9 research outputs found

    Modulation Of Thrombospondin-1 In The Lung Microenvironment: Implications For Targeting Metastasis

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    Metastasis of cancer to distant organs is the major cause of its lethality. With recent studies highlighting the role of the premetastatic niche in supporting metastatic progression, we focused our efforts on understanding the interplay between reprogrammed microenvironments in distal organs and tumor cells, to determine novel mechanisms that contribute to metastasis progression and identify potential targets for metastasis suppression. Our investigations unraveled a critical role of the anti-tumorigenic factor thrombospondin-1 (Tsp-1) in the reprogrammed lung microenvironment. Premetastatic niches generated in the lungs by metastasis-incompetent primary tumors showed elevated Tsp-1 expression. Interestingly, Gr1+ myeloid cells in the lungs were the major producers of Tsp-1, and they responded to the tumor-derived factor prosaposin (psap), abundantly secreted by metastasis-incompetent tumors compared to their metastatic counterparts. Further analysis led to the identification of the pentapeptide sequence, DWLPK, within psap that retains Tsp-1-inducing activity. Using DWLPK, recapitulated Tsp-1 induction in Gr1+ cells in the lungs and suppressed metastasis of Lewis lung carcinoma cells. These results suggest that DWLPK can potentially serve as a therapeutic agent for metastasis suppression. Studying metastasis in the setting of pre-existing lung inflammation, we observed loss of Tsp-1 in the lung microenvironment. Intranasal administration of bacterial lipopolysaccharide (LPS) induced lung inflammation, characterized by recruitment of Ly6G+ neutrophils, and it enhanced metastatic burden. Inflammation was accompanied by an increase in neutrophil elastase (NE) and cathepsin G (CG) protease activity, and loss of Tsp-1 protein in lungs. Our studies confirmed that NE and CG degrade Tsp-1 in lungs in response to LPS-induced inflammation, thus promoting metastasis. Interestingly, blocking the neutrophil proteases pharmacologically using the dual protease inhibitor Sivelestat suppressed metastasis, indicating that the proteases are clinically viable anti-metastatic therapeutic targets. Our studies demonstrate the novel finding that metastasis-incompetent tumors render the distal site metastasis-suppressive, by systemically inducing Tsp-1 expression in recruited myeloid cells. On the other hand, we have described a novel mechanism, where inflammation leads to Tsp-1 degradation via neutrophil proteases, generating a metastasis-promoting microenvironment. Our findings highlight the context-dependent plasticity of Ly6G+ cells at distal sites, functioning as the main source of anti-tumorigenic Tsp-1 and the proteases that degrade it

    Identification of Reprogrammed Myeloid Cell Transcriptomes in NSCLC

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    <div><p>Lung cancer is the leading cause of cancer related mortality worldwide, with non-small cell lung cancer (NSCLC) as the most prevalent form. Despite advances in treatment options including minimally invasive surgery, CT-guided radiation, novel chemotherapeutic regimens, and targeted therapeutics, prognosis remains dismal. Therefore, further molecular analysis of NSCLC is necessary to identify novel molecular targets that impact prognosis and the design of new-targeted therapies. In recent years, tumor ÔÇťactivated/reprogrammedÔÇŁ stromal cells that promote carcinogenesis have emerged as potential therapeutic targets. However, the contribution of stromal cells to NSCLC is poorly understood. Here, we show increased numbers of bone marrow (BM)-derived hematopoietic cells in the tumor parenchyma of NSCLC patients compared with matched adjacent non-neoplastic lung tissue. By sorting specific cellular fractions from lung cancer patients, we compared the transcriptomes of intratumoral myeloid compartments within the tumor bed with their counterparts within adjacent non-neoplastic tissue from NSCLC patients. The RNA sequencing of specific myeloid compartments (immature monocytic myeloid cells and polymorphonuclear neutrophils) identified differentially regulated genes and mRNA isoforms, which were inconspicuous in whole tumor analysis. Genes encoding secreted factors, including osteopontin (OPN), chemokine (C-C motif) ligand 7 (CCL7) and thrombospondin 1 (TSP1) were identified, which enhanced tumorigenic properties of lung cancer cells indicative of their potential as targets for therapy. This study demonstrates that analysis of homogeneous stromal populations isolated directly from fresh clinical specimens can detect important stromal genes of therapeutic value.</p></div

    RNA-deep sequencing analysis unravels differentially regulated mRNA isoforms in intratumoral BM-derived myeloid cells.

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    <p>(A) List of stroma-specific genes differentially regulated at the mRNA isoform level. (B) Wiggle plots showing read coverage across the Flt-1 gene in IMMCs from adenocarcinoma of lung (n = 3 patients) and IMMCs form adjacent lungs (n = 3 patients). The status of sFLT1 and mFLT1 is shown. (C) RNA-seq analysis showing <i>FLT-1</i> isoform expression levels of total <i>FLT1</i>, soluble FLT1 (<i>sFLT1</i>), and membrane binding FLT1 (<i>mFLT1</i>) in myeloid cells sorted from adjacent lung and tumor. (D) RT-PCR validation of <i>FLT1</i> and <i>mFLT-1</i> isoform expression in myeloid cells sorted from adjacent lung and tumor.</p

    Depletion of Ly6G<sup>+</sup> neutrophils impair growth of lung adenocarcinoma in mice.

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    <p>(A) Quantitation of orthotopic lung tumors in mice treated with either anti-Ly6G (╬▒Ly6G) or control (IgG) antibodies as assessed by bioluminescence (BLI) measurements as a function of time (in days, n = 7 and 6 per group respectively). Arrows indicate days at which antibody was administered. Data represent mean ┬▒ SEM. (B) Representative BLI images of animals. Color scale bar depicts the photon flux (photons per second) emitted from these mice at day 22. (C) Representative micro-CT slice images of lungs showing tumors in mice treated with anti-Ly6G or control IgG antibody at day 25. Axial, coronal, and sagittal views shown. Bright objects are high density (bone) and black represents air voids within the animal. Axial images were taken from same relative position in each animal; cross-hair points to the same location in all views. D, dorsal; V, ventral; L, left; R, right. (D) Quantification of lung tumor burden by micro-CT analysis (n = 3 per group) HU, Hounsfield Unit (where -1000 is air, -700 is lung, and +100 to +300 is soft tissue). Data represent mean ┬▒ SEM. (E) Flow cytometry scatter plots of peripheral blood showing depletion of Ly6G<sup>+</sup> cells in anti-Ly6G treated mice as compared with control IgG treated mice at 3 days post-treatment. (F) Flow cytometry analysis of peripheral blood showing numbers (cells per ul of blood) of Ly6G<sup>+</sup> and CD11b<sup>+</sup> myeloid cells, CD4<sup>+</sup> and CD8<sup>+</sup> T cells, B220<sup>+</sup> B cells, and Ly6C<sup>+</sup>Ly6G<sup>-</sup> monocytes. n = 4 per group. Data represent mean ┬▒ SEM.</p

    Increased number of bone marrow hematopoietic cells infiltrate lung compared to matched adjacent non-neoplastic lung.

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    <p>(A) H&E staining of lung tissue from an adenocarcinoma patient. The dotted line separates the tumor from the adjacent non-neoplastic lung. Image is at 20X magnification. (B) Representative immunofluorescence image of tumor and matched adjacent non-neoplastic lung of adenocarcinoma patient stained for epithelial cells (EpCAM<sup>+</sup>, red) and BM-derived hematopoietic cells (CD45<sup>+</sup>, green). DAPI (blue) was used to label cell nuclei. (C) Flow cytometry scatter plots showing CD45<sup>+</sup>EpCAM<sup>-</sup> BM hematopoietic cells and CD45<sup>-</sup>EpCAM<sup>+</sup> epithelial cells in tumor and matched adjacent lung. (D) Quantitation of CD45<sup>+</sup>EpCAM<sup>-</sup> BM-derived hematopoietic cells in NSCLC patients (n = 5). Data represents mean ┬▒ SEM. (E) Flow cytometry scatter plots showing EpCAM<sup>-</sup>CD11b<sup>+</sup>CD33<sup>-</sup> BM-derived neutrophils and EpCAM<sup>-</sup>CD11b<sup>+</sup>CD33<sup>+</sup> BM-derived immature myeloid cells in tumor and matched adjacent lung. (F) Flow cytometry scatter plots showing CD11b<sup>+</sup>CD33<sup>-</sup> neutrophils are CXCR2<sup>+</sup> while CD11b<sup>+</sup>CD33<sup>+</sup> immature myeloid cells are CXCR2<sup>-</sup> (left panel). Microscopy of flow cytometry sorted cells stained with H&E showing nuclear morphology CD11b<sup>+</sup>CD33<sup>-</sup> and CD11b<sup>+</sup>CD33<sup>+</sup> cells (right panel).</p

    RNA-seq analysis of BM immature monocytic myeloid cells and epithelial cells from NSCLC patients and controls.

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    <p>(A) Summary of RNA-seq reads from adenocarcinoma (tumor)- and adjacent lung-derived IMMCs (immature monocytic myeloid cells), neutrophils, and epithelial cells isolated from 6 adenocarcinoma patients and mapped to known human mRNA, genome, and novel mRNA of Aceview gene model. P, unique patient identifier; A, adjacent non-neoplastic lung tissue; T, neoplastic tumor tissue. (B) Spearman correlation analysis showing clustering of stromal cells derived from IMMCs, neutrophils, and epithelial cells based on global RNA-seq gene expression profiles into distinct tumor and adjacent lung groups. P, unique patient identifier; A, adjacent non-neoplastic lung tissue; T, neoplastic tumor tissue. (C) Venn diagrams showing total number of differentially expressed genes in immature IMMCs, neutrophils, and epithelial cells from adenocarcinoma compared to non-neoplastic adjacent lung. Cutoff of at least 50 unique mapped reads and FDR <5%. The genes in the list show differential expression with p<0.05, and fold change >2. (D) Differentially regulated stromal genes from (C), enriched for potential paracrine functions as determined by Gene Ontology annotation as secreted, extracellular space, or membrane (except membranes of organelles including golgi and endoplasmic reticulum). Of these, genes with the functions in key tumorigenic pathways including angiogenesis, ECM breakdown, cell migration, proliferation, invasion, cytokine function, chemokine function, and chemotactic function were selected. Genes selected for analysis are denoted in blue, transmembrane; red, secreted.</p