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The Effect of Human Platelets on Lung Cancer Stem Cell Invasion

  • Author / Creator
    Yan, Meng Jie
  • Background: The cancer stem cell (CSC) theory suggests there are small populations of cancer cells that has stem cell-like characteristics, such as self-renewal. These CSCs are thought to be responsible for initiating new tumors following metastasis. Platelets have also been implicated in cancer metastasis, in part by stimulating cancer cell invasion. Moreover, studies show cancer cells can activate platelets. Stromal derived factor-1 alpha (SDF-1), which may be secreted from activated platelets, can mobilize stem and CSCs via increased matrix metalloproteinase (MMP) expression. Thus, I hypothesize that SDF-1from activated platelets can preferentially stimulate CSC invasion by binding to its receptor C-X-C chemokine receptor type 4 (CXCR-4) on CSC surfaces. Additionally, I hypothesize that the preferential stimulation of CSC invasion by platelets occurs due to an SDF-1α stimulated increase in CSC MMP production. Methods: CSCs from the A549 lung carcinoma cell line were identified as Hoechst 33342-negative side population (SP) or identified and sorted using fluorescence activated cell sort (FACS) based on CD133-surface expression. Invasion assays using gelatin-coated Boyden chambers were used to compare the invasion of CSCs, non-CSCs, and total A549 population in response to collagen-aggregated human platelet releasates and quantified by flow cytometry and confocal microscopy. A549 CSC vs non-CSC MMP-2 and -9 protein levels were compared using gelatin zymography, and their MMP-2 mRNA levels were investigated using real time qPCR. Results: A Hoechst-negative SP was identified within the A549 cell line and this SP was enriched with CD133-positive cells. Incubation of A549 cells with platelets or platelet releasates did not increase A549 cell CD133-surface expression, indicating platelets do not cause the conversion of CD133-negative into CD133-positive cells. Collagen-aggregated platelet releasates preferentially stimulated invasion of both SP and CD133-positive cells. The CXCR-4 antagonist AMD3100 (10M) failed to inhibit SP invasion, but inhibited total A549 invasion. AMD3100 decreased the invasion of CD133-positive cells. Although there was a trend toward reduced inhibition of total A549 invasion (p = 0.07), AMD3100 failed to cause a statistically significant decrease in this set of experiments. A549 CD133-positive cells expressed higher basal MMP-2 levels than CD133-negative cells. 24-hour incubation with washed platelets increased MMP-2 protein in CD133-negative cells to CD133-positive cell levels, but did not change MMP-9 levels in either population. Real time qPCR revealed that CD133-negative cells increased MMP-2 mRNA expression to a greater extent than CD133-positive cells in response to incubation with platelets. Conclusions: Within the A549 lung carcinoma cell line, subpopulations of cancer cells with CSC markers exist as the A549 SP is enriched with CD133-expressing cells. Aggregated platelet releasates preferentially stimulate invasion of A549 SP- and CD133-identified CSCs. This preferential stimulation of invasion likely involves platelet-derived SDF-1α signalling. CD133-positive cells have higher basal levels of MMP-2. Incubation with washed platelets increases MMP-2 levels in CD133-negative cells to CD133-positive cell levels. Further experiments are required to delineate the role of SDF-1-CXCR-4 signalling in platelet-stimulated lung CSC invasion.

  • Subjects / Keywords
  • Graduation date
    2016-06
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/R3BR8MQ3W
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Master's
  • Department
    • Faculty of Pharmacy and Pharmaceutical Sciences
  • Specialization
    • Pharmaceutical Sciences
  • Supervisor / co-supervisor and their department(s)
    • Jurasz, Paul (Pharmacy and Pharmaceutical Sciences)
  • Examining committee members and their departments
    • Barakat, Khaled (Pharmacy and Pharmaceutical Sciences)
    • Velazquez, Carlos (Pharmacy and Pharmaceutical Sciences)
    • Marsh, Sharon (Pharmacy and Pharmaceutical Sciences)