Growing evidence suggests that selectin/selectin-ligand interactions are involved in mediating the adhesion of bloodborne circulating tumor cells at distant sites during the later stages of cancer metastasis. Presently, immunostaining is the standard method for detecting selectin ligands expressed on tissue, but this approach cannot ascertain if the selectin ligands are functional. That is, detection of purported selectin ligands by static (no-force) immunostaining under equilibrium conditions does not necessitate that the reactive antigens are able to mediate cell adhesion with hemodynamic flow (force) under non-equilibrium conditions. As a direct consequence, the relevance of functional selectin ligands as biomarkers is presently unclear because of the lack of adequate detection methods. Therefore, we developed a method, dynamic biochemical tissue analysis (DBTA), to detect and characterize functional selectin ligands in situ by perfusing selectin-coated microspheres (probes) over cancer tissues in a microfluidic device. DBTA using P-selectin microspheres was performed on cancer tissue sections in conjunction with static biochemical tissue analysis (SBTA) using P-selectin chimera, antibodies against purported selectin ligand carbohydrate moieties sLeX and sLeA (HECA-452, KM231, and CSLEX), and antibodies against P-selectin ligands (CD24, CD44, and PSGL-1). Using DBTA, calcium-dependent selectin/selectin ligand adhesive interactions in the form of P-selectin microsphere rolling were observed on multiple, distinct cases of colorectal cancer tissue. Examination of serial sections with SBTA for purported selectin ligand carbohydrate structures in the same regions of tissue displaying P-selectin microsphere rolling revealed significant inconsistencies; not all regions in which sLeX and sLeA moieties were detected using SBTA displayed DBTA P-selectin probe adhesion while some regions that lacked sLeX and sLeA SBTA detection displayed DBTA P-selectin probe adhesion. Furthermore, in the regions of tissue that displayed P-selectin microsphere adhesion, CD24 and PSGL-1 epitopes were not detected in SBTA of serial sections. While one case displayed CD44 expression that was in agreement with DBTA, not all regions of this tissue section that displayed specific reactivity with the P-selectin microspheres used in DBTA were recognized with the CD44 antibody in SBTA. Follow-up CD45 SBTA ruled out the possibility of microsphere interaction with infiltrated leukocytes, in agreement with the lack of PSGL-1 detection. These results imply the presence of less well-recognized, potentially novel P-selectin ligands in the tissue sections.
To further characterize these potentially-novel, functional P-selectin ligands, a more refined method of interaction analysis that discretizes DBTA probe rolling into a sequence of succinct pauses (i.e., brief stationary adhesion) was used. In this pause time analysis, microsphere rolling recorded at 200 frames per second was assessed using a cross correlation tracking algorithm to obtain a robust point estimate that represents the off-rate (inverse lifetime) bond breakage of the ensemble of receptor ligand complexes that mediate microsphere adhesion to the tissue surface. This analysis revealed the inverse lifetime of P-selectin DBTA probe adhesion to functional selectin ligands expressed on colorectal cancer tissue is force-dependent, with a global minimum off-rate observed at 0.75 dyne/cm2. More specifically, increasing levels wall shear stress initially decreased (range: 0.12 to 0.75 dyne/cm2) then increased (range: 0.75 to 1.5 dyne/cm2) P-selectin DBTA probe off-rate to the tissue surface. Appropriately, this method of adhesion inverse lifetime analysis was then subsequently applied to the aforementioned colorectal cancer tissue section that revealed the presence of CD44 (a P-selectin ligand heavily associated with colorectal cancer). Characterization of P-selectin DBTA probe adhesion in a 0.01 mm2 region of tissue that displayed CD44 recognition using SBTA was compared to an adjacent region of equal size that displayed no recognition of CD44 using SBTA. The maximum likelihood off-rate point estimate of 40 separate P-selectin DBTA probes perfused over a CD44-positive region at 0.50 dyne/cm2 was 0.6 inverse seconds while the corresponding CD44-negative region value was 0.4 inverse seconds, resulting in a U-value of 1152.5 (equivalent to P<0.001), further supporting the presence of distinguishable P-selectin ligands. Taken together, these results unequivocally demonstrate DBTA's ability to detect functional P-selectin ligands and generate an adhesion parameter that lays the framework for evaluating aberrantly expressed functional selectin ligands as functional cancer biomarkers.
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