Supplementary Methods and Figures from A Unique Nonsaccharide Mimetic of Heparin Hexasaccharide Inhibits Colon Cancer Stem Cells via p38 MAP Kinase Activation
Authors: Boothello Rio S. , Patel Nirmita J. , Sharon Chetna , Abdelfadiel Elsamani I. , Morla Shravan , Brophy Donald F. , Lippman H. Robert , Desai Umesh R. , Patel Bhaumik B. 
Published: April 2023

<p>The 16 page composite document outlines supplemental methods as well as Figures. Fig S1: a- In vivo tumor formation assay with subcutaneous injections of (2,000) Dual hi HT-29 cells show high tumor forming efficiency of Dual hi cells compared to unsorted cells in athymic NCr nude mice. b- Line graph showing tumor volume progression in NCr nude mice injected subcutaneously with varying amount of (100,000 - 2,000) of FACS-sorted Dual hi (CD133+/CXCR4+) HCT 116 colon cancer cells. The data shows that FACS-sorted CSCs are capable of inducing tumor even at a much lower number of cells. Fig S2: a- The Kaplan-Meier survival curves indicates significant survival in NCr nude mice bearing Dual hi induced HT-29 xenografts treated with 100mg/Kg (p=0.0025) or 200mg/Kg (p=0.014) of G2.2 compared to vehicle treated control. b- Scatter plot representation of flow cytometric analyses of Dual hi CSCs in HT-29 spheroids (100 ÂµM) and xenograft (200 mg/kg) derived cells following labeling with anti-CD133 and anti-CXCR4 antibodies. G2.2-treated HT-29 spheroids (100 ÂµM) and xenograft (200 mg/kg) derived cells show significant inhibition of Dual hi CSCs compared to respective vehicle controls. c- G2.2-treated HT-29 xenograft-derived cells show significant inhibition of CSC self-renewal (2{degree sign} and 3{degree sign} sphere formation) compared to vehicle controls. d- Scatter plot representation of flow cytometric analyses of Dual hi CSCs-induced xenograft derived cells following labeling with anti-CD133, anti-CXCR4, and anti-Annexin V antibodies as well as propidium iodide counterstaining. The Panel 1 shows significant increase in Annexin V+ (apoptotic) cells in G2.2 treated xenografts compared to vehicle controls. Upon further analyses of the G2.2 treated xenograft cells (Panel 2) to determine Annexin V+ cells in each of Dual hi (CSCs) and Dual lo (non-CSCs) population revealed a robust increase in apoptotic cells in Dual hi population only, suggesting that G2.2 selective targets CSC population. e- G2.2-treated primary HT-29 xenograft-derived cells 3{degree sign} colonospheres were retreated with G2.2 in vitro and spheroid growth was measure in the presence of the drug (4{degree sign} spheroids) as well as following withdrawal of the drug (5{degree sign} & 6{degree sign} spheroids) to determine growth and self-renewal respectively of HT-29 xenograft-derived spheroids following a re-challenge with the drug. The box-plot show a robust inhibition of CSC growth/self-renewal by G2.2 following in vivo exposure to the drug (4{degree sign}, 5{degree sign} and 6{degree sign} sphere formation) compared to vehicle controls. f- G2.2 treated secondary HT-29 xenografts-derived cells show significant inhibition of CSC self-renewal (2{degree sign}, 3{degree sign} and 4{degree sign} sphere formation) compared to vehicle controls. g- Western blot analyses of xenografts show robust inhibition of CSC makers (LGR5, DCLK1) and self-renewal factor (BMI1) in G2.2 treated HCT 116 xenografts compared to vehicle controls at days 42 post treatment initiation. The associated bar-graph represents relative densitometry value normalized to GAPDH (housekeeping protein). h- G2.2 treated HCT 116 xenograft-derived cells show significant inhibition of CSC self-renewal (4{degree sign} sphere formation) compared to vehicle controls.Fig S3: a- Flow cytometry analyses of xenograft-derived cells labelled with anti-CD133-APC and CXCR4-PE antibody demonstrating higher proportion of CSCs in Chemotherapy treated mice compared to vehicle treated controls.. b- FUOX treated xenograft-derived cells (CVeh) showed significant increase in CSC self-renewal (2{degree sign}, 3{degree sign} sphere formation) compared to Veh. The xenografts were harvested on day 21 post-FUOX treatment initiation.. c- Scatter plot representation of flow cytometry analyses of HT-29 xenograft-derived cells to determine proportion of Dual hi (CSCs) cells shows, a robust and sustained inhibition of Dual hi cells by CG2.2 compared to CVeh at days 30 and 42 post-FUOX treatment initiation. Numbers in the yellow box represent % of total events in a given quadrant.. d- Unedited uncut western blots along with molecular weight markers. e- FUOX treated xenograft-derived cells (CVeh) showed significant increase in CSC self-renewal (2{degree sign}, 3{degree sign} sphere formation) which was promptly inhibited by CG2.2 compared to both veh. and cVeh treatments. Fig S4: a- Line graph showing activation kinetics of two key mitogen-activated protein kinases, p38 and ERK1/2 (15min â†’ 720min) following G2.2 (100 ÂµM) treatment of HT-29 spheroids - shows early and sustained activation of p38MAPK. Whereas ERK1/2 showed brief activation followed by modest inhibition with G2.2. The dotted line represents basal activation state. b- Western-blot analyses of MAPK shows selective activation (increased levels of phosphorylated form) of p38 but modest inhibition of related MAPK members - JNK and ERK1/2 by G2.2 in HT-29 spheroids. Moreover, activation of p38 by G2.2 (but not its inactive analogs G1.4 and G4.1) was observed selective in HT-29 spheroids enriched in CSCs but not in their monolayer counterparts, suggesting selective activation of p38 MAPK in CSCs by G2.2. Numbers under the blot represent relative densitometric values normalized to vehicle treated controls. c- Immuno-precipitation (IP) with anti-pp38 followed by western blotting (WB) with isoform specific anti-p38 antibody as well as the corresponding bar graph representation of relative densitometry values shows activation of Î± and Î² isoforms, inhibition of Î' isoform, and no significant change in Î³ isoform upon G2.2 treatment (100 Î¼M for 3hrs) in HT-29 spheroids. d- Western Blot analysis shows depletion of p38Î± in HT-29 cells transfected with p38Î± siRNA (KD) without any significant effect on the levels of other isoforms. Moreover, p38Î± KD significantly inhibited G2.2 induced upregulation of pp38. e- Western-blot analyses of CSC (CD133, LGR5, CD44, EpCAM) and self-renewal (CMYC, BMI1) makers shows significant inhibition of these markers by G2.2 (100ÂµM) in scrambled transfected HT-29 spheroids. However, these effects of G2.2 were near-completely reversed in HT-29 spheroids transfected with p38Î± siRNA, suggesting a critical role of p38Î± in mediating anti-CSC effects of G2.2. GAPDH served as housekeeping control. The numbers under the blot represent relative densitometric values normalized to GAPDH and vehicle treated controls. f- Western blot analysis and the corresponding bar graph representation of relative expression of CSC markers (CD133, LGR5) and pp38 shows near-complete reversal G2.2's inhibition of levels of CSC markers (24 hours) and its induction of pp38 (3 hours) in the presence of p38Î± agf. (dominant negative). GAPDH was used as loading control. Fig S5: a- Scatter-plot of flow-cytometric analyses of Dual hi (CD133+/CXCR4+) cells in xenografts treated with vehicle, G2.2 (200 mg/kg), SB (10mg/kg) or SB followed by G2.2 (SG). The data shows that SB, a p38 inhibitor, reversed the effect of G2.2 caused inhibition of CSCs in vivo. Fig S6: a- Average weight of mice treated with 200mg/Kg of G2.2, 3 times a week for 5 weeks compared to vehicle treated controls.. b- High resolution (20X) photomicrograph of G2.2 treated liver shows minor (~ 5%) dropout of hepatocytes (shown with bold black arrows) compared to vehicle controls.</p>