Journal: Neoplasia (New York, N.Y.)

Article Title: Assessing axitinib-induced differential responses in tumor vascularization and oxygenation with combined optoacoustic angiography and diffuse optical spectroscopy

doi: 10.1016/j.neo.2026.101303

Figure Lengend Snippet: Axitinib inhibits Colo320 growth. ( a ) The experimental timeline: time points of axitinib administration are indicated by black triangles, time points of OA and DOS investigation - by white triangles, time point of morphological study – by red triangle. ( b ) Dynamics of the Colo320 tumor volumes during treatment with axitinib. Individual values, 25-75 percentiles, medians, minimum and maximum of the data set (n = 6). *, p < 0.05; **, p < 0.01 for treated versus control group (Wilcoxon test). # , p < 0.05 for current values versus initial level (Mann-Whitney test).

Article Snippet: All the experiments were conducted on subcutaneously implanted xenograft model based on the Colo320 colon adenocarcinoma cell line (Colo320DM, ATCC No CCL-220).

Techniques: Control, MANN-WHITNEY

Journal: Neoplasia (New York, N.Y.)

Article Title: Assessing axitinib-induced differential responses in tumor vascularization and oxygenation with combined optoacoustic angiography and diffuse optical spectroscopy

doi: 10.1016/j.neo.2026.101303

Figure Lengend Snippet: Axitinib-induced reduction of tumor vascularity. ( a ) Examples of OA images of Colo320 vasculature before and after treatment with axitinib. Bar is 3 mm. Dashed lines contour the tumor zones. ( b ) Volumetric vessel fraction of the Colo320 tumors during treatment with axitinib. ( c ) The corresponding projected vessel area. Individual values and M ± SD (n = 6). *, p < 0.05; **, p < 0.01 for treated versus control group (unpaired t-test). # , p < 0.05; # # , p < 0.01 for current values versus initial level (paired t-test).

Article Snippet: All the experiments were conducted on subcutaneously implanted xenograft model based on the Colo320 colon adenocarcinoma cell line (Colo320DM, ATCC No CCL-220).

Techniques: Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Assessing axitinib-induced differential responses in tumor vascularization and oxygenation with combined optoacoustic angiography and diffuse optical spectroscopy

doi: 10.1016/j.neo.2026.101303

Figure Lengend Snippet: Axitinib-induced decreases of CD31 positive blood vessels. ( a ) Examples of microimages from the tumor sections (scale bar 100 µm). ( b ) The numbers of CD31+ microvessels in the untreated and axitinib-treated Colo320 tumors after immunohistochemical staining for CD31. Individual values and M ± SD. ***, p < 0.001 for treated versus control group (unpaired t-test). ( c ), The values of CD31+ microvessels versus vascular fraction in the treated and untreated tumors.

Article Snippet: All the experiments were conducted on subcutaneously implanted xenograft model based on the Colo320 colon adenocarcinoma cell line (Colo320DM, ATCC No CCL-220).

Techniques: Immunohistochemical staining, Staining, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Assessing axitinib-induced differential responses in tumor vascularization and oxygenation with combined optoacoustic angiography and diffuse optical spectroscopy

doi: 10.1016/j.neo.2026.101303

Figure Lengend Snippet: Axitinib-induced increase of pimonidazole-positive areas. ( a ) Examples of LSM images taken from the tumor sections. Bar 2 mm. ( b ) Relative hypoxic fraction (RHF) of the untreated and axitinib-treated Colo320 tumors after immunofluorescent staining for hypoxia with pimonidazole. Individual values and M ± SEM. *, p < 0.05 for treated versus control group (unpaired t-test). ( c ) The RHF values versus StO 2 .

Article Snippet: All the experiments were conducted on subcutaneously implanted xenograft model based on the Colo320 colon adenocarcinoma cell line (Colo320DM, ATCC No CCL-220).

Techniques: Staining, Control

Journal: Nature Communications

Article Title: DKC1 promotes colorectal cancer progression and therapy resistance by dysregulating sphingolipid biosynthesis

doi: 10.1038/s41467-026-72800-2

Figure Lengend Snippet: a Heatmap depicting DKC1 expression in CRC patients across CMS categories and its association with WNT target genes; data derived and reanalyzed from TCGA-COADREAD. The color bar indicates z-score values. b Relative expression of DKC1 in CRC patient samples ( N = 76) categorized based on CMS classification; data retrieved from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). c Box and whiskers plot showing Dkc1 expression in murine colon tissue ( n = 3) based on Apc alteration status ( GSE65461 ) across different time points of Apc floxing. d Bar plot showing DKC1 transcript upon siRNA-mediated silencing of CTNNB1 (β-catenin) in SW620, WiDr, and Colo320HSR cell lines. e Same as in ( d ), except protein expression. CD44 was used as a positive control. f Immunoblots showing subcellular DKC1 expression upon WNT signaling stimulation across three independent CRC patient-derived organoids (PDOs). Lamin A was immunoblotted separately as a nuclear loading control, and α-tubulin was used as a cytosolic loading control, respectively. p-β-Catenin (Ser675) was used as a positive control for assessing active WNT signaling. g Schema depicting the chromosomal location of β-Catenin Binding Motif (CBM 1 to 4) at the DKC1 promoter (top). ChIP-qPCR data showing enrichment of β-catenin on DKC1 promoter using SW620 cells ( P < 0.0001); LGR5 and ANKARD5 were used as positive controls, and CD70 was used as a negative control (bottom). h Schema representing the proposed canonical WNT signaling-driven DKC1 oncogenicity and the positive feedback loop. For panels, ( b ) One-way ANOVA, Dunnett’s multiple comparisons test was used; c One-way ANOVA, Sidak’s multiple comparisons test was used; ( d , g ) Unpaired Student’s two-tailed t test was applied. For panels, ( b , c ), data are presented as box-and-whisker plots indicating median (middle line), 25th and 75th percentile (box) and minimum and maximum values (whiskers). Data shown in the panels ( 3d–g ) are from one representative experiment out of three independent replicates with similar results, each experiment includes 3 biological replicates per group. Data expressed as mean ± SEM. Figure 3h was created in BioRender. KUNDU, S. (2026) https://BioRender.com/h9o2vi8 . Source data are provided as a Source Data file.

Article Snippet: Colo320HSR cells were maintained in RPMI1640 media, whereas WiDr and SW620 cells were maintained in MEM and Leibovitz’s L-15 medium, respectively, supplemented with 10% FBS and 0.5% Penicillin/Streptomycin and grown in culture conditions as per ATCC recommendations.

Techniques: Expressing, Derivative Assay, Positive Control, Western Blot, Control, Binding Assay, ChIP-qPCR, Negative Control, Two Tailed Test, Whisker Assay

Journal: bioRxiv

Article Title: Dynamic optimization of extrachromosomal DNA copy number drives tumour evolution

doi: 10.64898/2026.03.20.713026

Figure Lengend Snippet: (a) Representative images of DNA FISH for MYC and EGFR in COLO 320 and GBM39 isogenic cell line pairs, respectively (top). Scale bars, 10 µm. Quantification of ecDNA copy number measured by DNA FISH area for COLO 320 and GBM39 isogenic cell line pairs (bottom). (b) Heatmap depicting Pearson correlation coefficients between amplicon copy number and transcript expression from single-cell multiomics sequencing (scATAC-seq and scRNA-seq) in several cell lines. Strength of correlation ( R ) depicted only for statistically significant correlations ( p < 0.05) (c) Shannon indices of the expression of genes amplified on ecDNA (top) and hallmark pathways (bottom) between ecDNA(+) and HSR(+) cells in COLO 320 (left) and GBM39 (right) isogenic cell line pairs. Wilcoxon rank-sum tests were performed to compare the mean gene and pathway expression between ecDNA(+) and HSR(+) cells. COLO 320DM vs. COLO 320HSR ecGene expression (p = 0.0016) and hallmark pathway scores (p = 0.024). GBM39-EC vs. GBM39-HSR ecGene expression (p = 0.22) and hallmark pathway scores (p=0.13). (d) Distributions of pathway expression scores for MYC-V2 targets (top) and PI3K/AKT-mTOR (bottom) expression between cells with high and low ecDNA copy numbers in cell lines with MYC ecDNA (top) and EGFR ecDNA (bottom). Statistical significance was assessed using a two-sided Wilcoxon rank-sum test. (e) Distributions of pathway expression scores for DNA damage response and unfolded protein response between cells with high and low ecDNA copy numbers in cell lines with MYC ecDNA (top) and EGFR ecDNA (bottom). Statistical significance was assessed using a two-sided Wilcoxon rank-sum test. ( f ) Representative images of combined FISH for EGFR ecDNA and IF for EGFR protein, phospho-Akt S473, and γH2AX (left). Scale bars, 5 µm. Pearson correlation scatterplot between EGFR copy number (measured by EGFR DNA FISH area) and EGFR protein expression (measured by EGFR intensity); n=97 cells (top right). Distribution of phospho-Akt Ser473 expression (measured by pAkt intensity); n=56 cells (middle right) and γH2AX expression (measured by foci area); n=818 cells (bottom right) across EGFR ecDNA copy number quintiles. (g) Distributions of EGFR protein abundance and pathway expression scores between cells with high and low EGFR ecDNA copy numbers in a human patient glioblastoma sample. Statistical significance was assessed using a two-sided Wilcoxon rank-sum test. (h) Schematic summarizing impact of ecDNA copy numbers on cancer cell phenotypes.

Article Snippet: The TG19 HSR line was engineered similarly from COLO 320HSR cells obtained from ATCC and detailed as follows.

Techniques: Amplification, Expressing, Single Cell, Sequencing, Quantitative Proteomics

Journal: bioRxiv

Article Title: Dynamic optimization of extrachromosomal DNA copy number drives tumour evolution

doi: 10.64898/2026.03.20.713026

Figure Lengend Snippet: (a) Table of cell lines used for single-cell multiomics analyses. (b) Pearson correlation scatterplot demonstrating concordance between ecDNA oncogene copy number estimated by whole genome sequencing and by scATAC-seq. Colors used to match point to text label. (c) Pearson correlation scatterplots between copy number (measured by scATAC-seq) and transcript expression (measured by scRNA-seq) for MYC and housekeeping gene ACTB in COLO 320DM and COLO 320HSR. (d) Pearson correlation scatterplots between copy number and transcript expression for EGFR and ACTB in GBM39-EC and GBM39-HSR. (e) Pearson correlation scatterplots between copy number and transcript expression for EGFR and ACTB in a human patient glioblastoma sample. (f) Distributions of pathway expression scores for MYC-V2 targets, PI3K_AKT_MTOR expression, DNA damage response, and unfolded protein response between cells with high and low ecDNA copy numbers across different cell cycle stages in the SNU16-m1 cell line, which has both MYC and FGFR2 amplified on ecDNA. Statistical significance was assessed using a two-sided Wilcoxon rank-sum test. (g) Representative image of combined IF for γH2AX and DNA FISH for EGFR in GBM39-EC cells with Edu labeling to distinguish replicating cells (in S-phase) from non-replicating cells. Scale bar, 20 µm (left). Distribution of γH2AX expression (right) across EGFR ecDNA copy number quintiles among Edu-positive (n=220) and Edu-negative (n=605) cells. (h) Volcano plot demonstrating differentially expressed genes between cells with low and high ecDNA copy numbers in COLO 320DM. Genes found on ecDNA and genes in the MYC-V2 targets, DNA damage response, and unfolded protein response pathway signatures are labeled.

Article Snippet: The TG19 HSR line was engineered similarly from COLO 320HSR cells obtained from ATCC and detailed as follows.

Techniques: Single Cell, Sequencing, Expressing, Amplification, Labeling

Journal: bioRxiv

Article Title: Dynamic optimization of extrachromosomal DNA copy number drives tumour evolution

doi: 10.64898/2026.03.20.713026

Figure Lengend Snippet: (a) Schematic of live-cell imaging cell line engineering and image acquisition and analysis pipeline. The engineered live-cell imaging cell lines originating from COLO 320DM and COLO 320HSR were named TG19 ec and TG19 HSR, respectively. (b) Representative images from live-cell imaging capturing a cancer cell as it divides and expands to a population of eight cells in TG19 ec (top) and TG19 HSR (bottom). Scale bars, 20 µm. (c) Quantification of TetR-mNeonGreen foci area in each cell in the TG19 ec and TG19 HSR lineages shown in (b). Each node represents a single cell, with connections relating parent cells to their daughter cells in the subsequent generation. TetR-mNeonGreen foci area was used as a proxy for MYC copy number. TetR-mNeonGreen foci area is likely underestimated in the parent generation due to condensed chromatin configuration at the time of image acquisition. (d) Representative images (left) and quantification (right) TetR-mNeonGreen foci area and time to division (T2D) from live-cell imaging and lineage tracking of TG19 ec. P = parent, F1_x = daughter cells (Generation 1), F2_x = granddaughter cells (Generation 2), F3 = great granddaughter cells (Generation 3). Red dashed outlines indicate the most recent division since the last timeframe. Scale bars, 5 µm. (e) Representative images (left) and quantification (right) of TetR-mNeonGreen foci area and T2D from live-cell imaging and lineage tracking of TG19 HSR. P = parent, F1_x = daughter cells (Generation 1), F2_x = granddaughter cells (Generation 2). Scale bars, 5 µm. (f) Quantification of TetR-mNeonGreen foci area difference between sister cells in TG19 ec (n=20 sister cell pairs) and TG19 HSR (n=18 sister cell pairs). Each dot represents the difference between two sister cells. (g) Quantification of T2D difference (hours) between sister cells in TG19 ec (n=24 sister cell pairs) and TG19 HSR (n=24 sister cell pairs). Each dot represents the difference between two sister cells. (h) Relationship between TetR-mNeonGreen foci area and T2D in TG19 ec cells (n=53 divisions, 106 cells). Horizontal linear, linear, sigmoidal, and quadratic models were applied to fit the data.

Article Snippet: The TG19 HSR line was engineered similarly from COLO 320HSR cells obtained from ATCC and detailed as follows.

Techniques: Live Cell Imaging, Single Cell

Journal: bioRxiv

Article Title: Dynamic optimization of extrachromosomal DNA copy number drives tumour evolution

doi: 10.64898/2026.03.20.713026

Figure Lengend Snippet: (a) Schematic depicting CRISPR-Cas9-mediated knock-in of a tetO array (96x) into the intergenic region between MYC and PCAT1 and subsequent visualization of tetO upon binding to TetR-mNeonGreen. We named the engineered COLO 320DM- tetO and COLO 320HSR- tetO lines TG19 ec and TG19 HSR, respectively. (b) Representative images of metaphase DNA FISH ( tetO in red and MYC in green) in TG19 ec and TG19 HSR. Scale bars, 10 µm (c) Pearson correlation scatterplot depicting correlation between tetO area and MYC area measured from TG19 ec (n=19) and TG19 HSR (n=20) metaphase DNA FISH images. (d) Fraction of overlap between tetO and MYC foci for each TG19 ec metaphase DNA FISH image. (e) Pearson correlation scatterplot depicting correlation between MYC copy number (measured by MYC DNA FISH foci area) and overlap fraction. (f) Frequency histograms depicting proportion of MYC inherited (measured by TetR-mNeonGreen foci area) to daughter cells in dividing TG19 ec cells (n=60 divisions) and TG19 HSR cells (n=30 divisions) captured by live-cell imaging (left). Frequency histograms depicting proportion of MYC inherited (measured by MYC DNA FISH foci area) to daughter cells (indicated by Aurora B Kinase IF) in fixed COLO 320DM (n=76 divisions) and COLO 320HSR cells (n=105 divisions) (right).

Article Snippet: The TG19 HSR line was engineered similarly from COLO 320HSR cells obtained from ATCC and detailed as follows.

Techniques: CRISPR, Knock-In, Binding Assay, Live Cell Imaging

Journal: bioRxiv

Article Title: Dynamic optimization of extrachromosomal DNA copy number drives tumour evolution

doi: 10.64898/2026.03.20.713026

Figure Lengend Snippet: (a) Schematic illustrating competition assay workflow with COLO 320DM-mCherry and COLO 320HSR-GFP cells. (b) Change in cell composition, expressed as the percentage of COLO 320DM-mCherry and COLO 320HSR-GFP cells, at the start and end of competition assays performed in culture and in xenograft mouse models. (c) Tumourigenicity plots depicting rate of tumour growth in COLO 320DM (n=5 mice) and COLO 320HSR (n=5 mice) xenograft mouse models. Initial seeding density of 125,000 cells. (d) Mean tumour volume on day 20 in COLO 320DM (n=5 mice) and COLO 320HSR (n=5 mice) xenograft mouse models. Statistical significance was assessed using a two-sample independent t-test. (e) Copy number fold change of genes encoded on ecDNA between xenograft tumour cells and cultured cells in COLO 320DM and COLO 320HSR. Gene copy numbers were estimated from whole genome sequencing data. Statistical significance was assessed using a two-sided Wilcoxon rank-sum test. (f) Representative images from multiplexed FISH for MYC and IF for Ki67 on FFPE sections of COLO 320DM and COLO 320HSR xenograft mouse tissue. Initial seeding density of 1000 cells. Scale bars, 20 µm. (g) Proportion of COLO 320DM and COLO 320HSR cells by Ki67 expression. Ki67 status was classified as high if the area of Ki67 signal per cell was above 3.079 µm 2 (2 median absolute deviations) . Difference in the proportion of highly Ki67-expressing cells between samples was assessed using a chi-squared test. (h) Density curves depicting proportion of cells vs. MYC copy number (measured by MYC DNA FISH area) for COLO 320DM (n=2327) and COLO 320HSR (n=1159) tissue cells, stratified by Ki67 expression status. Dashed lines indicate the mean copy number for each group. (i) Schematic summarizing that the increased tumourigenicity of COLO 320DM cells in vivo can be accounted for by their ability to shift to a higher optimal MYC copy number.

Article Snippet: The TG19 HSR line was engineered similarly from COLO 320HSR cells obtained from ATCC and detailed as follows.

Techniques: Competitive Binding Assay, Cell Culture, Sequencing, Expressing, In Vivo

Journal: bioRxiv

Article Title: Dynamic optimization of extrachromosomal DNA copy number drives tumour evolution

doi: 10.64898/2026.03.20.713026

Figure Lengend Snippet: Flow cytometry plots of single and mixed COLO 320DM-mCherry and COLO 320HSR-GFP populations from (a) in vitro and (b) in vivo competition assays. Gating strategy was established from single populations.

Article Snippet: The TG19 HSR line was engineered similarly from COLO 320HSR cells obtained from ATCC and detailed as follows.

Techniques: Flow Cytometry, In Vitro, In Vivo

Journal: bioRxiv

Article Title: Dynamic optimization of extrachromosomal DNA copy number drives tumour evolution

doi: 10.64898/2026.03.20.713026

Figure Lengend Snippet: (a) Metaphase DNA FISH ( MYC ) in dissociated cells from COLO 320DM and COLO 320HSR mouse xenograft tissue. (b) Density curve of Ki67 expression from multiplexed IF (Ki67) and FISH ( MYC ) in COLO 320DM and COLO 320HSR FFPE mouse xenograft tissue sections. The dashed red line at 3.079 µm 2 (2 median absolute deviations) marks the threshold for Ki67-High versus Ki67-Low status.

Article Snippet: The TG19 HSR line was engineered similarly from COLO 320HSR cells obtained from ATCC and detailed as follows.

Techniques: Expressing