Journal:

Article Title: Stepwise Deletions of PolyA Sequences in Mismatch Repair-Deficient Colorectal Cancers

doi:

Figure Lengend Snippet: A: Examples of BAT26 and BAT40 deletions in MSI+ colorectal cancers. Lengths of the alleles are estimated from the densest band (open circle, normal; filled circle, tumor). B: Total deletions in BAT20, 25, 26, and 40 were variable for the 20 MSI+ colorectal cancers (specimens 1 to 20). For comparison, estimated total deletions for MMR-deficient (specimens 21 to 23; HCT116, SW48, HCT15) and MMR-proficient (specimens 24 to 26; HT29, SW480, SW837) colorectal cancer cell lines are illustrated. Deletions for the cell lines are estimates because matching normal tissues were unavailable and their starting germline sizes were assumed to be equal to the average lengths found in the 20 MSI+ tumor patients. C: Normal (open circles) and tumor (filled circles) allele lengths for MSI+ cancers and cell lines (triangles) at the four polyA loci.

Article Snippet: Colorectal cancer cell lines HT29, SW480, and SW837 (MMR-proficient); HCT116 and SW48 (MMR (MLH1)-deficient 16 ); and HCT15 [MMR (MSH6)-deficient 17 ] were obtained from American Type Culture Collection (Manassas, VA).

Techniques: Comparison

Journal: Cell Reports Methods

Article Title: Genome-wide profiling of unmodified DNA using methyltransferase-directed tagging and enrichment

doi: 10.1016/j.crmeth.2025.101187

Figure Lengend Snippet: Schematic of the Active-Seq workflow and initial method validation using synthetic oligonucleotides (A) Schematic showing the single-tube, Active-Seq workflow. Purified DNA (cfDNA or fragmented gDNA) is tagged using a CpG-targeting methyltransferase (M.MpeI) and a synthetic cofactor analog. The enzyme catalyzes DNA alkylation with azide-terminated tags exclusively at unmodified CpG sites. Tagged DNA molecules are subsequently subject to standard library end repair and adapter ligation, followed by affinity tagging and isolation using streptavidin-coated magnetic beads. Tagged (unmodified CpG sites, Active-Seq) and untagged (5mCpG and other modified CpG sites and CpG-free fragments, unbound fraction) DNA can be separately amplified and sequenced. (B) Plot showing the efficiency for DNA binding and release (recovery) of tagged and affinity-labeled DNA, using 10 ng target DNA input, as a function of target DNA CpG site density. Binding efficiency is calculated based on the assumption that any reduction in DNA concentration after DNA incubation with beads is as a result of DNA binding to the beads. (C) Plot showing the efficiency for binding and release (recovery) of tagged and affinity-labeled DNA for a decreasing amount of input target DNA against a background of 24 ng of non-target DNA (containing no CpG sites). Note that the recovered DNA concentration was determined by qPCR, and reported recoveries are measured against DNA we expect to be bound to the beads (input DNA quantified via Qubit). (D) Plot showing number of reads of spiked-in DNA oligos (5, 25, and 50 pg in total) in the enriched (unmethylated) fraction with four unmethylated CpG sites (4 uMe CpG), four methylated CpG sites (4 Me CpG), and no target CpG sites (0 CpG) from a typical Active-Seq experiment. (E) Plot showing number of reads of spiked-in DNA oligos (5, 25, and 50 pg in total) in the unbound fraction with four unmethylated CpG sites (4 uMe CpG), four methylated CpG sites (4 Me CpG), and no target CpG sites (0 CpG) from a typical Active-Seq experiment. Data in bar-plots are presented as mean ± SD of three independent replicates. Two-way ANOVA test: ∗ p < 0.05, ∗∗ p < 0.01 , ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001.

Article Snippet: Human gDNA: SW48 , ATCC , cat#: CCL-231DQ.

Techniques: Biomarker Discovery, Purification, Adapter Ligation, Isolation, Magnetic Beads, Modification, Amplification, Binding Assay, Labeling, Concentration Assay, Incubation, Methylation

Journal: PLoS ONE

Article Title: CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

doi: 10.1371/journal.pone.0149099

Figure Lengend Snippet: (A) Schematic of KRAS isogenic cell lines generation. KRAS mutations were introduced into the parental cell lines via r-AAV-mediated homologous recombination. A general structure of the targeting construct is represented. The resulting mutant KRAS allele is expressed from its endogenous promoter. The Neo cassette is removed from the genome of the targeted cells by Cre recombinase-mediated excision. AAV, adeno-associated virus; ITR, inverted terminal repeat; Neo, geneticin-resistance gene; P, SV40 promoter; triangles, loxP sites (Figure adapted from ). (B) RAS activation status of LIM1215 KRAS isogenic cell lines. Western blot showing active RAS (RAF1 GTP-bound) levels for LIM1215 KRAS isogenic cell lines. The RAF1 RAS binding domain (RBD) was used to precipitate GTP-RAS. The RAS activation status was tested for each clone with mutated KRAS. Precipitated RAS-GTP was detected by western blot using anti-RAS antibody. As a positive control, HeLa cells (RAS wild-type) were stimulated with epidermal growth factor (EGF) to activate the RAS pathway. HeLa and MCF7 cells (unstimulated) were used as negative controls. Total lysates were also immunoblotted with anti-β-Actin antibody as loading control. (C) and (D) KRAS dependence in the LIM1215 KRAS isogenic cell line models, obtained from the HT siRNA screen described in . Bar graph of KRAS siRNA Z-score values across the LIM1215 KRAS WT and mutant isogenic cell lines, C and D respectively. KRAS dependence was greater in the cell lines carrying KRAS mutations than in WT cells. Error bars represent SEM from three independent experiments. (E) Western blot of KRAS in SW48 cells expressing KRAS -specific siRNAs. Multiple KRAS siRNA oligos and a pool efficiently suppressed KRAS expression showing that the siRNAs were on-target.

Article Snippet: For assessment of the in vivo efficacy of AZD5438, 5x10 6 of SW620 cells, or SW48 KRAS WT or p.G12V isogenic cells were injected into the flank regions of female athymic Balb/C mice, twenty mice per cell line (Harlan Laboratories).

Techniques: Homologous Recombination, Construct, Mutagenesis, Virus, Activation Assay, Western Blot, Binding Assay, Positive Control, Control, Expressing

Journal: PLoS ONE

Article Title: CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

doi: 10.1371/journal.pone.0149099

Figure Lengend Snippet: (A) GTP-RAS assay showing the RAS activation status of SW48 KRAS isogenic cell lines. (B) KRAS dependence in the SW48 KRAS isogenic cell lines (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Student’s t-test for comparison between each KRAS mutant and the WT cell lines). (C) CDK1-specific siRNAs suppress CDK1 expression. Cell viability after CDK1 depletion in SW48 isogenic KRAS cell lines (ns, not statistically significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Student’s t-test for comparison between each KRAS mutant and the WT cell lines). Error bars represent SEM from three independent experiments. (D) Western blot of CDK1 in SW48 parental cells expressing CDK1-specific siRNAs.

Article Snippet: For assessment of the in vivo efficacy of AZD5438, 5x10 6 of SW620 cells, or SW48 KRAS WT or p.G12V isogenic cells were injected into the flank regions of female athymic Balb/C mice, twenty mice per cell line (Harlan Laboratories).

Techniques: Activation Assay, Comparison, Mutagenesis, Expressing, Western Blot

Journal: PLoS ONE

Article Title: CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

doi: 10.1371/journal.pone.0149099

Figure Lengend Snippet: (A) Exposure of SW48 isogenic cell lines to RO-3306 in a fifteen-day colony formation assay. (B) Exposure of SW48 isogenic cell lines to inhibitor AZD5438, in a fifteen-day colony formation assay. (C) Drug-dose response curves of CRC cells after AZD5438 exposure in a fifteen-day colony formation assay. ****P<0.0001, Two-way ANOVA. Error bars represent SEM of three technical replicates. All the experiments were performed two independent times with three technical replicates.

Article Snippet: For assessment of the in vivo efficacy of AZD5438, 5x10 6 of SW620 cells, or SW48 KRAS WT or p.G12V isogenic cells were injected into the flank regions of female athymic Balb/C mice, twenty mice per cell line (Harlan Laboratories).

Techniques: Colony Assay

Journal: PLoS ONE

Article Title: CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

doi: 10.1371/journal.pone.0149099

Figure Lengend Snippet: (A—C). CDK1 phosphorylation levels in KRAS mutant and WT cells as shown by Western blot analysis of total cell protein lysates from SW48 KRAS isogenic (A), non-isogenic pancreatic tumour cell lines (B) and non-isogenic colorectal cell lines (C). Western blots were probed for CDK1 (pThr161 CDK1 and total CDK1). β-actin detection was used as a loading control. (D and E) Bar graphs illustrating the percentage of cells in G1, S and G2/M cell cycle phases in SW48 KRAS WT or p.G12V mutant cell lines after AZD5438 exposure. SW48 KRAS WT (D) and p.G12V (E) were exposed to 0.3 μM AZD5438 or DMSO for 16, 24 and 48 hours after which cell cycle profiles were assessed by propidium iodide (PI) staining and flow cytometry. The KRAS p.G12V mutant cells showed a decrease in S and G2-fractions after exposure to AZD5438 when compared to control (DMSO) treated cells and to KRAS WT cells (AZD5438 and DMSO). (F—H) DNA synthesis in SW48 KRAS WT and p.G12V cell lines after AZD5438 exposure. (F) and (G) 5-ethynyl-2'-deoxyuridine (EDU)/ PI FACS plots in SW48 KRAS WT (F) and p.G12V mutant cells exposed to AZD5438 0.3 μM and 0.75 μM, or DMSO for 24 and 48 hours. After AZD5438 exposure, EDU/PI profiles were assessed by flow cytometry. EDU stained cells are represented in blue. (H) Bar graph illustrating the percentage of cells stained with EDU over time for both SW48 KRAS WT and p.G12V mutant cells. (I) Western blot illustrating the phosphorylation of Retinoblastoma protein (pRb) in SW48 KRAS WT and p.G12V mutant cell lines after AZD5438 exposure. Cells were exposed to AZD5438 for two hours after which total cell lysates were generated and western blotted as shown. Detection of β-Actin was used as a loading control. The levels of Rb phosphorylation on Ser807/811 were decreased in the KRAS p.G12V cells when compared to the WT cells, after AZD5438 2 hours exposure. (J) Western blot illustrating PARP1 cleavage in SW48 KRAS WT and p.G12V mutant cells after 72h of AZD5438 exposure. Cells were exposed to AZD5438 for two hours after which total cell lysates were generated and western blotted as shown. Exposure to camptothecin was used as a positive control.

Article Snippet: For assessment of the in vivo efficacy of AZD5438, 5x10 6 of SW620 cells, or SW48 KRAS WT or p.G12V isogenic cells were injected into the flank regions of female athymic Balb/C mice, twenty mice per cell line (Harlan Laboratories).

Techniques: Phospho-proteomics, Mutagenesis, Western Blot, Control, Staining, Flow Cytometry, DNA Synthesis, Generated, Positive Control

Journal: Heliyon

Article Title: Exercise potentially prevents colorectal cancer liver metastases by suppressing tumor epithelial cell stemness via RPS4X downregulation

doi: 10.1016/j.heliyon.2024.e26604

Figure Lengend Snippet: Knockdown of RPS4X expression reduced tumor stemness. (A) The RPS4X gene expression level was assessed by qPCR. GAPDH served as an internal parameter (n = 3). (B) The cell proliferation ability of SW48 was determined using the CCK-8 assay (n = 3). (C–D) Cell migration and invasion ability of SW48 were evaluated (n = 3). Data are presented as mean ± SD. *P < 0.05 (E–F) Cell apoptotic rate of SW48 was evaluated (n = 3). Data are presented as mean ± SD. **P < 0.01. (G) Tumor growth curve and RPS4X down-regulation in tumors from mice (n = 5 per group). Data are presented as mean ± SD. *P < 0.05 **P < 0.01 ***P < 0.001. (H) Representative images showing the tumors harvested from SW48-bearing mice (n = 5 per group). (I)Weight of the harvested tumors from tumor-bearing mice (n = 5 per group). Data are presented as mean ± SD. **P < 0.01.

Article Snippet: SW48 (catalog KG536) cancer cell lines were purchased from KeyGEN.

Techniques: Knockdown, Expressing, Gene Expression, CCK-8 Assay, Migration

Journal: Oncoimmunology

Article Title: Generation of neoantigen-specific T cells for adoptive cell transfer for treating head and neck squamous cell carcinoma

doi: 10.1080/2162402X.2021.1929726

Figure Lengend Snippet: Evaluation of the in vivo activities of MAGOHB G17A - TCR-engineered T cells and KIAA1429 D1358E - TCR-engineered T cells . (a) NCG mice were burdened with subcutaneous tumors carrying MAGOHB G17A mutation or KIAA1429 D1358E mutation for eight days, and treated with intravenous injections of corresponding TCR-engineered T cells, respectively. 400,000 IU systemic IL-2 was given daily by intraperitoneal injection for 3 days. For mice treated with MAGOHB G17A - TCR-engineered T cells: (b) tumor sizes, (c) tumor area of tumor growth over time, (d) tumor weights and (e) proportions of human CD3 + T cell in the peripheral blood at the end of this experiment were recorded. For mice treated with KIAA1429 D1358E - TCR-engineered T cells: (f) tumor sizes, (g) tumor area of tumor growth over time, (h) tumor weights and (i) proportions of human CD3 + T cell in the peripheral blood at the end of this experiment were recorded

Article Snippet: Five weeks-old NCG mice (GemPharmatech Co., Ltd., China) were inoculated subcutaneously with 2 × 10 6 SW480-KIAA1429 D1358E tumor cells or SW480-MAGOHB G17A tumor cells.

Techniques: In Vivo, Mutagenesis, Injection

Journal: Oncotarget

Article Title: Therapeutic efficacy of SYM004, a mixture of two anti-EGFR antibodies in human colorectal cancer with acquired resistance to cetuximab and MET activation

doi: 10.18632/oncotarget.18749

Figure Lengend Snippet: (A) Western blot analysis of protein expression in GEO, SW48, GEO-CR and SW48-CR cells treated with cetuximab (5 μg/ml) and SYM004 (5 μg/ml) was performed. Total cell protein extracts were subjected to immuneblotting with the indicated antibodies, as described in Materials and Methods. (B) Two mg of GEO cell or of GEO-CR cell protein extracts were immune-precipitated with a specific anti-MET antibody and then were immune-blotted with a specific anti-EGFR antibody, as described in Materials and Methods. (C) Western blot analysis of protein expression in SW48, SW48-CR, SW48H2 and SKBR3 was performed. Total cell protein extracts were subjected to immuneblotting with the indicated antibodies, as described in Materials and Methods. (D) HER2 gene amplified SW48 cells line (SW48H2) are exposed to different concentration of cetuximab (range, 0.001 to 10 μg/ml) and SYM004 (range, 0.001 to 10 μg/ml) for 96 hours and evaluated for proliferation by MTT staining, as described in Materials and Methods.

Article Snippet: The human SW48 (catalogue number: HTL99020) ( KRAS, NRAS, BRAF and PIK3CA WT), colon cancer cell line was obtained from IRCCS “Azienda Ospedaliera Universitaria San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genova” Italy.

Techniques: Western Blot, Expressing, Amplification, Concentration Assay, Staining

Journal: Oncotarget

Article Title: Therapeutic efficacy of SYM004, a mixture of two anti-EGFR antibodies in human colorectal cancer with acquired resistance to cetuximab and MET activation

doi: 10.18632/oncotarget.18749

Figure Lengend Snippet: (A-B) Mice were injected subcutaneously in the right flank with SW48 cells as described in the Materials and Methods. After two weeks (average tumor size 200-300 mm 3 ) mice were treated intraperitoneally with: PBS control, cetuximab (1 mg twice a week), SYM004 (50 mg/kg twice a week). The treatment was continued for 30 weeks. Each group consisted of 10 mice. Tumor volumes were measured three times a week. Animals were sacrificed when tumors achieved 2.000 mm 3 in size. Abbreviations: CTR, control; A, median tumor volume (mm 3 ); B, alive mice/total mice; C, number of mice without clinical evidence of progression.

Article Snippet: The human SW48 (catalogue number: HTL99020) ( KRAS, NRAS, BRAF and PIK3CA WT), colon cancer cell line was obtained from IRCCS “Azienda Ospedaliera Universitaria San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genova” Italy.

Techniques: Injection, Control

Journal: Oncotarget

Article Title: Therapeutic efficacy of SYM004, a mixture of two anti-EGFR antibodies in human colorectal cancer with acquired resistance to cetuximab and MET activation

doi: 10.18632/oncotarget.18749

Figure Lengend Snippet: (A-B) SW48 cells were injected s.c. into the right flank of seven nude mice. After two weeks mice were treated with Cetuximab (1 mg twice a week) by i.p. injection. Treatment was continued until disease progression. The black arrows indicate the time of progression to cetuximab. At progression phase mice were assigned to SYM004 treatment (50 mg/Kg twice a week) by i.p. injection. The treatment was continued until 30 weeks. At week 30 four out of seven mice were still responding to SYM004 (as indicated by double asterisk). Abbreviations: PD, progression disease; PR, partial response; SD, stable disease.

Article Snippet: The human SW48 (catalogue number: HTL99020) ( KRAS, NRAS, BRAF and PIK3CA WT), colon cancer cell line was obtained from IRCCS “Azienda Ospedaliera Universitaria San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genova” Italy.

Techniques: Injection, Biomarker Discovery