CDNA expression profiling of 4T1 breast cancer cells isolated from tumor-draining lymph node (4T1LN) or primary tumors (4T1PT)
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE86971 | GEO | 2017/09/10
SECONDARY ACCESSION(S): PRJNA343116
REPOSITORIES: GEO
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Project description:The microenvironment of lymph node metastasized tumors (LNMT) determines tumor progression and response to therapy, but a systematic study of LNMT is lacking. Here, we generate single-cell maps of primary tumors (PTs) and paired LNMTs in 8 breast cancer patients. We demonstrate that the activation, cytotoxicity, and proliferation of T cells are suppressed in LNMT compared with PT. CD4+CXCL13+ T cells in LNMT are more likely to differentiate into an exhausted state. Interestingly, LAMP3+ dendritic cells in LNMT display lower T cell priming and activating ability than in PT. Additionally, we identify a subtype of PLA2G2A+ cancer-associated fibroblasts enriched in HER2+ breast cancer patients that promotes immune infiltration. We also show that the antigen-presentation pathway is downregulated in malignant cells of the metastatic lymph node. Altogether, we characterize the microenvironment of LNMT and PT, which may shed light on the individualized therapeutic strategies for breast cancer patients with lymph node metastasis.
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Project description:Although the primary organ has been the subject of intense investigation in the field of organ fibrosis over the past several decades, the presence of lymph node fibrosis due to persistent activation of the immune response in its partner organ remains largely unknown. Previously, we demonstrated that activation of the immune response following ischemia-reperfusion injury (IRI) and crescentic glomerulonephritis (CGN) in the kidney was associated with extracellular matrix (ECM) production by fibroblastic reticular cells (FRCs) of the kidney-draining lymph node (KLN). Here, we sought to determine whether FRCs in the KLN become similarly fibrogenic following unilateral ureteral obstruction (UUO) of the kidney. We subjected 6-8-week-old C57BL/6J mice to UUO for 2, 7, and 14 days. We examined the microarchitecture of the kidney and KLN by immunofluorescence staining at each timepoint, and we quantified immune cell populations in the KLN by flow cytometry. The contralateral kidney unaffected by UUO and its partner KLN were used as controls. We found through immunofluorescence staining that FRCs increased production of ECM fibers and remodeled the microarchitecture of the UUO KLN, contributing to fibrosis that mirrored the changes in the kidney. We also observed by flow cytometry that the populations of CD11b+ antigen-presenting cells, CD11c+ dendritic cells, and activated CD4+ and CD8+ T cells were significantly higher in the UUO KLN than the KLN draining the unaffected contralateral kidney. Expression of the TGFβ/TGFβR signaling pathway was upregulated and colocalized with FRCs in the UUO KLNs, suggesting a possible mechanism behind the fibrosis. Both release of ureteral ligation at 2 days following UUO and depletion of FRCs at the time of injury onset halted the progression of fibrosis in both the kidney and the KLN. These findings for the first time highlight the association between fibrosis both in the kidney and the KLN during UUO, and they lay the groundwork for future studies that will investigate more deeply the mechanisms behind the connection between FRCs and KLN fibrosis.
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Project description:Lymph node metastasis is associated with tumor aggressiveness and poor prognosis in patients. Despite its significance in cancer progression, how immune cells in the tumor-draining lymph node (TDLN) participate in cancer immune regulation remains poorly understood. It has been reported that both anti-tumor and exhausted tumor-specific T cells can be induced in the TDLNs; however, B cell activation and maturation in the TDLN has received far less attention. In our studies using C57BL/6 mouse syngeneic E0771 breast cancer or B16F10 melanoma cell lines, tumor-associated antigens were found colocalized with the follicular dendritic cells (FDCs) in the germinal centers (GCs), where antigen-specific B cell maturation occurs. LN conduits and the subcapsular sinus (SCS) macrophages are two major routes of antigen trafficking to FDCs. Tumor growth induced LN conduit expansion in the B cell zone and disrupted the SCS macrophage layer, facilitating both the entry of tumor-associated antigens into the B cell zone and access to FDCs located in the GCs. Regional delivery of clodronate liposome specifically depleted SCS macrophages in the TDLN, increasing GC formation, and promoting tumor growth. Our study suggests that TDLN reconstruction creates a niche that favors B cell activation and maturation during tumor growth.
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Project description:BACKGROUND: We previously identified tumor-reactive lymphocytes in the first lymph nodes that drain the primary tumor. In this study, we performed lymphatic mapping to investigate the possibility of finding the first lymph nodes that drain metastases, and of learning whether these lymph nodes contained tumor-reactive lymphocytes suitable for adoptive immunotherapy. METHODS: Nineteen patients were studied. The primary tumor site was colorectal cancer in seven patients, malignant melanoma in four, ovarian cancer and breast cancer in two, and one each with pancreatic cancer, cholangiocarcinoma, leiomyosarcoma, and squamous cellular cancer of the tongue. By injection of Patent blue dye or radioactive tracers around the metastases, we identified draining lymph nodes from liver metastases (n = 9), intra-abdominal local recurrences (n = 3), and regional lymph node metastases (n = 7). In six patients, a preoperative lymphoscintigraphy was performed. RESULTS: We located the first draining lymph node or nodes from metastases or local recurrences; we named them "metinel nodes." Lymphocytes from the metinel nodes proliferated, showed clonal expansion, and produced interferon gamma (via in vitro expansions on stimulation with tumor homogenate) and interleukins, all of which demonstrate the characteristics of tumor-reactive lymphocytes. Eight of the nineteen patients received immunotherapy on the basis of tumor-reactive T cells derived from the metinel nodes. CONCLUSIONS: We demonstrate that it is possible to locate the first lymph nodes draining subcutaneous, lymphatic, and visceral metastases, the so-called metinel nodes. Metinel node-derived lymphocytes may be used to treat disseminated solid cancer, and clinical trials should evaluate the effect of such treatment.
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Project description:BackgroundLymph node metastasis status is a prognostic factor for further lymph node involvement and for patient survival in breast cancer patients. Frozen section analysis of lymph nodes is a reliable method for detection of macro-metastases. However, this method is far less effective in detecting micro-metastases, requesting improved diagnostic procedures.MethodsWe investigated expression and truncation of ezrin in (i) sentinel lymph node metastases, (ii) unaffected axillary lymph nodes, (iii) primary breast tumors, and (iv) healthy glandular breast tissues using 2D gel electrophoresis, SDS-PAGE, and mass spectrometry in addition to Western blotting.ResultsFull-length ezrin (E1; amino acids 1-586) is present in all four investigated tissues. Two truncated ezrin forms, one missing about the first hundred amino acids (E2a) and the other lacking about 150 C-terminal amino acids (E2b) were detectable in primary tumor tissues and in sentinel lymph node metastases but not in glandular tissues. Strikingly, an ezrin truncation (E3) which consists approximately of amino acids 238-586 was found strongly expressed in all sentinel lymph node metastases. Moreover, an N-terminal ezrin fragment (E4) that consists approximately of amino acids 1-273 was identified in sentinel lymph node metastases as well.ConclusionsWe show for the first time the existence of tissue-dependent specific ezrin truncations. The distinguished strong Western blot staining of ezrin E3 in sentinel lymph node metastases underlines its capability to substantiate the occurrence of lymph node (micro)metastases in breast cancer patients.
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Project description:Epithelial-mesenchymal transition (EMT) was shown to enhance metastatic abilities of cancer cells, but it remains elusive in clinical samples. Moreover, EMT is rarely studied in lymph node metastases (LNM), thus limiting our understanding of its role outside of the primary tumors (PT). We collected a set of samples including triplets - PT, circulating tumor cells (CTCs)-enriched blood samples and LNM from 108 early breast cancer patients. With immunohistochemistry we analyzed levels of EMT effectors - E-cadherin, vimentin and N-cadherin in LNM, central areas and margins of PT. Additionally, expression of EMT core regulators TWIST1, SNAI1, SNAI2 was measured with RT-qPCR. Patients with E-cadherin loss had CTCs in 45% of the cases in comparison to 23% with normal E-cadherin level (P = 0.05). Mesenchymal phenotype of CTCs-enriched blood fractions was five-times more frequent in patients with E-cadherin loss in PT compared to PT with normal E-cadherin levels (P = 0.01). Epithelial/mesenchymal status of matched samples at different stages of dissemination was frequently discordant, especially for pairs involving CTCs, indicating high plasticity of tumor cells. LNM showed increased expression of TWIST1, SNAI1, SNAI2 accompanied by decreased Ki67 labeling index, with median Ki67 of 15% in PT and 10% in LNM (P = 0.0002). Our findings demonstrate that E-cadherin loss, not only in PT margin, might lead to seeding of especially malignant CTCs with mesenchymal phenotype. In comparison to PT, cells in LNM re-express E-cadherin, upregulate EMT transcription factors and reduce cell division rate, which could be viewed as their long-term survival strategy.
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Project description:Cancer patients with malignant involvement of tumor-draining lymph nodes (TDLNs) and distant metastases have the poorest prognosis. A drug delivery platform that targets the primary tumor, TDLNs, and metastatic niches simultaneously, remains to be developed. Here, we generated a novel monoclonal antibody (MHA112) against peripheral node addressin (PNAd), a family of glycoproteins expressed on high endothelial venules (HEVs), which are present constitutively in the lymph nodes (LNs) and formed ectopically in the tumor stroma. MHA112 was endocytosed by PNAd-expressing cells, where it passed through the lysosomes. MHA112 conjugated antineoplastic drug Paclitaxel (Taxol) (MHA112-Taxol) delivered Taxol effectively to the HEV-containing tumors, TDLNs, and metastatic lesions. MHA112-Taxol treatment significantly reduced primary tumor size as well as metastatic lesions in a number of mouse and human tumor xenografts tested. These data, for the first time, indicate that human metastatic lesions contain HEVs and provide a platform that permits simultaneous targeted delivery of antineoplastic drugs to the three key sites of primary tumor, TDLNs, and metastases.
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Project description:BackgroundProgrammed cell death ligand 1 (PD-L1) is a potential predictive biomarker of the response to anti-PD-L1/anti- programmed cell death 1 (PD-1) therapy in multiple cancers, including triple negative breast cancer(TNBC). The purpose of this study was to investigate whether PD-L1 expression is homogenous in primary tumors(PTs) and synchronous axillary lymph node metastases(LNMs) of TNBC.MethodsPD-L1 expression was immunohistochemically evaluated in 101 TNBC patients' PTs and paired LNMs. PD-L1 expression in tumor cells and infiltrating immune cells or node lymphocytes in the PTs and associated LNMs was scored separately and was correlated with patients' clinical parameters and prognoses.ResultsPD-L1 expression exhibited spatial heterogeneity in both the tumor cells and the infiltrating immune cells or node lymphocytes of PTs and LNMs. The PD-L1 expression levels were significantly higher in the lymphocytes and tumor cells of the LNMs than in the PTs. PD-L1 expression was also more frequent among the LNMs. PD-L1 expression was associated with high grade and more stromal tumor-infiltrating lymphocytes(TILs). Furthermore, the disease-free survival and overall survival were similar between the PT- negative/LNM- positive and PT- positive/LNM- positive patients, both of which exhibited worse disease-free survival(DFS) thanPT -negative/LNM -negative patients.ConclusionsThe differential expression of PD-L1 between the PTs and LNMs suggests that LNMs PD-L1 status may be used to indicate whether PD-1/PD-L1-targeted therapy would be suitable for a node-positive TNBC patient in the future.
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