CD44s Assembles Hyaluronan Coat on Filopodia and Extracellular Vesicles and Induces Tumorigenicity of MKN74 Gastric Carcinoma Cells

Abstract

CD44 is a multifunctional adhesion molecule typically upregulated in malignant, inflamed and injured tissues. Due to its ability to bind multiple ligands present in the tumor microenvironment, it promotes multiple cellular functions related to tumorigenesis. Recent data has shown that CD44 and its principal ligand hyaluronan (HA) are carried by extracellular vesicles (EV) derived from stem and tumor cells, but the role of CD44 in EV shedding has not been studied so far. To answer this question, we utilized CD44-negative human gastric carcinoma cell line MKN74 manipulated to stably express CD44 standard form (CD44s). The effect of CD44s expression on HA metabolism, EV secretion, morphology and growth of these cells was studied. Interestingly, HAS2 and HYAL2 expression levels were significantly upregulated in CD44s-expressing cells. Cell-associated HA levels were significantly increased, while HA levels in the culture medium of CD44s-positive cells was lower compared to CD44s-negative MOCK cells. CD44s expression had no significant effect on the proliferation capacity of cells, but cells showed diminished contact inhibition. Superresolution imaging revealed that CD44s and HA were accumulated on filopodia and EVs secreted from CD44s-positive cells, but no differences in total numbers of secreted EV between CD44s-negative and -positive cells was detected. In 3D cultures, CD44s-expressing cells had an enhanced invasion capacity in BME gel and increased spheroidal growth when cultured in collagen I gel. No significant differences in mitotic activity, tumor size or morphology were detected in CAM assays. However, a significant increase in HA staining coverage was detected in CD44s-positive tumors. Interestingly, CD44s-positive EVs embedded in HA-rich matrix were detected in the stromal areas of tumors. The results indicate that CD44s expression significantly increases the HA binding capacity of gastric cancer cells, while the secreted HA is downregulated. CD44s is also carried by EVs secreted by CD44s-expressing cells. These findings highlight the potential usefulness of CD44s and its ligands as multipurpose EV biomarkers, because they are upregulated in inflammatory, injured, and cancer cells and accumulate on the surface of EVs secreted in these situations.

The Density and Length of Filopodia Associate with the Activity of Hyaluronan Synthesis in Tumor Cells

“…To study the effect of hyaluronan receptor CD44 on filopodial growth, two versions of human gastric cancer cell line MKN74, MOCK (CD44-negative) and CD44 (CD44 overexpression), were utilized ( Figure 6 A). The characterization of hyaluronan metabolism of MKN74 has shown that CD44 overexpression induces binding of hyaluronan to the plasma membrane, especially around filopodia [ 22 ]. However, the level of secreted hyaluronan was slightly higher in MOCK cells, the average amount being 2.5 ng/10,000 cells, while CD44+ cells produced 1.9 ng/10,000 cells of hyaluronan during 24 h [ 22 ].…”

“…The characterization of hyaluronan metabolism of MKN74 has shown that CD44 overexpression induces binding of hyaluronan to the plasma membrane, especially around filopodia [ 22 ]. However, the level of secreted hyaluronan was slightly higher in MOCK cells, the average amount being 2.5 ng/10,000 cells, while CD44+ cells produced 1.9 ng/10,000 cells of hyaluronan during 24 h [ 22 ]. The FiloQuant analysis showed no significant difference in length ( Figure 6 B) or density ( Figure 6 C) of filopodia between the two cell lines.…”

“…However, as a protein that increases cell motility and invasion of cancer cells, the role of cell surface adhesion protein CD44 in the formation of filopodia has been minimally studied. To study the effect of CD44 on filopodial growth, human gastric cancer cell line MKN74 was utilized [ 22 ]. The results of this study showed that CD44 expression had no effect on the formation of filopodia of MKN74 cells.…”

“…MKN74 human gastric cancer cell lines, MOCK and CD44 [ 22 ] were cultured in RPMI 1640 medium (Lonza, Walkersville, MD, USA) supplemented with 10% FBS (Thermo Fisher Scientific), 100 μg/mL streptomycin sulphate (EuroClone), and 100 U/mL penicillin (EuroClone). Both cell lines were passaged twice a week at a 1:5 split ratio using 0.05% trypsin (w/v) 0.02% EDTA (w/v) (Biochrom AG, Berlin, Germany).…”

The role of CD44 in pathological angiogenesis

“…CD44 was also observed to be localized and accumulated in filopodia of gastric and squamous carcinoma cells together with HA, but CD44 does not appear to be essential in the formation of filopodia. 80,81 Downstream of CD44, activation of PKC, actin cytoskeleton reorganization, and ECM remodeling processes may be responsible for the regulatory roles of HA-CD44 pathway in promoting EC adhesion and migration during angiogenesis. We will discuss the molecular basis of the HA-CD44 pathway below.…”