Genetically Encoded Toolbox for Glycocalyx Engineering: Tunable Control of Cell Adhesion, Survival, and Cancer Cell Behaviors

Abstract

The glycocalyx is a coating of protein and sugar on the surface of all living cells. Dramatic perturbations to the composition and structure of the glycocalyx are frequently observed in aggressive cancers. However, tools to experimentally mimic and model the cancer-specific glycocalyx remain limited. Here, we develop a genetically encoded toolkit to engineer the chemical and physical structure of the cellular glycocalyx. By manipulating the glycocalyx structure, we are able to switch the adhesive state of cells from strongly adherent to fully detached. Surprisingly, we find that a thick and dense glycocalyx with high O-glycan content promotes cell survival even in a suspended state, characteristic of circulating tumor cells during metastatic dissemination. Our data suggest that glycocalyx-mediated survival is largely independent of receptor tyrosine kinase and mitogen activated kinase signaling. While anchorage is still required for proliferation, we find that cells with a thick glycocalyx can dynamically attach to a matrix scaffold, undergo cellular division, and quickly disassociate again into a suspended state. Together, our technology provides a needed toolkit for engineering the glycocalyx in glycobiology and cancer research.

Keywords: Muc1; glycobiology; mechanobiology; mucin; podocalyxin; synthetic biology.

Figure 1

Vector for stable expression. (A) Graphic illustration of the lentiviral and transposon stable incorporation systems. (B) Representative immunoblot (left) and lectin blot (right) comparison of stable Muc1 expression and PNA binding in lentiviral infection versus transposon integration, n = 3. (C) Mean integrated signal density from α-Muc1 immunoblots in B normalized to lentiviral samples; error bars represent the SD, n = 3. (D) Immunoblot (left) and lectin blot (right) of Muc1 expression in w.t. MCF10A cells compared to stable expression lines uninduced and after 24 h induction with 0.2 µg mL⁻¹ doxycycline, n =1. Cell lines were prepared with the transposon incorporation system. (E) Fold change in Muc1 analyzed by flow cytometry upon induction with various doxycycline concentrations, n = 3. * p < 0.05; ** p < 0.01 (two-tailed t test).

Figure 2

Genetically encoded glycoproteins for glycocalyx editing. (A) Schematic representation of the components and features of the native and engineered glycoproteins used in this study. S, signal sequence; VNTR, variable number tandem repeat; SEA, sea-urchin sperm protein, enterokinase, and agrin domain; TM, transmembrane domain; CT, cytoplasmic tail; FP, fluorescent protein; TM21, synthetic transmembrane domain, 21 amino acids; MP, membrane proximal domain; and ED, O-glycosylation rich ecto-domain region. (B) Cartoon illustration of the approximate relative size and features of various engineered glycoproteins. (C) Immunoblot (left) and lectin blot (center) showing the molecular weights and expression levels of Muc1 mutants in mammary epithelial cells (MECs) stably expressing the indicated gene, n = 3. Flow cytometry histograms (right) of the α-Muc1 antibody binding in cells expressing each of the engineered mutants compared to knockdown (shRNA) and w.t. cells, > 50,000 cells measured per condition. (D) Immunoblot (left) showing the relative size and expression level of Podxl mutants in stable MEC cell lines, n = 3. Flow cytometry histograms (right) of α-Podxl antibody binding in the same cell lines; >50,000 cells measured per condition.

Figure 3

Glycoproteins of tunable length. (A) Schematic illustration of the restriction sites introduced via mutation (*), reassembly fragments, and relative lengths of the engineered SynPodxl variants. (B) Lectin blot (left) of the relative O-glycosylation level of each mutant transiently expressed in HEK293T cells and an increased exposure time of the same lectin blot (right), n = 2.

Figure 4

Engineering the chemical and physical properties of the glycocalyx. (A) Confocal images of Muc1 (left), O-glycan (center left), and sialic acid (center right) showing cell surface localization in control and stably expressing MECs for each indicated construct (scale bar 25 µm), n = 3. (B) Representative flow cytometry histograms showing cell surface O and N-glycan and sialic acid levels of Muc1 and Podxl ΔCT and SynMucin mutants; >20,000 cells measured per condition and n = 3. (C) Representative fluorescence maximum intensity projections of SAIM image sequences (top left, second row, scale bar 10 µm) and membrane height reconstructions (top right, scale bar 10 µm, bottom row, scale bar 5 µm) of the ROIs boxed in red. (D) Average membrane height from 2k pixels in each of 5 cells per condition. Boxes correspond to SD and whiskers to extrema. Asterisks indicate statistical comparison of each mucin-expressing cell line to shRNA Muc1 expressing cells. * p < 0.05; ** p < 0.01; *** p < 0.001 (one-way ANOVA).

Figure 5

Expression of a bulky glycocalyx inhibits cellular adhesion. (A) Representative phase-contrast images of MECs expressing Muc1 ΔCT, uninduced, and induced for 24 h (scale bar 100 µm). (B) Representative epifluorescence images of MECs expressing Muc1 ΔCT moxGFP 4, 12, and 20 h postinduction (scale bar 100 µm), n = 2. (C) Ratio of detached MECs to adherent as a function of relative Muc1 expression measured by Muc1 ΔCT moxGFP fluorescence intensity, n = 2. (D) Concentration of live, detached MECs expressing Muc1 ΔCT as a function of time postinduction, quantified by flow cytometry with live/dead cell stain, n = 3. (E) Percentage of live cells in total detached population at various time points after induction quantified by flow cytometry with live/dead cell stain, n = 3. All error bars are SD.

Figure 6

Expression of a bulky glycocalyx enhances cell survival in suspension. (A) Percentage live cells as a function of time for MECs growing in suspension culture, n = 3. The solid curves represent a single exponential decay fit, $t_{w.t}^{1/2}=0.797d,t_{\mathit{\text{Muc}}1}^{1/2}=3.12d$. (B) Receptor tyrosine kinase signaling screen for MECs grown in suspension in full serum for 24 h, n = 2. Visible signal from positive controls. See Supporting Figure 2 for full details. (C) Percent live MECs expressing Muc1 ΔCT grown in suspension with 10 µM MAPK inhibitor U-0126 or DMSO control, n = 2. (D) Representative flow cytometry histogram showing incorporation of EdU into MECs under adherent or in suspension culture conditions; > 100,000 cells measured per condition, n = 2. Percentage indicates the fraction of cells with signal >10⁴ (gray line) after background subtraction. (E) Epifluorescence images of an MEC undergoing division with increasing expressing of Muc1 ΔCT moxGFP. Following separation, one of the new cells retains attachment to the substrate (white arrow), while the other detaches and drifts out of frame (red arrow) (scale bar 50 µm). All error bars are SD. Timestamp shown in hour:min.

Figure 7

Cellular division is associated with adhesion. (A) Live cell time-course images of a detached cell reattaching to the substrate (top), a division event wherein one of the new cells detaches from the substrate (top center, white arrow), a division event wherein a new cell briefly detaches then reattaches to the substrate (bottom center, white arrow), and a detached cell attaches to the substrate, divides, then one of the new cells detaches (bottom, white arrow) (scale bars 50 µm). (B) Ratio of detached, floating cells to adherent as a function of time in an initially attached population expressing Muc1 ΔCT. (C) The same measurement as B for an initially detached population. Error bars in B and C represent one SD, n = 3 for both conditions. (D) Epifluorescence image of a mixed population of MECs expressing Muc1 ΔCT and either cytoplasmic EGFP (green, initially detached) or mCherry (magenta, initially attached) demonstrating the exchange of states occurs in both directions (scale bar 100 µm).