Abstract
Cancer cell glycocalyx is a major line of defence against immune surveillance. However, how specific physical properties of the glycocalyx are regulated on a molecular level, contribute to immune evasion and may be overcome through immunoengineering must be resolved. Here we report how cancer-associated mucins and their glycosylation contribute to the nanoscale material thickness of the glycocalyx and consequently modulate the functional interactions with cytotoxic immune cells. Natural-killer-cell-mediated cytotoxicity is inversely correlated with the glycocalyx thickness of the target cells. Changes in glycocalyx thickness of approximately 10 nm can alter the susceptibility to immune cell attack. Enhanced stimulation of natural killer and T cells through equipment with chimeric antigen receptors can improve the cytotoxicity against mucin-bearing target cells. Alternatively, cytotoxicity can be enhanced through engineering effector cells to display glycocalyx-editing enzymes, including mucinases and sialidases. Together, our results motivate the development of immunoengineering strategies that overcome the glycocalyx armour of cancer cells.
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Data availability
Raw data are available from the corresponding author upon request. Source data are provided with this paper.
Code availability
Packages for fitting the SAIM image sequences with the above model have been implemented in C++ and Julia and are available via GitHub at https://github.com/mjc449/SAIMscannerV3 and https://github.com/paszeklab/SAIMFitKit.jl.
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Acknowledgements
This investigation was supported by the National Science Foundation (NSF) grant 1752226 (M.J.P.), the Breast Cancer Coalition of Rochester predoctoral fellowship (S.P.) and the following National Institutes of Health (NIH) grants: CA276398 (M.J.P.), GM229133 (M.J.P.), CA210184 (M.J.P. and C.F.), CA193043 (M.J.P. and W.R.Z.), GM138692 (M.J.P.), GM137314 (M.J.P and M.D.), AI147362 (M.K.), T32AI007285 (C.J.S.) and CA273349 (S.N.). NGM138692 supported the glycoengineered cell-line development and glycocalyx material characterization. Work was performed at the Cornell Nanoscale Facility (NSF NNCI-2025233), Biotechnology Resource Center (RRID:SCR_021740) and Imaging Facility (RRID:SCR_021741) with NYSTEM (C029155) and NIH (S10OD018516) funding the ZEISS LSM880 instrument. The SAIM instrument development was supported by the Kavli Institute at Cornell for Nanoscale Science and project no. CA193043. We thank X. Su for providing the CD19 CAR cDNA and S. Malaker for helpful insights.
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All authors contributed to the design of experiments, interpretation of results and preparation of the manuscript. Construction of plasmids, preparation of cell lines and execution of experiments were performed by S.P., M.J.C., J.H.P., C.R.S., A.S., E.J.S., C.J.S., L.-T.H., J.C.-H.K, M.C.G. and J.S. M.J.C. built the SAIM microscope. C.J.S. and M.K. generated the CAR T cells. A.S. and S.N. conducted the CORA analysis. S.P. and M.J.P. wrote the manuscript with feedback from all authors.
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Cornell’s Center for Technology Licensing has submitted a patent related to the findings of this work with two authors (S.P. and M.J.P.) listed as the inventors (PCT/US2022/080937). All other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Additional analysis of Muc1-mediated protection against NK cell cytotoxic function.
a,b, Representative confocal and brightfield images of two clonally expanded MCF10A cell lines expressing Muc1-GFP with 42 tandem repeats (TRs) under the control of a tetracycline inducible promoter at the indicated doxycycline induction level. Scale bars, 10 µm. c, Representative confocal and brightfield images of Muc1-expresssing B2 clonal cells labelled with anti-Human Muc1 Janelia Fluor 549; the doxycycline induction level is indicated. Scale bars, 10 µm. d, NK-92 cell-mediated cytotoxicity against the polyclonal Muc1-GFP and three Muc1-GFP expressing clonal cell lines (1E7, 2E4, and 2G9) expressing MCF10A line from which the clonal lines were isolated, and a clonal MCF10A cell line expressing the Muc1 construct without the GFP reporter (B2 clone); the doxycycline induction levels are indicated. Results are the mean ± s.d of n =3 experimental replicates. NK cell to target cell ratio is 5:1. e. Mean fluorescence intensity of cell-surface Muc1-GFP in 1E7 cells treated with the indicated concentration of StcE mucinase; cell-surface Muc1-GFP probed with Alexa Fluor 647 conjugated GFP nanobody. Results are shown for induction of Muc1-GFP at 100 ng/ml and 1000 ng/ml doxycycline. Results are the mean ± s.d. of at least 14 cells from one representative of 3 independent experiments. Statistical analysis by one-way ANOVA with multiplicity-adjusted P values from Tukey’s multiple comparisons test.
Extended Data Fig. 2 Additional data supporting Siglec-independent protection by the mucin layer.
a, b, Surface expression levels of CD3 (a) and CD56 (b) in NK-92 cells and primary NK cells analyzed by flow cytometry, validating isolation of CD56+/CD3- NK cells. c, d, Surface levels of Siglec-7 (c) and Siglec-9 (d) on NK-92 and primary human NK cells evaluated by flow cytometry, demonstrating minimal to no detectable levels of the receptors on NK-92. e, Western blot analysis confirming UDP-N-acetylglucosamine 2-epimerase (GNE) knockout (KO) in 1E7 cells to ablate sialylation. Flow cytometry data for primary NK cells are representative results for one of n =3 human blood donors.
Extended Data Fig. 3 Measurement of the nanoscale glycocalyx thickness with Ring Scanning Angle Interference Microscopy (Ring-SAIM).
a, Muc1-GFP size standards with biopolymer domains comprised of 0, 10, 21, and 42 tandem repeats (TRs). b, Western blot analysis of Muc1-GFP constructs stably expressed in MCF10A epithelial cells; arrows indicate fully glycosylated Muc1 with 10, 21, or 42 TRs; the primary antibody reacts with the Muc1 TRs and does not probe the 0TR Muc1. c, Flow cytometry analysis of cell-surface Muc1-GFP probed with Alexa Fluor 647 conjugated GFP nanobody. d, Examples of the pixelwise SAIM interferograms for Muc1-GFP constructs labelled with Alexa Fluor 647 conjugated GFP nanobody. e, Representative widefield image and glycocalyx thickness map of live MCF10A epithelial cells expressing the indicated Muc1 constructs labeled with Alexa Fluor 647 conjugated GFP nanobody; the Muc1 x42 mutant construct is comprised of 42 TRs of the mutated Muc1 tandem repeat – PDARPAPGATAPPAHGVTAA – which has three of the five serine/threonine O-glycosylation sites mutated to alanine. Scale bars, 10 µm. f, g, Quantification of glycocalyx thickness in cells expressing the indicated Muc1 constructs (f) and the Muc1-expressing B2 clonal line induced at the indicated doxycycline levels (b). Boxes and whiskers show the first and third quartiles (boxes), median, and range of the data. Each condition includes a minimum of 19 cells from a representative experiment (n=3 independent experiments). Statistical analysis by one-way ANOVA with Tukey’s post-hoc tests. h, Log of the mean glycocalyx thickness versus log of the mucin biopolymer length from (f); best-fit line to a power-law model with slope = 0.16 and R2= 0.62. i, Log of the mean glycocalyx thickness in 1E7 cells expressing Muc1-GFP at varying doxycycline induction levels versus the log of the mean GFP nanobody signal, a proportionate measure of Muc1 surface density; plotted data from Fig. 2f and Fig. 1d; best-fit line to a power-law model has slope = 0.06 and R2 = 0.79. j, Quantification of glycocalyx thickness in cells expressing the Muc1 x42 and the Muc1 x42 mutant construct. Boxes and whiskers show the first and third quartiles (boxes), median, and range of the data. Each condition includes a minimum of 23 cells from a representative experiment (n=3 independent experiments). Statistical analysis by two-tailed t tests. Unless otherwise indicated, doxycycline induction of mucin expression is at 1,000 ng/ml.
Extended Data Fig. 4 Validation of glycoengineered cell lines.
a, Agarose gel electrophoresis of PCR-amplified and EcoRI-digested segment of C1GALT1 showing successful homozygous knockout (KO) in 1E7 cells (See 2A7 sub-clone of 1E7); the homology directed repair (HDR) template for CRISPR/Cas9 mediated C1GALT1 KO introduces a unique EcoRI site. b, Sanger-sequencing results of a PCR-amplified segment of C1GALT1 with insertion of EcoRI restriction site (GAATTC) and stop codon (TAA) in the 1E7 C1GALT1 KO clone, verifying homozygous KO. (Top: 1E7; bottom: 1E7 C1GALT1 KO) c, Fluorescence images of Glucosaminyl (N-Acetyl) Transferase 1 (GCNT1) in wild-type and GCNT1 overexpressing (GCNT1 OE) 1E7 cells with Muc1-GFP expression induced at 1,000 ng/ml of doxycycline; GCNT1 is localized in the Golgi. d, Relative composition of Core O-glycans in wild-type, 1E7 GCNT1 OE, and 1E7 GNE KO cells measured with the Cellular O-glycome Reporter (CORA) method e, Flow cytometry analysis of wild-type and glycoengineered 1E7 cells. Results for the parental MCF10A epithelial cells that do not express Muc1-GFP (-Muc1-GFP) are included as a control. f, Western blot showing the relative size of Muc1 in the wild-type and glycoengineered 1E7 cells. g, sWGA lectin blot after extended SDS-PAGE run time showing increased molecular weight of Muc1-GFP in 1E7 GCNT1 OE cells compared to wild-type 1E7 cells. h, Rate of proliferation of wild-type and glycoengineered 1E7 cells. Results are the mean of 2 independent experiment replicates. i, Flow cytometry analysis of 1E7 cells and their glycoengineered progeny induced at 0 ng/ml and 1,000 ng/ml of doxycycline.
Extended Data Fig. 5 Galectin interactions with Muc1 O-glycans.
a, Quantification of recombinant human galectin-3 (rhGal-3) binding to wild-type, C1GALT1 knockout (KO), and GCNT1 overexpressing (OE) 1E7 cells; data normalized to the Muc1 signal intensity and presented as the mean and ± s.d. of 20 cells from one representative of n = 3 independent experiments. b, Same as in a, with 5,000 U/mL PNGase F treatment to selectively remove N-glycans prior to analysis. c, Quantification of endogenous cell-surface galectin-3 (Gal-3) and Muc1 before and after treatment with 200 nM StcE mucinase. Results are the mean ± s.d. of at least 11 cells from one representative of n = 3 independent experiments. Statistical analysis by two-tailed t tests. d, Representative confocal images of Muc1-GFP and immuno-labelled Gal-3 in wild-type and LGALS3 overexpressing (LGALS3 OE) 1E7 cells. Scale bars, 10 µm. e, Quantification of endogenous Gal-3 on the 1E7 cell surface following treatment with the indicated concentration of galectin inhibitor, TD139; data normalized to the Muc1 signal intensity and presented as the mean ± s.d. of 10 cells from one representative of n = 3 independent experiments. f, Representative confocal images of endogenous galectin-3 on the cell membrane in presence and absence of 50µM TD139. Scale bars, 10 µm. g, Quantification of recombinant human galectin-1 (rhGal-1) binding to wild-type and glycoengineered 1E7 cells; data normalized to the Muc1 signal intensity and presented as the mean ± s.d. of 20 cells from one representative of n = 3 independent experiments. h, Same as in g with 5,000 U/mL PNGase F treatment to selectively remove N-glycans prior to analysis. i, FRAP analysis of Muc1-GFP in wild-type and 1E7 GCNT1 OE cells treated with control buffer, as well as 1E7 GCNT1 OE cells treated with 10 µM rhGal-1. Results are the mean ± s.d. of intensity profiles of n= 11, 10, 9 cells, respectively. j, k, Quantification of mobile fraction (j) and half recovery time (k) from (i). In all experiments, Muc1-GFP expression was induced with 1,000 ng/ml of doxycycline. Unless otherwise indicated, statistical analysis by one-way ANOVA with multiplicity-adjusted P values from Tukey’s multiple comparisons test.
Extended Data Fig. 6 Apoptosis is not inhibited by Muc1-GFP expression.
a, Quantification of 1E7 cell death resulting from treatment with the indicated concentrations of recombinant perforin for 30 minutes; Muc1-GFP expression induced at 1, 100, 1,000 ng/ml doxycycline. Results are the mean ± s.d. of n = 3 technical replicates for one representative of three independent experiments. b, Quantification of cell viability (%) of uninduced (0 ng/ml) and doxycycline-induced (1,000 ng/ml) 1E7 cells following treatment with indicated concentration of doxorubicin for 48 hours. Results are the mean ± s.d. of n = 3 technical replicates.
Extended Data Fig. 7 Validation of endogenous labeling of Granzyme B (GzmB) in NK-92 cells.
Using CRISPR/Cas9 and an appropriate homology directed repair template, the gene sequence for a flexible GGSGGSGGS linker and the pH-tolerant green fluorescent protein, Gamillus, was inserted immediately before the endogenous GZMB stop codon in NK-92 cells. a, Representative brightfield and fluorescence images of NK-92 GzmB-Gamillus cells stained with Lysotracker Deep Red. Scale bar, 10µm. b, Pearson’s coefficients of correlation between GzmB-Gamillus and Lysotracker. Results are the mean ± s.d. of 20 cells. c, d, NK-92 GzmB-Gamillus mediated cytotoxicity against 1E7 with Muc1-GFP expression induced at the indicated doxycycline concentration. Results are the mean ± s.d. of n =3 independent experimental replicates. NK cell to target cell ratio is 10:1. Statistical analysis by one-way ANOVA with multiplicity-adjusted P values from Tukey’s multiple comparisons test. e, Western blot showing the relative expression level of Granzyme B (GzmB) in the NK-92 cells and NK-92 GzmB-Gamillus knock-in (KI) cells. Note that the acid-labile linker between GzmB and Gamillus is expected to undergo cleavage in the low pH of lysosomes and cytolytic granules.
Extended Data Fig. 8 Supporting characterization for StcE NK-92 and Zip-NK-92 cells.
a, Representative confocal images of Muc1-GFP on 1E7 cells treated with control buffer or 100 nM StcE, StcE ΔX409, StcE ΔX409 fused to the EE leucine zipper (StcE-EE); Alexa Fluor 647 conjugated GFP nanobody is used to probe cell-surface Muc1-GFP constructs. Scale bar, 10 µm. b, Viability of NK-92 and Zip-NK-92 after treatment with indicated concentration of StcE-EE. Propidium iodide (20 μg/ml) was used to detect dead cell population by flow cytometry. c, Cytotoxicity mediated by Zip-NK-92 cells coupled with the indicated concentration of Sialidase-EE zipper fusion protein (Sialidase-EE) against B2 cells at the indicated doxycycline induction level; NK cell to target cell ratio is 5:1. Results are the mean ± s.d. of n = 3 technical replicates for each doxycycline concentration.
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Park, S., Colville, M.J., Paek, J.H. et al. Immunoengineering can overcome the glycocalyx armour of cancer cells. Nat. Mater. 23, 429–438 (2024). https://doi.org/10.1038/s41563-024-01808-0
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DOI: https://doi.org/10.1038/s41563-024-01808-0
