Introduction: Current therapies with tyrosine kinase inhibitors (such as Imatinib) against Chronic Myeloid Leukemia (CML) inhibit the production of fast-dividing cells. However, there is a small portion of cells that is insensitive to these drugs and impedes the eradication of the disease[1]. Part of this aberrant process is attributed to an altered adhesion via integrin receptors of CML cells to fibronectin (FN) present in the extracellular matrix of the bone marrow[2]. It is likely that adhesion of CML cells to bone marrow confers protection of cancer cells to most recent molecular drug therapy. To understand the importance of this adhesion process, this study was designed to immobilize FN on functionalized PTFE films and investigate the response of attached K562 cells to exploratory siRNA therapy.
Methods: FN was bound to PTFE films through glutaric anhydride or sulfo-succinimidyl-4-(p-maleimidophenyl-butyrate (S-SMPB), as described elsewhere[3]. PTFE films fitted in 96-well plates were seeded with ~15,000 cells and fixed and stained with 0.1% of Coomassie Blue where indicated. Cell viability assay (MTT) was performed 1, 5 and 7 days after seeding. siRNA delivery was undertaken using linoleic acid-substituted 2 kDa polyethyleneimine (PEI2-LA)[4]. siRNA/polymer particles were prepared with siRNA and polymer at 60 nM siRNA and polymer:siRNA ratio of 12:1. For silencing, Green Fluorescence Protein (GFP)-positive K562 cells (300,000 cells) were seeded on films and cells were transfected with GFP-siRNA/polymer particles after 1 week, and GFP silencing was evaluated by measuring the reduction in GFP fluorescence.
Results and Discussion: The extent of cell attachment on Clean PTFE and PTFE+GA films was almost negligible. Adsorbed FN showed sporadic and inconsistent cell adhesion. FN grafted via GA showed slightly higher cell density in comparison, although not uniform among replicates. Grafting of FN with SMPB allowed much higher cell density than GA+FN. Moreover, CRGDS peptide grafting via SMPB also showed high cell attachment (comparable to PTFE+SMPB+FN) (Fig 1A). Based on MTT analysis, the cell numbers were similar on day 1 for adsorbed and grafted FN, as well as for grafted peptides. The subsequent cell growth however, varied among groups, where cell growth was higher on films where FN and peptides were covalently grafted (SPMB or GA, Fig 1B).
The GFP silencing on K562 attached on PTFE+SMPB+FN was evaluated and compared to that of K562 cells transfected in suspension and the cells adhered (if any) on Clean PTFE films. The decrease in GFP fluorescence (silencing) of cells on Clean PTFE was -7.6±3.5%, whereas the silencing for cells attached on PTFE+SMPB+FN films was 25.1±18.9%, and for the cells transfected in suspension was 36.4±9.0% (p>0.05, Fig 2).
Conclusions: FN grafting with SMPB was more efficient in promoting cell adhesion and cell growth on films than the others films. Moreover, adhesion of leukemia cells through FN did not seem to affect significantly the siRNA silencing efficiency in comparison with the cells grown in suspension, suggesting that the siRNA delivery therapy with lipid-polymeric carriers can transfect similarly the leukemia cells regardless of their suspension or adhesion state. The FN grafted covalently through SMPB on PTFE films is potentially a good model for assessing the effects FN-mediated CML adhesion have on the response to siRNA and other therapies.
Alberta Cancer Foundation (ACF); Natural Sciences and Engineering Research Council of Canada (NSERC); Canadian Institutes of Health Research (CIHR); NSERC CREATE Program; Alberta Innovates - Health Solution (AIHS)
References:
[1] Sloma I, et al. Nature Publishing Group; 2010 Sep 23;24(11):1823–33.
[2] Weisberg E, et al. Open Journal of Hematology. 2012 Feb 21;3(1).
[3] Vallières K, et al. Macromol Biosci. 2007 May 10;7(5):738–45.
[4] Remant Bahadur KC, et al. Acta Biomaterialia. 2011 May;7(5):2209–17.