Metastatic cancer cell attachment to endothelium is promoted by endothelial glycocalyx sialic acid degradation

Abstract

While it is known that cancer cell interactions with vascular endothelial cells (ECs) drive metastatic cancer cell extravasation from blood vessels into secondary tumor sites, the mechanisms of action are still poorly understood. Here, we tested the hypothesis that neuraminidase-induced degradation of EC surface glycocalyx (GCX), particularly the sialic acid (SA) residue components of the GCX, will substantially increase metastatic cancer cell attachment to ECs. To our knowledge, our study is the first to isolate the role of GCX SA residues in cancer cell attachment to the endothelium, which were found to be differentially affected by the presence of neuraminidase and to indeed regulate metastatic cancer cell homing to ECs. We hope that this work will eventually translate to identification of EC GCX-based cancer markers that can be therapeutically targeted to hinder the progression of metastasis.

Keywords: endothelial glycocalyx; metastatic cancer cells; secondary tumor; sialic acid.

Figure 1

(a) Drawing shows endothelium with intact GCX. As metastatic cancer cells move with blood flow, the healthy GCX blocks the adhesion ligands on the cancer cells from attaching to the adhesion receptors on the endothelium lining of the blood vessel wall. We hypothesize that cancer cell attachment to the endothelium is caused by endothelial GCX degradation. (b, c) These drawings illustrate the conclusions of the findings reported herein. Our observations provide evidence that the systemic increase in Neur (b), which coincides with metastatic cancer conditions, degrades the GCX as a whole and as applied to its∝‐2,6‐linked and ∝‐2,3‐linked SA residues (c). This GCX degradation leads to increased cancer cell attachment to ECs, and we speculate that this is mediated by exposure of adhesion receptors on the endothelium which become accessible to adhesion ligands on cancer cells (c). ECs, endothelial cells; GCX, glycocalyx; SA, sialic acid

Figure 2

Images show the effect of exposure to various concentrations of Neur enzyme on the GCX, endothelium integrity, and cancer cell attachment to ECs. As shown, Neur concentrations include 0, 15, 135, 1,215, and 3,645 mU/mL. (a–e) ×5 magnification phase contrast images merged with red fluorescence micrographs, to confirm integrity of the EC layer in all conditions and show cancer cells attached to the endothelium. Scale bar equal to 500 μm is shown. (f–t) ×63 magnification confocal micrographs of GCX labeled with green fluorescence conjugated to the following lectins: WGA (f–j), SNA (k–o), and MAL II (p–t). The blue is DAPI, which labels EC nuclei. Scale bar equal to 20 μm is shown. (u–y) ×5 magnification red fluorescence micrographs clarify the presence of CellTracker Red labeled cancer cells. Scale bar equal to 500 μm is shown. DAPI, 4′6‐diamidino‐2‐phenylindole; ECs, endothelial cells; GCX, glycocalyx; MAL II, Maakia amurensis lectin II; SNA, Sambucus nigra (elderberry bark) lectin; WGA, wheat germ agglutinin

Figure 3

Comparing the coverage (a) and thickness (b) of GCX components that bind WGA, SNA, and MAL II at various Neur concentrations. Compared to untreated baseline conditions as indicated by the dashed line (‐ ‐ ‐), Neur concentration of 15 mU/mL slightly reduced thickness of and coverage of the endothelium by WGA‐labeled GCX, SNA‐labeled ∝‐2,6‐linked SA residue, and MAL II‐labeled ∝‐2,3‐linked SA residue. SNA‐labeled ∝‐2,6‐linked SA residue was reduced most statistically significantly. 135 mU/mL of Neur slightly reduced WGA‐labeled GCX but statistically significantly reduced SNA‐labeled ∝‐2,6‐linked SA residue and MAL II‐labeled ∝‐2,3‐linked SA residue. 1,215 mU/mL and 3,654 mU/mL of Neur further reduced WGA‐labeled GCX, SNA‐labeled ∝‐2,6‐linked SA residue, and MAL II‐labeled ∝‐2,3‐linked SA residue. Results are normalized based on 0 mU/mL conditions. Significance differences between groups are denoted as *****p < .0001, and “ns” denotes nonsignificance. GCX, glycocalyx; MAL II, Maakia amurensis lectin II; SA, sialic acid; SNA, Sambucus nigra (elderberry bark) lectin; WGA, wheat germ agglutinin

Figure 4

The extent of EC coverage by GCX and the thickness of GCX, as assessed by quantifying WGA labeled GCX, are inversely proportional to the number of cancer cells that attach to endothelium. Results are normalized to 0 mU/mL baseline conditions, which are indicated by the dashed lines (‐ ‐ ‐). Significance is denoted as *p < .05, **p < .01, and ****p < .0001. (a) Compared to 0 mU/mL Neur conditions, WGA‐labeled GCX coverage of ECs only becomes statistically low at high Neur doses of 1,215 and 3,645 mU/mL. N = 3, and representative en face images are shown in Figure 2a–j. (b) Compared to 0 mU/mL Neur conditions, WGA‐labeled GCX thickness is statistically significantly affected by Neur doses of 135, 1,215, and 3,645 mU/mL. N = 3, and representative cross‐section images used for this data are shown in Figure 2a–j. (a, b) Exponential increase in cancer attachment was observed with the increasing Neur concentration. At 0 mU/mL, N = 9; at 15 mU/mL, N = 8; at 135 mU/mL, N = 8; at 1,215 mU/mL, N = 9; and at 3,645 mU/mL, N = 9, and representative images used for this data are shown in Figure 2u–y. EC, endothelial cell; GCX, glycocalyx; WGA, wheat germ agglutinin

Figure 5

The extent of EC coverage by SA and the thickness of SA, as assessed by quantifying SNA‐labeled ∝‐2,6‐linked SA residue, are compared to the number of cancer cells that attach to endothelium. 0 mU/mL baseline conditions are indicated by the dashed lines (‐ ‐ ‐). Significance is denoted as *p < .05, **p < .01, and ***p < .001. (a) Compared to 0 mU/mL Neur conditions, SNA‐labeled ∝‐2,6‐linked SA residue coverage of ECs becomes statistically low at Neur doses of 135, 1,215, and 3,645 mU/mL. N = 3, and representative en face images are shown in Figure 2k–o. (b) Compared to 0 mU/mL Neur conditions, SNA‐labeled ∝‐2,6‐linked SA residue thickness is statistically significantly affected by Neur doses of 15, 135, 1,215, and 3,645 mU/mL. N = 3, and representative cross‐section images used for this data are shown in Figure 2k–o. (a, b) The observed Neur‐induced increase in cancer attachment as shown in Figure 3 is shown again, for comparison to expression of SNA‐labeled ∝‐2,6‐linked SA residue. At 0 mU/mL, N = 9; at 15 mU/mL, N = 8; at 135 mU/mL, N = 8; at 1,215 mU/mL, N = 9; and at 3,645 mU/mL, N = 9, and representative images used for this data are shown in Figure 2u–y. EC, endothelial cell; SA, sialic acid; SNA, Sambucus nigra (elderberry bark) lectin

Figure 6

The extent of EC coverage by SA and the thickness of SA, as assessed by quantifying MAL II‐labeled ∝‐2,3‐linked SA residue, are compared to the number of cancer cells that attach to endothelium. Results are normalized to 0 mU/mL baseline conditions, which are indicated by the dashed lines (‐ ‐ ‐). Significance is denoted as *p < .05, **p < .01, and ****p < .0001. (a) Compared to 0 mU/mL Neur conditions, MAL II‐labeled ∝‐2,3‐linked SA residue coverage of ECs becomes statistically low at Neur doses of 135, 1,215, and 3,645 mU/mL. N = 3, and representative en face images are shown in Figure 2p–t. (b) Similarly, compared to 0 mU/mL Neur conditions, SNA‐labeled ∝‐2,6‐linked SA residue thickness is statistically significantly affected by Neur doses of 135, 1,215, and 3,645 mU/mL. N = 3, and representative cross‐section images used for this data are shown in Figure 2p–t. (a, b) The observed Neur‐induced increase in cancer attachment as shown in Figures 3 and 4 are shown again, for comparison to expression of MAL II‐labeled ∝‐2,3‐linked SA residue. At 0 mU/mL, N = 9; at 15 mU/mL, N = 8; at 135 mU/mL, N = 8; at 1,215 mU/mL, N = 9; and at 3,645 mU/mL, N = 9, and representative images used for this data are shown in Figure 2u–y. EC, endothelial cell; MAL II, Maakia amurensis lectin II; SA, sialic acid

Figure 7

Preliminary data was collected regarding human ECs (HUVEC), their GCX, and the extent of their recruitment of 4T1 breast cancer cells in comparison with RFPEC controls. This human EC data confirms and validates the rat EC data that was the focus of this report. (a) Phase image shows that an untreated HUVEC layer is healthy. (b) WGA‐labeled untreated HUVEC reveals the presence of GCX even in the absence of physiological flow stimulation, which is usually required for in vitro human EC studies. (c) Low expression of HS is observed when these HUVEC, which lack flow stimulation, are labeled with HS antibody. The limited HS is presumably insufficient to expose the EC surface adhesion molecules to 4T1 breast cancer cells, because WGA is abundant enough to compensate and provide adequate coverage. (d) As expected, the level of attachment of 4T1 breast cancer cells to untreated HUVEC is low, similar to what was observed in untreated RFPEC. (e) Phase image shows healthy untreated RFPEC monolayer. (f) Fluorescent image of WGA‐labeled untreated RFPEC monolayer shows intact GCX. (g) Expression of HS is abundant in RFPEC without flow stimulation. (h) Picture shows attachment of 4T1 breast cancer cells to RFPEC monolayers. (i) Plot shows that the difference between RFPEC and HUVEC adhesiveness to 4T1 breast cancer cells is statistically not significant (ns). N = 3 for both cell types that were studied. EC, endothelial cell; GCX, glycocalyx; HS, heparan sulfate; RFPEC, rat fat pad endothelial cell; WGA, wheat germ agglutinin