Journal: iScience

Article Title: CHK1 inhibitor sensitizes resistant colorectal cancer stem cells to nortopsentin

doi: 10.1016/j.isci.2021.102664

Figure Lengend Snippet: CR-CSphCs are endowed with innate chemoresistance (A) Cell growth kinetics of CR-CSphCs treated with a vehicle or 5-FU in combination with oxaliplatin, up to 4 days. Data represent the mean ± SD (n = 3) using 2 different CR-CSphC lines for each subgroup (wt, #7 and #21; Braf , #3 and #5; Kras #8 and #9; Res #R3 and #R4). Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ns, nonsignificant. (B) Cell cycle analysis in CR-CSphCs treated as in (A) for 24 h. Data show percentage of cell number in G0–G1, S, and G2–M phases. Data are expressed as mean ± SD of three independent experiments performed in CR-CSphCs isolated from patients with wt (#7 and #21), Braf (#3 and #5), Kras (#8 and #9) and chemoresistant (#R3 and #R4) CRC. (Right panels) Representative cell cycle analysis of CR-CSphCs treated with a vehicle or 5-FU in combination with oxaliplatin, for 24 h (blue color = G0-G1; yellow color = S; green color = G2-M). (C) Percentage of viability in flow-cytometry-sorted TOP-GFP CR-CSphCs, treated as in (A) up to 120 h. Data are expressed as mean ± SD of three independent experiments performed using two different CR-CSphC lines (#8, #9). (Right panel) Representative flow cytometry analysis of TOP-GFP expression in spared CR-CSphCs, at 5 days. Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ∗∗p ≤ 0.01. (D) Cell viability percentage of CR-CSphCs enriched for CD44v6 expression and treated with vehicle or 5-FU in combination with oxaliplatin up to 120 h. Data are expressed as mean ± SD of three independent experiments performed using four different CR-CSphCs lines (#3, #9, #21, #R4). (Right panel) Representative flow cytometry analysis of the percentage of CD44v6 positivity in spared CR-CSphCs, at 5 days. Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ∗p ≤ 0.05. (E) Tumor size of CR-CSphCs subcutaneously injected into immunocompromized mice, treated for 4 weeks (from sixth to ninth week) with vehicle or 5-FU in combination with oxaliplatin. Data represent the mean ± SD of tumor size measured in six mice per group, using 2 different CR-CSphC lines (#8, #21). Black arrowheads indicate weeks of treatment. Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ns, nonsignificant. (F) Cell viability analysis of healthy cells (IMEC and AD-MSCs) treated as in (D), for 3 days. Data are expressed as mean ± SD of three independent experiments. Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed).∗∗p ≤ 0.01; ∗∗∗∗p ≤ 0.0001. See also Figure S1 .

Article Snippet: Double staining was performed using antibodies against CD44v6 (2F10 APC, mouse IgG1, R&D systems) and p-CHK1 (Ser345, 133D3, Rabbit IgG, Cell Signaling Technology), revealed by the MACH 2 double stain 2 kit conjugated goat antimouse polymer horseradish peroxidase (HRP) and the conjugated goat antirabbit polymer alkaline phosphatase (Biocare Medical), and detected by DAB and Vulcan Fast Red chromogen.

Techniques: Cell Cycle Assay, Isolation, Flow Cytometry, Expressing, Injection

Journal: iScience

Article Title: CHK1 inhibitor sensitizes resistant colorectal cancer stem cells to nortopsentin

doi: 10.1016/j.isci.2021.102664

Figure Lengend Snippet: CD44v6 + CR-CSCs acquire resistance to NORA234 treatment (A) Kinetics of cell growth of CR-CSphCs treated with vehicle or NORA234, up to 4 days. Data represent the mean ± SD (n = 3) using CR-CSphCs isolated from wt (#7 and #21), Braf (#3 and #5), Kras (#8 and #9) and chemoresistant(#R3 and #R4) CRC patients. Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ∗p ≤ 0.05; ∗∗p ≤ 0.01. (B) Viability percentage of low and high TOP-GFP cell fraction treated as in (A) up to 120 h. Data are expressed as mean ± SD of three independent experiments performed using two different CR-CSphC lines (#8, #9). (C) Viability percentage of CR-CSphCs enriched for CD44v6 expression and treated as in (A) up to 120 h. Data are expressed as mean ± SD of three independent experiments performed using four different CR-CSphC lines (#3, #9, #21, #R4). (D) Colony-forming assay of CR-CSphCs treated with a vehicle or NORA234, at 21 days. The number of colonies represents mean ± SD of 3 independent experiments performed with cells isolated from 4 different patients with CRC (CR-CSphCs #3, #9, #21, #R4). Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ns, nonsignificant; ∗∗p ≤ 0.01. See also Figure S1 .

Article Snippet: Double staining was performed using antibodies against CD44v6 (2F10 APC, mouse IgG1, R&D systems) and p-CHK1 (Ser345, 133D3, Rabbit IgG, Cell Signaling Technology), revealed by the MACH 2 double stain 2 kit conjugated goat antimouse polymer horseradish peroxidase (HRP) and the conjugated goat antirabbit polymer alkaline phosphatase (Biocare Medical), and detected by DAB and Vulcan Fast Red chromogen.

Techniques: Isolation, Expressing

Journal: iScience

Article Title: CHK1 inhibitor sensitizes resistant colorectal cancer stem cells to nortopsentin

doi: 10.1016/j.isci.2021.102664

Figure Lengend Snippet: Inhibition of CHK1 activity sensitized CR-CSphCs to NORA234 by synthetic lethality (A) Fold variation of cell number in CR-CSphCs transduced with shCHK1 or ns shRNA and treated with vehicle, 5-FU in combination with oxaliplatin, or NORA234, up to 72 h. Data are mean ± S.D. of 3 independent experiments performed with cells isolated from patients with wt (#21), Braf (#3), Kras (#9), or chemoresistant (#R4) CRC. (B) Cell viability analysis of cells treated as in (A) for 72 h. Data are mean ± S.D. of 3 independent experiments performed with cells isolated from 4 different CRC patients (CR-CSphCs #3, #9, #21, #R4). Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ns, nonsignificant; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. (C) Representative colony forming assay of CR-CSphCs (R#4) transduced with shCHK1 or ns shRNA and treated with vehicle or NORA234, at 21 days. n represents the number of colonies. Scale bars, 1000 μm. (D) Percentage of Annexin V positivity in CR-CSphCs treated with vehicle or rabusertib (LY2603618), for 24 h. Data represent mean ± S.D. of 3 independent experiments performed with cells isolated from patients with wt (#21), Braf (#3), Kras (#9) or chemoresistant (#R4) CRC. Statistical significance between 2 groups was determined by unpaired Student's t-test (2-tailed). ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001. (E) Cell cycle analysis in CR-CSphCs treated with vehicle or NORA234 in combination with Rabusertib (LY2603618), for 48 h. Data show percentage of cell number in subG0 (red color), G0–G1 (blue color), S (brow color), and G2–M (green color) cell cycle phase. Data are expressed as mean ± SD of three independent experiments using cells isolated from 4 different patients with CRC (CR-CSphCs #3, #9, #21, #R4). (F) Flow cytometry analysis of GFP and CD44v6 positivity percentage in CR-CSphCs transduced with TOP-GFP and treated with a vehicle, NORA234, alone or in combination with rabusertib (LY2603618), for 48 hr. Data are expressed as mean ± SD of three independent experiments using cells isolated from 2 different patients with CRC (CR-CSphCs #8, #9). (G) 3D synergy map of viability in CR-CSphCs treated alone or in combination with NORA234 and rabusertib (LY2603618) at the indicated doses, for 48 h. Data are mean of 3 independent experiments using cells isolated from patients with wt (#21), Braf (#3), Kras (#9) or chemoresistant (#R4) CRC. See also and .

Article Snippet: Double staining was performed using antibodies against CD44v6 (2F10 APC, mouse IgG1, R&D systems) and p-CHK1 (Ser345, 133D3, Rabbit IgG, Cell Signaling Technology), revealed by the MACH 2 double stain 2 kit conjugated goat antimouse polymer horseradish peroxidase (HRP) and the conjugated goat antirabbit polymer alkaline phosphatase (Biocare Medical), and detected by DAB and Vulcan Fast Red chromogen.

Techniques: Inhibition, Activity Assay, Transduction, shRNA, Isolation, Cell Cycle Assay, Flow Cytometry