Journal: bioRxiv
Article Title: The E2F4 transcriptional repressor is a key mechanistic regulator of colon cancer resistance to irinotecan (CPT-11)
doi: 10.1101/2025.01.22.633435
Figure Lengend Snippet: To understand whether colon cancer cells with a cancer stem cell (CSC) phenotype displayed preferential resistance to chemotherapy, we tested whether, in immune-deficient mice engrafted with a human colon cancer patient-derived xenograft (PDX) line, in vivo treatment with irinotecan (CPT-11) was more capable of inducing apoptosis in cancer cells with a top-of-the-crypt (EpCAM + , CD44 neg , CD166 neg ) phenotype, which are non-tumorigenic, as compared to cancer cells with a bottom-of-the-crypt phenotype (EpCAM + , CD44 + , CD166 + ), which are enriched in cancer stem cells (CSCs). Adult, female, NOD/SCID/IL2Rg -/- (NSG) immune-deficient mice were engrafted sub-cutaneously (s.c.) with the PDX-COLON-8 line, which is known to contain populations with both top-of-the-crypt (EpCAM + , CD44 neg , CD166 neg ) and bottom-of-the-crypt (EpCAM + , CD44 + , CD166 + ) phenotypes. Tumor-bearing mice were then treated with either irinotecan (50 μg/g, once weekly x 4 weeks, i.p.) or a placebo control (1 ml of saline solution, once weekly x 4 weeks, i.p.) and sub-cutaneous tumors harvested 48 hours after the last treatment (day 24). The percentage of apoptotic cells was quantified by flow cytometry, by measuring the percentage of Annexin-V + cancer cells in each of the two phenotypic sub-populations. In vivo treatment with irinotecan (CPT-11) induced preferential apoptosis among cancer cells with a top-of-the-crypt phenotype (EpCAM + , CD44 neg , CD166 neg ) as compared to cancer cells with a bottom-of-the-crypt phenotype (EpCAM + , CD44 + , CD166 + ). Differences in the percentage of Annexin-V + cancer cells between populations were tested for statistical significance using; 1) a Welch ANOVA across the full dataset (assuming unequal variance), followed by a Dunnett’s T3 test for pre-specified pairwise comparisons; and 2) a two-way ANOVA test for interaction (cell phenotype vs. chemotherapy). Increases in the percentage of Annexin-V + cancer cells caused by treatment with irinotecan were tested for statistical significance using a one-tailed Mann-Whitney U-test. Error bars: mean +/-standard deviation.
Article Snippet: The antibodies used to visualize cancer cells with bottom-of-the-crypt (EpCAM + , CD44 + , CD166 + ) and top-of-the-crypt (EpCAM + , CD44 neg , CD166 neg ) phenotypes included: anti-human-EpCAM-AlexaFluor488 (clone 9C4; BioLegend), anti-human CD44-PE-Cy7 (clone G44-26; BD Biosciences) and anti-human-CD166-PE (clone 105902; R&D Systems).
Techniques: Derivative Assay, In Vivo, Control, Saline, Flow Cytometry, One-tailed Test, MANN-WHITNEY, Standard Deviation
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![(A) Analysis by RT-qPCR of E2F4 mRNA expression in colorectal cancer (CRC) cells infected with lentivirus vectors encoding for shRNA constructs targeting the E2F4 mRNA. The analysis confirmed the capacity of both shRNA constructs used in this study, E2F4-shRNA[#1] and E2F4-shRNA[#2], to cause a reduction in E2F4 expression levels, as compared to what observed in parental cells infected with an empty vector (reference standard). The analysis was conducted on 6 independent models, including: a) four patient derived xenograft (PDX) lines (PDX-COLON-8, PDX-COLON-60, PDX-COLON-C441, PDX-COLON-136); and b) two conventional cell lines grown as two-dimensional (2D) monolayers (HT29, HCT116). In both cases, the analysis was conducted on purified preparations of lentivirus-infected malignant cells, isolated by FACS from primary tissues based on the expression of a green fluorescent reporter (copGFP) encoded by the lentivirus construct. Results are reported as fold-changes relative to cells infected with an empty vector, after ΔΔCt normalization to ACTB mRNA expression levels and calculation of 2 -ΔΔCt values (Livak & Schmittgen, Methods , 25:402-408, 2001). Differences in expression levels were tested for statistical significance using a two-way ANOVA test on ΔΔCt values (cell line vs. lentivirus construct) followed by Dunnett’s test on pairwise comparisons. Error bars: mean +/-standard deviation. (B) Analysis by Western blot of E2F4 protein expression in cancer cells with a bottom-of-the-crypt phenotype (EpCAM + , CD44 + , CD166 + ) purified by FACS from the PDX-COLON-8 line following infection with either an empty lentivirus vector or lentivirus vectors encoding for the E2F4-shRNA [#1] or E2F4-shRNA [#2] constructs. The analysis confirmed the capacity of both shRNAs to cause a visually detectable reduction in E2F4 protein levels in the population with a bottom-of-the-crypt phenotype (EpCAM + , CD44 + , CD166 + ), which is enriched in cells with cancer stem cell (CSC) properties. ACTB (β-Actin) protein levels were analyzed in parallel and used as a visual standard to normalize for the total amount of protein loaded onto the gel.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_35/10__1101_slash_2025__01__22__633435/10__1101_slash_2025__01__22__633435___F7.large.jpg)