Fig 1.
SPRM Principle and illustration of pancreatic cancer cell binding kinetics.
A) Schematic showing simultaneous SPR detection and bright field visualization of individual cells. A transparent flow cell facilitates the optical imaging and precise delivery of sample solutions. B) bright field and SPR image of pancreatic cancer cells and samples of derived unfitted sensorgrams from bare area and single cell area, from which hundreds of sensorgrams from hundreds of cells on several different regions on the cell can be collected simultaneously. Black squares indicate sensorgram is derived from 1) bare area and 2) response from a single cell area. C) Adherent cell on sensor surface model for profiling of N- and O-linked glycan kinetics with Con A and HPA, respectively, and antibody kinetics with anti-MUC4 monoclonal antibody.
Fig 2.
Con A lectin binding kinetics on BxPC3 pancreatic cancer cell surface.
A) bright field image of BxPC3 pancreatic cancer cells. B) Corresponding SPR image. Overlaid red highlights indicating areas of detected Con A binding interaction, which overlap very closely with the cell regions and indicate high specificity. C) A small sampling of sensorgrams taken from hundreds responsive ROIs is shown. Arrows from cells point to their respective binding response sensorgrams. Serial injections of Con A lectin solutions (0.102, 0.307, 0.923, 2.77, 8.33, 25, and 75 Nm) were exposed to the cells. Sensorgrams were fitted using ImageSPR software.
Fig 3.
Con A binding affinity and heterogeneity increases upon enzymatic N-linked deglycosylation of BxPC3 cells.
A) Affinity isotherm plot extracted from hundreds of responsive ROIs showing Con A binding population on glycosylated BxPC3 cells. B) Histogram describing total statistic kinetic interactions and distributions for Con A on glycosylated BxPC3 cells. C) Affinity isotherm plot extracted from hundreds of responsive ROIs for Con A on deglycosylated BxPC3 cells. D) Histogram describing total statistic of the kinetic interactions and distributions for Con A on deglycosylated BxPC3 cells. Diagonal lines of isoaffinity plots represent KD. a, b, and c represent binding interaction modes. Histogram data were graphed and fitted using Origin 2023b. Affinity isotherm plots were acquired using ImageSPR software.
Table 1.
Kinetic parameters of all lectin and antibody interactions.
Fig 4.
Binding affinity of Anti-MUC4 monoclonal antibody increases on deglycosylated BxPC3 pancreatic cancer cells.
A) Affinity isotherm plot extracted from hundreds of responsive ROIs for anti-MUC-4 on glycosylated BxPC3 cells which produces two distinct binding interactions, labeled as modes a and b. B) Histograms describing total statistic kinetic interactions and distributions for anti-MUC-4 on glycosylated BxPC3 cells. C) Affinity isotherm plot extracted from hundreds of responsive ROIs for anti-MUC-4 on deglycosylated BxPC3 cells which produces higher affinity and faster association for the two binding interaction modes labeled as modes a and b. D) Histograms describing total statistic kinetic interactions and distributions for anti-MUC-4 on N-linked deglycosylated BxPC3 cells. a and b represent binding interaction modes. Histogram data were graphed and fitted using Origin 2023b. Affinity isotherm plots were acquired using Image SPR software.
Fig 5.
Removal of N-linked glycans unmasks HPA binding heterogeneity.
A) Affinity isotherm plot extracted from hundreds of responsive ROIs showing four distinct HPA binding population modes on glycosylated BxPC3 cells. B) Affinity isotherm plot extracted from hundreds of responsive ROIs for HPA on deglycosylated BxPC3 cells showing four distinct binding populations. Mode e highlights the appearance of a new binding population mode upon enzymatic deglycosylation. a, b, c, d, and e represent binding interaction modes. Affinity isotherm plots were acquired using ImageSPR software.