory relationship with NFATC2. This study clarifies that NFATC2 may represent a therapeutic target for early metastasis breast cancer. Citation Format: Xue J, Chi Y, Chen J, Wu J. NFATC2 suppresses the metastatic cascade in breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-09-11.