(335a) Dynamic Biochemical Tissue Analysis of P-Selectin Ligands Expressed By Colon Cancer

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-positiveregion 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.