The Role of Surface Receptor Density in Surface-Initiated Polymerizations for Cancer Cell Isolation

Abstract

Fluid biopsies potentially offer a minimally invasive alternative to traditional tissue biopsies for the continual monitoring of metastatic cancer. Current established technologies for isolating circulating tumor cells (CTCs) suffer from poor purity, yield, and require fixatives that preclude the collection of viable cells for longitudinal analyses of biological function. Antigen Specific Lysis (ASL) is a rapid, high-purity method of cell isolation based on targeted protective coatings on antigen-presenting cells and lysis depletion of unprotected antigen-negative cells. In ASL, photoinitiators are specifically labeled on cell surfaces that enable subsequent surface-initiated polymerization. Critically, the significant determinants of process yield have yet to be investigated for this emerging technology. In this work, we show that the labeling density of photoinitiators is strongly correlated with the yield of intact cells during ASL by flow cytometry analysis. Results suggest ASL is capable of delivering ~25% of targeted cells after isolation using traditional antibody labeling approaches.. Monomer formulations of two molecular weights of PEG-diacrylate (Mn~575 and 3500) are examined. The gelation response during ASL polymerization is also investigated via protein microarray analogs on planar glass. Finally, a density threshold of photoinitiator labeling required for protection during lysis is determined for both monomer formulations. These results indicate ASL is a promising technology for high yield CTC isolation for rare-cell function assays and fluid biopsies.

Comparison of eosin and fluorescein conjugates for the photoinitiation of cell-compatible polymer coatings

“…The effect of irradiation intensity and photoinitiator density on gelation thickness is also shown. Polymerizations initiated by 30 mW/cm 2 and SA-EITC yielded a mean gelation thickness of 145 nm at the highest initiator labeling density studied of approximately 18,000 eosin molecules per μm 2 , which is consistent thickness with previous studies at similar polymerization conditions and photoinitiator densities [ 17 , 26 ]. For SA-EITC, polymerization with the higher irradiation intensity of 30 mW/cm 2 generally resulted in thicker films than irradiation with 20 mW/cm 2 , and this is most distinct above 2,000 molecules per μm 2 .…”

“…The UV-Vis absorbance spectra of fluorescent dye conjugates dissolved in PBS were also obtained in triplicate. Using the generated linear fit equations detailed in our previous study [ 26 ], the degree of labeling for each fluorescent conjugate was then determined to be 2.2 (SA-Cy3), 5.1 (SA-EITC), and 4.3 (SA-FITC). Additionally, absorbance peaks at 280 nm were used to determine the overall protein concentration in stock dye solutions, and solutions were adjusted to between 0.5 and 1 mg/ml for storage of all dye conjugates.…”

“…Antibodies are coupled with eosin labels to localize eosin to the cell membrane of antigen positive cells. When these eosin-labeled cells are irradiated in a monomer and triethanol amine solution, polymerization proceeds only at the eosin-primed cell surfaces, yielding a ~100 nm polymer coating at high cell viabilities (~90%) [ 16 , 19 , 20 ]. A critical limitation to this coating approach is the requirement of each researcher to prepare custom eosin-biomolecule conjugates to localize the initiator at the cell membrane.…”

Genetically Encoded Stimuli-Responsive Cytoprotective Hydrogel Capsules for Single Cells Provide Novel Genotype–Phenotype Linkage

“…Several early demonstrations of cellular encapsulation technology utilized formation of multicellular aggregates on the scale of 0.1-1 mm by relying on self-assembly of oppositely charged polyelectrolyte chains 12,13 or incorporation of cells in agarose emulsions 14 . Polymerization-based approaches have further been developed which rely on cross-linking polymer materials from monomeric precursor solutions [15][16][17][18][19][20][21] .…”

50th Anniversary Perspective: Polymeric Biomaterials: Diverse Functions Enabled by Advances in Macromolecular Chemistry

“…186, 187 Photocleavage of o -NB linkages in the polymer then allows retrieval of highly purified cell populations post-lysis. 186 The sequential application of two photopolymerization steps to thiolene-based polymers has allowed the capture of a population of cells, followed by selective encapsulation of undesirable cells and retrieval of the desired cell population. 188 Polymer-based cell-sorting strategies have thus enabled both analysis of individual cell types and enrichment of specific cell populations for therapeutic use.…”