Anti-Tumor Effects of Biomimetic Sulfated Glycosaminoglycans on Lung Adenocarcinoma Cells in 2D and 3D In Vitro Models

Abstract

Lung cancer development relies on cell proliferation and migration, which in turn requires interaction with extracellular matrix (ECM) components such as glycosaminoglycans (GAGs). The mechanisms through which GAGs regulate cancer cell functions are not fully understood but they are, in part, mediated by controlled interactions with cytokines and growth factors (GFs). In order to mechanistically understand the effect of the degree of sulfation (DS) of GAGs on lung adenocarcinoma (LUAD) cells, we synthesized sulfated alginate (AlgSulf) as sulfated GAG mimics with DS = 0.0, 0.8, 2.0, and 2.7. Human (H1792) and mouse (MDA-F471) LUAD cell lines were treated with AlgSulf of various DSs at two concentrations 10 and 100 µg/mL and their anti-tumor properties were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypan blue exclusion, and wound healing assays for 2D models and sphere formation assay for the 3D model. The proliferation and number of live MDA-F471 cells at the concentration of 100 µg/mL decreased significantly with the increase in the DS of biomimetic GAGs. In addition, the increase in the DS of biomimetic GAGs decreased cell migration (p < 0.001 for DS = 2.0 and 2.7 compared to control) and decreased the diameter and number of spheres formed (p < 0.001). The increased DS of biomimetic GAGs attenuated the expression of cancer stem cell (CSC)/progenitor markers in the 3D cultures. In conclusion, GAG-mimetic AlgSulf with increased DS exhibit enhanced anti-proliferative and migratory properties while also reducing growth of KRAS-mutant LUAD spheres in vitro. We suggest that these anti-tumor effects by GAG-mimetic AlgSulf are possibly due to differential binding to GFs and consequential decreased cell stemness. AlgSulf may be suitable for applications in cancer therapy after further in vivo validation.

Keywords: biomimetic; cancer stem cells; glycosaminoglycans; lung adenocarcinoma; sulfated alginates.

Figure 1

The effect of the DS of biomimetic sulfated GAGs on the activity of human (H1792) and murine (MDA-F471) LUAD cell lines. The metabolic activity of (A) H1792 treated with 10 µg/mL of AlgSulf_n (B) H1792 treated with 100 µg/mL of AlgSulf_n (C) MDA-F471 treated with 10 µg/mL of AlgSulf_n and (D) MDA-F471 treated with 100 µg/mL of AlgSulf_n. For each graph, heparin was added at the same concentration of the polysaccharides as positive controls, and untreated cells (media only) were used as a negative control. Results are expressed as the percentage of the treated group compared to control after 24 h and 48 h of culture. Data represent the average of five independent experiments and are reported as mean ± SEM (* p < 0.05; ** p < 0.01) using two-way ANOVA followed by Bonferroni’s multiple comparison test.

Figure 2

The effect of the DS of biomimetic sulfated GAGs on the viability of human and murine LUAD cell lines (A) H1792 treated with 10 µg/mL of AlgSulf_n, (B) H1792 treated with 100 µg/mL of AlgSulf_n, (C) MDA-F471 treated with 10 µg/mL of AlgSulf_n, and (D) MDA-F471 treated with 100 µg/mL of AlgSulf_n assessed using a trypan blue exclusion assay. For each graph, heparin was added at the same concentration of the polysaccharides as a positive control, and untreated cells (media only) were used as a negative control. Results are expressed as the percentage of the treated group compared to the control after 24 h and 48 h of culture. Data represent the average of five independent experiments and are reported as mean ± SEM (* p < 0.05; ** p < 0.01; *** p < 0.001) using two-way ANOVA followed by Bonferroni’s multiple comparison test.

Figure 3

The effect of the DS of biomimetic sulfated GAGs on the migratory abilities of human (H1792) and murine (MDA-F471) LUAD cell lines. A scratch was made on a 12-well plate of confluent H1792 and MDA-F471 cells using a 200 µL micropipette tip and images were taken at T = 0, 6, 18, 24, and 48 h. (A) Representative bright-field images of H1792 and MDA-F471 wounds with different AlgSulf_n. Results are expressed as the percentage of the wound distance in each group compared to its condition at T = 0 h for (B) H1792 and (C) MDA-F471 treated with AlgSulf_n. Untreated cell layers were used as negative controls and heparin was used as a sulfated GAG positive control. Data represent the average of three independent experiments and are reported as mean ± SEM (** p < 0.01; *** p < 0.001) using two-way ANOVA followed by Bonferroni’s multiple comparison test. Scale = 100 µm.

Figure 3

The effect of the DS of biomimetic sulfated GAGs on the migratory abilities of human (H1792) and murine (MDA-F471) LUAD cell lines. A scratch was made on a 12-well plate of confluent H1792 and MDA-F471 cells using a 200 µL micropipette tip and images were taken at T = 0, 6, 18, 24, and 48 h. (A) Representative bright-field images of H1792 and MDA-F471 wounds with different AlgSulf_n. Results are expressed as the percentage of the wound distance in each group compared to its condition at T = 0 h for (B) H1792 and (C) MDA-F471 treated with AlgSulf_n. Untreated cell layers were used as negative controls and heparin was used as a sulfated GAG positive control. Data represent the average of three independent experiments and are reported as mean ± SEM (** p < 0.01; *** p < 0.001) using two-way ANOVA followed by Bonferroni’s multiple comparison test. Scale = 100 µm.

Figure 4

The effect of the DS of biomimetic sulfated GAGs on the sphere-forming capacities of human and murine LUAD cell lines. (A and B) Quantification of the sphere-forming unit (SFU) for H1792 and MDA-F471 spheres at G2 under different treatment conditions. (C and D) Quantification of the average area (µm²) of 30 spheres at G2. (E) Representative bright-field images of H1792 and MDA-F471 spheres at G2 with different DS of biomimetic GAGs. Data represent the average of three independent experiments and are reported as mean ± SEM (*** p < 0.001) using one-way ANOVA followed by Bonferroni’s multiple comparison test. Scale = 50 µm.