Fig 2: Enhanced sialylation and reduced sialidases in pancreatic cancer sphere-forming cells (PCS). (A) Cell lysates were prepared from both PCS and adherent cancer cells, separated electrophoretically using 10% polyacrylamide gel and further processed for Western blotting. Representative blots show enhanced Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MALII) binding in PCS. Ponceau S stained blots were used as loading control. (B) Flow cytometry analysis revealed higher surface binding of SNA and MALII with PCS (1 × 105) from both MIAPaCa2 and AsPC1 cells as described in materials and methods. PCS and adherent cells were washed and resuspended in lectin-binding buffer. Cells were incubated separately with FITC-conjugated SNA and MALII for 1 h (5.0 µg/mL at 4 °C), and FITC positivity was acquired by flow cytometry. (C) Immunohistological staining shows the reduced expression of Neu2 in pancreatic tumors tissues compared to adjacent normal tissues (20X magnification). Optical densitometry score showed a significant reduction in the expression level of Neu2 in tumor tissues. Scores were measured by ImageJ software. (D) qPCR analysis of all four sialidases, namely Neu1, Neu2, Neu3 and Neu4, in PCS vs. adherent cells was determined by using specific primers as described in Supplementary Table S1. Error bars represent the mean (±) SD; p-values (ns p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001) calculated using Two-tailed Student’s t-test.

Fig 3: Neu2-overexpressed pancreatic cancer sphere-forming cells (N-PCS) exhibited reduced sialylation and stemness-like property. (A) Phase-contrast images showed reduced sphere formation of N-PCS compared to PCS generated from both MIAPaCa2 and AsPC1 cells. (B) Bar graph of qPCR data showed decreased expression of pluripotent stem cell markers (OCT4, SOX2, NANOG) and CD133 in N-PCS than PCS from both MIAPaCa2 and AsPC1 cells. (C) Confocal microscopy images showed enhanced expression of Oct4, Sox2 and Nanog in PCS compared to adherent cancer cells. Those expressions were again reduced in N-PCS. (D) Representative blots exhibited reduced SNA and MALII binding with N-PCS compared to PCS. Cell lysates were prepared and electrophoretically separated using 10% polyacrylamide gel and further processed for Western blotting. Ponceau S stained blots were used as loading control. (E) Cell viability assay showed reduced viability upon Neu2 overexpression in PCS. Error bars represent the mean (±) SD; p-values (** p < 0.01; *** p < 0.001) calculated using Two-tailed Student’s t-test. (F) Western blot analysis illustrated higher expression of pro-caspase proteins such as Caspase 7, 8, cleaved caspase 3 and Bax along with reduced expression of Bcl2 in N-PCS. No change of expression level was found in Caspase 9. ß-actin served as a loading control.

Fig 4: Overexpressed Neu2 demonstrated preferential specificity toward a2,6-linked sialic acids on OC cells.A–C Genetic expression levels of Neu1, Neu2, Neu3, and Neu4 in PA1 (A), OVCAR3 (B), and SKOV3 (C) ovarian cancer cell lines by real-time PCR analysis. Relative mRNA expression was expressed in comparison to 18S rRNA. D Relative fold change in the Neu2 mRNA level in PA1 and OVCAR3 after overexpression with Neu2 plasmid (24 h) compared to mock cells was analyzed by real-time PCR analysis. E, F Western blot analysis was performed to confirm the enhanced Neu2 protein levels in transfected PA1 (E) and OVCAR3 (F) cells compared to mock using an anti-Neu2 antibody. ß-Actin was used as a loading control. G–J FACS analysis showing that Neu2 overexpression decreased the FITC-SNA binding in PA1 (G) and OVCAR3 (I) compared to mock cells. Bar graphs represent the MFI values of FITC-SNA-stained mock and Neu2-overexpressed PA1 (H) and OVCAR3 (J) cells. K, L Binding of FITC-MALII with mock and Neu2-overexpressed PA1 (K) by flow cytometry. The MFI values are compared by plotting a bar graph (L). The results are represented as mean ± SD from independent experiments and p values (*p < 0.05; **p < 0.01; ***p < 0.001; two-tailed t test) represented the significant differences between the means of the two test groups.

Fig 5: Neu2 overexpression reduced Hedgehog pathway (Hh) activity in PCS. (A) Immunohistological staining of pancreatic tumors and adjacent normal tissues showed the enhanced expression of Shh in cancer tissues. The tissue specimens were photographed at 20× magnification. Optical densitometry score showed significant changes in the expression level of Shh. Scores were measured using ImageJ software. Error bars represent the mean (±) SD; p-value (*** p < 0.001) calculated using Two-tailed Student’s t-test. (B) Representative immunoblots exhibited higher expression of several Hedgehog pathway proteins like Patched1, Gli1, Gli2, Smo, Shh and Sufu in PCS than in adherent cells in both MIAPaCa2 and AsPC1 cells. ß-actin was used as a loading control. (C) Representative immunoblots demonstrated decreased expression of Gli1, Gli2, Smo, Shh, and Sufu in N-PCS than PCS from both MIAPaCa2 and AsPC1 cells. ß-actin used as a loading control.