No abstract

Malignant pleural effusion (MPE) can accompany advanced lung adenocarcinoma. Recent studies suggest that MPE could contain a heterogeneous subpopulation of cells with stem-like properties, such as tumorigenicity and self-renewal, indicating that they could be the source of metastasis. Although previous studies analyzed the correlation between cancer stem cell (CSC) marker expression and clinical outcomes using lung cancer tissues, investigations regarding the association of MPE with CSC marker expression are limited. We performed immunohistochemistry to examine the expression of aldehyde dehydrogenase 1 (ALDH1) and Sal-like 4 (SALL4) in 46 cell block samples of MPE from patients with lung adenocarcinoma. ALDH1-positive and SALL4-positive cancer cells in MPE were detected in 30 (65.2%) and 21 samples (45.7%), respectively. Cluster formation was detected in 26 samples (56.5%). The number of clusters was significantly higher in ALDH1-positive/SALL4-negative samples. SALL4 expression was inversely correlated with the cluster ratio (r = −0.356) and positively associated with the Ki-67 index (r = 0.326), suggesting that MPE cells with high SALL4 expression comprised the proliferative subpopulation. In conclusion, we demonstrated that MPE contains an ALDH1-positive/SALL4-negative subpopulation exhibiting cluster formation and a SALL4-positive proliferative subpopulation.

Purpose Heparan sulfate (HS) is one of the factors that has been suggested to be associated with angiogenesis and invasion of glioblastoma (GBM), an aggressive and fast-growing brain tumor. However, it remains unclear how HS of endothelial cells is involved in angiogenesis in glioblastoma and its prognosis. Thus, we investigated the effect of endothelial cell HS on GBM development. Methods We generated endothelial cell-specific knockout of Ext1, a gene encoding a glycosyltransferase and essential for HS synthesis, and murine GL261 glioblastoma cells were orthotopically transplanted. Two weeks after transplantation, we examined the tumor progression and underlying mechanisms. Results The endothelial cell-specific Ext1 knockout (Ext1CKO) mice exhibited reduced HS expression specifically in the vascular endothelium of the brain capillaries compared with the control wild-type (WT) mice. GBM growth was significantly suppressed in Ext1CKO mice compared with that in WT mice. After GBM transplantation, the survival rate was significantly higher in Ext1CKO mice than in WT mice. We investigated how the effect of fibroblast growth factor 2 (FGF2), which is known as an angiogenesis-promoting factor, differs between Ext1CKO and WT mice by using an in vivo Matrigel assay and demonstrated that endothelial cell-specific HS reduction attenuated the effect of FGF2 on angiogenesis. Conclusions HS reduction in the vascular endothelium of the brain suppressed GBM growth and neovascularization in mice.

Heparan sulfate (HS) is a glycocalyx component present in the extracellular matrix and cell-surface HS proteoglycans (HSPGs). Although HSPGs are known to play functional roles in multiple aspects of tumor development and progression, the effect of HS expression in the tumor stroma on tumor growth in vivo remains unclear. We conditionally deleted Ext1, which encodes a glycosyltransferase essential for the biosynthesis of HS chains, using S100a4-Cre (S100a4-Cre; Ext1f/f) to investigate the role of HS in cancer-associated fibroblasts, which is the main component of the tumor microenvironment. Subcutaneous transplantation experiments with murine MC38 colon cancer and Pan02 pancreatic cancer cells demonstrated substantially larger subcutaneous tumors in S100a4-Cre; Ext1f/f mice. Additionally, the number of myofibroblasts observed in MC38 and Pan02 subcutaneous tumors of S100a4-Cre; Ext1f/f mice decreased. Furthermore, the number of intratumoral macrophages decreased in MC38 subcutaneous tumors in S100a4-Cre; Ext1f/f mice. Finally, the expression of matrix metalloproteinase-7 (MMP-7) markedly increased in Pan02 subcutaneous tumors in S100a4-Cre; Ext1f/f mice, suggesting that it may contribute to rapid growth. Therefore, our study demonstrates that the tumor microenvironment with HS-reduced fibroblasts provides a favorable environment for tumor growth by affecting the function and properties of cancer-associated fibroblasts, macrophages, and cancer cells.

Osteoprotegerin (OPG) is a regulator of bone metabolism that is also produced by breast tumor cells. There is accumulating evidence that OPG has tumor-promoting effects in breast cancer. We have recently demonstrated that OPG can promote invasion and metastasis of triple negative breast cancer cells. We are now investigating the expression of OPG across major breast cancer subtypes. We cultured 3 breast cancer cells lines from each of the major subtypes: MCF-7, ZR-75-1 and T47-D (Estrogen Receptor positive; ER+); SKBR-3, HCC1954 and HCC202 (Human Epidermal Growth Factor Receptor 2 positive; HER2+) and MDA-MB-231, MDA-MB-436 and BT-549 (triple negative). We incubated cells for 24 hours and then measured OPG mRNA expression by real time RT-PCR and secreted OPG protein in cell culture supernatant by ELISA. The triple negative cells that we tested expressed the highest levels of OPG mRNA and protein. A much lower level of OPG expression was observed in the HER2+ cells. Contrasting results were observed with the ER+ cells. While there was no expression from the ZR-75-1 or T47-D cells after 24 hours incubation, the MCF-7 cells produced levels of OPG similar to those observed with the triple negative breast cancer cells. We also looked at whether OPG expression levels could be altered by incubating breast cancer cells with macrophages, a cell type found in the breast tumor microenvironment. We co-cultured cells from each subtype with THP-1 human macrophages for 8 hours and extracted mRNA from the breast cancer cells or incubated the breast cancer cells for an additional 16 hours alone to measure secreted OPG protein. We found that co-culture with macrophages significantly increased OPG mRNA and protein expression in MCF-7 (ER+) and SKBR-3 (HER2+) cells but there was no increase in the MDA-MB-231 cells (triple negative). In conclusion, while OPG was expressed at comparatively high levels in triple negative breast cancer cells, there were generally low levels or no expression in the ER+ and HER2+ cells tested. This suggests that the tumor-promoting effects of OPG may contribute to the more aggressive phenotype in triple negative cells. However OPG expression in ER+ and HER2+ cells can be stimulated through co-culture with macrophages. Thus conditions within the breast tumor microenvironment could amplify the effects of OPG in these tumor subtypes. Citation Format: Stephanie Tsang, Ashleigh Renaud, Kim Roseman, Yuko Imaizumi, Nalini Yadav, Michael Weichhaus, Linda Connelly. Level of Osteoprotegerin expression is breast cancer subtype specific. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3210. doi:10.1158/1538-7445.AM2015-3210