Lipid microdomains, ordered membrane phases containing cholesterol and glycosphingolipids, play an essential role in cancer cell adhesion and ultimately metastasis. Notably, elevated levels of cholesterol-rich lipid microdomains are found in cancer cells relative to their normal counterparts. Therefore, alterations of lipid microdomains through cholesterol modulation could be used as a strategy to prevent cancer metastasis. In this study, methyl-beta-cyclodextrin (MβCD), sphingomyelinase (SMase), and simvastatin (Simva) were used to investigate the effects of cholesterol on the adhesive behaviors of four non-small cell lung cancer (NSCLC) cell lines (H1299, H23, H460, and A549) and a small cell lung cancer (SCLC) cell line (SHP-77) on E-selectin, a vascular endothelial molecule that initiates circulating tumor cell recruitment at metastatic sites. Under hemodynamic flow conditions, the number of adherent NSCLC cells on E-selectin significantly decreased by MβCD and Simva treatments, while SMase treatment did not show a significant effect. Significant increases in rolling velocities were detected only for H1299 and H23 cells after MβCD treatment. In contrast, cholesterol depletion did not affect SCLC cell attachment and rolling velocities. Moreover, cholesterol depletion by MβCD and Simva, induced CD44 shedding and resulted in an enhanced membrane fluidity in the NSCLC cells, while it did not affect the membrane fluidity of the SCLC cells which lacked detectable expression of CD44. Our finding suggests that cholesterol regulates the E-selectin-mediated adhesion of NSCLC cells by redistributing the CD44 glycoprotein, and thus modulating the membrane fluidity.

Despite numerous advances in cancer research, lung cancer has remained the major cause of cancer-related mortality worldwide, mainly because of metastasis and the lack of curative systemic therapy. It has been well established that lipid rafts, the membrane microdomains containing cholesterol and glycosphingolipids, play an important role in cell adhesion and spreading. Notably, there are elevated levels of cholesterol-rich lipid rafts observed in cancer cells relative to normal ones. Thus, alterations in lipid rafts, such as through cholesterol depletion, have been considered as a strategy for cancer metastasis prevention. In this study three different treatments - methyl-beta-cyclodextrin (mβCD), sphingomyelinase (smase), and simvastatin (simva) - were used to investigate the effects of cholesterol depletion on the mechanics of adhesion and rolling velocities of multiple non-small cell lung cancer (NSCLC) cell lines (H1299, H23, H460, and A569) and a small cell lung cancer (SCLC) cell line (SHP-77) on E-selectin, a vascular endothelial molecule hypothesized to initiate recruitment of circulating tumor cells at metastatic sites. Under physiological conditions in a parallel plate chamber, it was observed that the number of adherent NSCLC cells significantly decreased through mβCD and simva treatments, while smase treatment did not show significant effect on those cells. In addition, only for NSCLC cells, significant increases in rolling velocities were detected after treatments with mβCD and simva, but not via smase. In contrast, cholesterol depletion did not affect the number of attached cells and rolling velocity of the SCLC cells. Our results show that cholesterol might regulate E-selectin-mediated adhesion of NSCLC cells to endothelium, most likely by disturbing functional E-selectin ligands expressed on the surface of the cancer cells. We have previously reported that CD44 expressed on colon cancer and breast cancer cells are functional E-selectin ligands that mediate cell adhesion and rolling on endothelium. Protein structure analysis by flow cytometry and western blot showed that all NSCLC cell lines strongly expressed CD44. After the cholesterol depletion through the various treatments, the molecular surface expression of CD44 was down regulated for all these cells. In contrast, the SCLC line did not show any expression of CD44. Although further studies must be performed to verify CD44 function as an E-selectin ligand, the data in total imply protein loss or absence due to the defection of the cell membrane integrity could impact cell adhesion and rolling, and consequently metastasis. Therefore, cholesterol depletion could have the potential to help prevent cancer metastasis through down regulation of relevant adhesion molecules. Citation Format: Amina Mohammadalipour, Christian Showalter, Harrison T. Muturi, Vishva Sharma, Amir M. Farnoud, Vishwajeet Puri, Sonia M. Najjar, Monica M. Burdick. Cell membrane cholesterol modulates lung cancer cell adhesion and rolling on E-selectin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 98.

Exosomes are small extracellular vesicles (40-150 nm in diameter) secreted by cells and initially proposed as a mechanism to eliminate cellular waste. More recently, exosomes have been reported to participate in cancer metastasis because of their ability to mediate local and distant cell-to-cell communication. Furthermore, exosomes create cancer-favored microenvironments, potentially enhancing cancer metastasis. However, it is unknown whether exosomes interact with E-selectin, an adhesion molecule expressed by vascular endothelium and shown to be involved in metastasis. Our lab has previously shown that BT-20 human breast cancer cells adhesively interact with endothelial E-selectin via several E-selectin ligands such as sialofucosylated CD44 variants, Mac-2BP, and gangliosides. Therefore, we hypothesize that exosomes secreted by the BT-20 human breast cancer cell line interact with E-selectin. To investigate this hypothesis, exosomes were generated by culturing cells in serum-depleted media and isolated by a series of ultracentrifugation steps. Molecular size analysis using dynamic light scattering revealed that the size range of the isolated particles was similar to the expected size of exosomes. To detect CD9 (an exosome marker) and E-selectin ligands, both breast cancer whole cell lysate and exosome lysate were tested by Western blot. The higher expression of CD9 in isolated particles than the whole cell lysates confirmed that the isolated particles were exosomes. When Western blots were labeled with recombinant E-selectin/Fc chimera with Ca2+, bands were detected at approximately 130 kDa, indicating the presence of E-selectin ligands. As a negative control, the divalent cation chelator EDTA was added to the primary labeling incubation. Bands were not detected in these negative control experiments, indicating no E-selectin interactions in the absence of Ca2+. Human IgG (hIgG) was also used as a negative control in Western blots and showed no signal with exosome lysates, as expected. In comparison, experiments were also performed with Hs578T breast cancer cells, a cell line that our lab has shown does not express E-selectin ligands. Exosomes isolated from Hs578T cells did not express E-selectin ligands, consistent with whole cells. Altogether, these results suggest that BT-20 human breast cancer exosomes express E-selectin ligands. Future work will focus on identifying and characterizing the exosome E-selectin ligands, as well as investigating other cancer cells that express E-selectin ligands. Ultimately, understanding the interaction between exosomes and E-selectin can lead to new diagnostic and therapeutic strategies for cancer metastasis. Citation Format: Yinan Huang, Christian A. Showalter, Monica M. Burdick. Exosomes derived from the BT-20 human breast cancer cell line express E-selectin ligands [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2626.

The epithelial-to-mesenchymal transition (EMT), and its reverse process, the mesenchymal-to-epithelial transition (MET), are associated with metastatic tumor progression in breast cancer (BC). E-selectin, a cell adhesion molecule expressed by cytokine-activated vascular endothelium, is also involved in metastasis via binding to counter-receptors (E-selectin ligands) on the surface of BC cells. Consequently, efforts have been made to investigate a link between the EMT/MET and the adhesive interactions between E-selectin and their ligands. Our lab previously demonstrated that BC cells with an epithelial phenotype exhibit more adhesive interactions between E-selectin/ligands under blood flow conditions than cells with a mesenchymal phenotype. Therefore, molecular factors in the blood that activate the EMT or the MET in circulating tumor cells (CTCs) have been of particular interest. These factors may regulate the potential for CTCs to traffic to bone marrow endothelium, a frequent site of BC metastasis that constitutively expresses E-selectin. To advance knowledge in this area, we hypothesized that miRNA-200c, associated with poor prognosis in metastatic BC patients, induces the MET in MDA-MB-231 BC cells and promotes adhesion between E-selectin/ligands. Microscopy images show that MDA-MB-231 cells transfected with miRNA-200c displayed a cell morphology similar to that of epithelial cells, whereas cells transfected with a negative control miRNA (NC miRNA) retained their mesenchymal phenotype. Furthermore, cells transfected with miRNA-200c exhibited greater mRNA expression of E-cadherin and lower expression of Vimentin compared to cells transfected with a NC miRNA by qRT-PCR. The expression of Zeb1/2, both targets for miRNA-200c, were down-regulated in cells transfected with miRNA-200c compared to cells transfected with a NC miRNA. Both the microscopy images and mRNA expression analysis indicate induction of MET in miRNA-200c transfected cells. Shear flow adhesion assays and shear flow detachment assays were performed at bone marrow shear stresses to examine adhesive interactions between E-selectin/ligands. Using these assays, MDA-MB-231 cells transfected with miRNA-200c demonstrated slower rolling velocities, greater firm adhesion, lower detachment, and equivalent tethering activity when compared to cells transfected with a NC miRNA. Moreover, analysis of the mRNA expression of α1,3- and α1,4- fucosyltransferases (FT) revealed increased expression of FT3 and FT6 in cells transfected with miRNA-200c compared to cells transfected with a NC miRNA. Altogether, these results suggest that miRNA-200c increases fluid-shear resistant adhesion and that factors present in the blood may impact the metastatic potential of cancer cells in circulation by regulating cellular phenotypes. Citation Format: Christian A. Showalter, Monica M. Burdick. MicroRNA-200c (miRNA-200c) increases adhesive interactions between E-selectin ligands expressed by breast cancer cells and E-selectin under physiological blood flow conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1878.

Cancers of the digestive tract cause nearly one quarter of the cancer deaths worldwide, and nearly half of these are due to cancers of the esophagus and colon. Early detection of cancer significantly increases the rate of survival, and thus it is critical that cancer within these organs is detected early. In this regard, endoscopy is routinely used to screen for transforming/cancerous (i.e. dysplastic to fully cancerous) tissue. Numerous studies have revealed that the biochemistry of the luminal surface of such tissue within the colon and esophagus becomes altered throughout disease progression. Molecular endoscopic imaging (MEI), an emerging technology, seeks to exploit these changes for the early detection of cancer. The general approach for MEI is as follows: the luminal surface of an organ is exposed to molecular ligands, or particulate probes bearing a ligand, cognate to biochemistry unique to pre-cancerous/cancerous tissue. After a wash, the tissue is imaged to determine the presence of the probes. Detection of the probes post-washing suggests pathologic tissue. In the current review we provide a succinct, but extensive, review of ligands and target moieties that could be, or are currently being investigated, as possible cognate chemistries for MEI. This is followed by a review of the biophysics that determines, in large part, the success of a particular MEI design. The work draws an analogy between MEI and the well-advanced field of cell adhesion and provides a road map for engineering MEI to achieve assays that yield highly selective recognition of transforming/cancerous tissue in situ.