Posted on May 6, 2021
Supplementary MaterialsSupplemental data jciinsight-3-99791-s023
Supplementary MaterialsSupplemental data jciinsight-3-99791-s023. primary mediators of OS lung tropism and suggest pleiotropic, redundant mechanisms by which they could impact metastasis. Combination therapy research demonstrate proof concept for focusing on these tumor-lung relationships to influence metastatic disease. = 10 test pairs. Single-sample testing against a theoretical suggest of 0 (for normalization to major tissue) were examined, controlling to get a false discovery price of 0.05 using the Benjamini-Hochberg method. *Denotes applicant genes whose manifestation differs between major tumors and metastases considerably. (B) Consultant IHC areas from 1 major tumorClung metastasis set showing adjustments in the staining strength and staining patterns from major to metastasis. Size pubs: 100 m and 25 m (insets). Extra examples are demonstrated in Supplemental Shape 2. Using formalin-fixed paraffin-embedded (FFPE) major tumor-lung metastasis pairs from cells surgically excised from individuals noticed at our hospital, we determined the relative expression of each candidate gene using qRT-PCR assays specifically designed for and validated against archival FFPE tissues (Figure 1). Examples of hematoxylin and eosinCstained (H&E-stained) specimens and the specific tissues selected for RNA extraction are shown in Supplemental Figure 1. Of the candidate genes tested, IL-6 and CXCL8 were among the genes most reliably enriched for in the metastatic tumors. Expression of these genes was many fold higher in the metastatic lesions than in matched primaries. In IHC analysis, expression was heterogeneous and strongest for both IL-6 and CXCL8 along the leading edges (Figure 1B and Supplemental Figure 2). Select clinical characteristics of the sample population are shown in Table 1. Table 1 Clinical characteristics of patients Open in a separate window Production of IL-6 and CXCL8 correlates with metastatic potential in murine xenograft models of lung Pluripotin (SC-1) colonization. We next tested a panel of OS cell lines for their ability to colonize mouse lung. We found that OS-17 cells, when introduced into circulation via tail vein, develop metastatic foci with very high efficiency, while OHS and OS-25 cell lines demonstrate much Pluripotin (SC-1) lower metastatic efficiency (Figure 2). This effect remained consistent across multiple passages of cells and multiple assays. We tested these cell lines for production of IL-6 and CXCL8 by subjecting cell-free supernatants to ELISA (Figure 2D), which revealed a strong correlation between tumor cell production of both cytokines and the cell lines capacity to colonize murine lung. Open in a separate window Figure 2 Expression of IL-6 and CXCL8 correlates with lung-colonization efficiency.CB-17 SCID mice inoculated with 1 106 osteosarcoma cells were euthanized 49 days after inoculation. (A) Gross appearance of lung blocks taken from those mice suggests markedly greater efficiency of colonization by OS-17 relative to the other 2 Rabbit Polyclonal to ZC3H4 cell lines. Scale bar: 2 mm. (B) H&E stains from sections of paraffin-embedded left lobes were counted to quantify the number of metastases per section. Scale bar: 2 mm. (C) Quantification reveals significantly higher numbers of metastases (mets) in the OS-17 sections relative to both OS-25 and OHS (= 15 OS-17 and OHS, = 6 OS-25). (D) Determination of Pluripotin (SC-1) IL-6 and CXCL8 concentrations in 72-hour supernatants from cultures of each cell line reveals significant expression of both cytokines in the metastatic OS-17 cells relative to either nonmetastatic Pluripotin (SC-1) cell line (= 3 samples per cell line, run in triplicate). (E) Evaluation of capacity to respond to IL-6 and CXCL8 signals using transwell migration assay. Cells were plated in the top chamber and RPMI alone or RPMI containing 50 ng/mL IL-6 or 100 ng/ml IL-8 was placed in the bottom chamber. After 24 hours, plates Pluripotin (SC-1) were harvested and processed as described to quantify the number of cells migrating (= 3 per condition). ** 0.01; *** 0.001; **** 0.0001 relative to OS-17 (C and D) or RPMI (E); 1-way ANOVA with Tukeys post hoc test. IL-6.