b16 f10 mouse melanoma tumor cell line  (ATCC)

Journal: bioRxiv

Article Title: Indomethacin exerts both cyclooxygenase inhibition-dependent and independent mechanisms to enhance chemo-immunotherapy in mice

doi: 10.64898/2026.01.07.698231

Figure Lengend Snippet: (A) CT26 tumor growth curves. The schema depicts the treatment timeline. CT26 cells were implanted s.c. into BALB/c mice. Once tumors reached 60–90 mm², mice were randomized into four groups and treated as indicated. Tumor growth curves are shown, with number of mice indicated. (B) Kaplan–Meier survival analysis of the same cohort. (C) MC38 tumor growth curves. MC38 cells were implanted s.c. into C57BL/6 mice. Mice with established tumors (60–90 mm²) were randomized to the indicated treatment groups. (D) Kaplan–Meier survival analysis. (E) 4T1 tumor growth curves. 4T1 cells were implanted into the fourth mammary fat pad of female BALB/c mice. Mice with palpable tumors (20–30 mm²) were randomized and treated as indicated. (F) Kaplan–Meier survival analysis. Data were pooled from two independent experiments for each tumor model. Statistics: (B, D, F) Log-rank (Mantel–Cox) test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Article Snippet: A20, 4T1, MC38, and CT26 mouse tumor cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques:

Journal: bioRxiv

Article Title: Indomethacin exerts both cyclooxygenase inhibition-dependent and independent mechanisms to enhance chemo-immunotherapy in mice

doi: 10.64898/2026.01.07.698231

Figure Lengend Snippet: (A) Indo fails to enhance CTX efficacy in immunodeficient NSG mice. The schema depicts the timeline of experimental procedures. NSG mice bearing established CT26 tumors (60–90 mm²) were randomized into four groups and treated as indicated. Tumor growth curves are shown. (B) Kaplan–Meier survival analysis of the same cohort. Data were pooled from two independent experiments. (C) The beneficial effect of Indo requires endogenous CD8⁺ T cells. The schema depicts the timeline of experimental procedures. BALB/c mice bearing established CT26 tumors (60–90 mm²) were treated with CTX+Indo. A subset of mice additionally received i.p. anti-CD8 Ab before and during treatment to deplete endogenous CD8⁺ T cells. Mice treated with CTX alone were included as controls. Tumor growth curves are shown. Mouse survival is summarized in the Kaplan-Meier plot (D). (E) Blockade of T cell trafficking by FTY720 diminishes the efficacy of CTX+Indo. BALB/c mice with established CT26 tumors (60–90 mm²) were treated with CTX+Indo. These mice also received either FTY720 or solvent by i.p. injection three times weekly for 4 weeks, starting one day prior to CTX administration. Tumor growth curves are shown. (F) Kaplan–Meier survival analysis. Data were pooled from two independent experiments. Data were pooled from two independent experiments. Statistics: (B, D, F) Log-rank (Mantel-Cox) test. **, p < 0.01; ***, p < 0.001; ns, not significant.

Article Snippet: A20, 4T1, MC38, and CT26 mouse tumor cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Solvent, Injection

Journal: bioRxiv

Article Title: Indomethacin exerts both cyclooxygenase inhibition-dependent and independent mechanisms to enhance chemo-immunotherapy in mice

doi: 10.64898/2026.01.07.698231

Figure Lengend Snippet: The schema depicts the timeline of experimental procedures. RAG2KO mice bearing established CT26 tumors (60–90 mm²) were randomized into four groups and treated as indicated. (A) Tumor growth curves are shown with mice numbers in each group indicated. (B) Kaplan–Meier survival analysis. Statistics: Log-rank (Mantel-Cox) test. **, p < 0.01; ns, not significant.

Article Snippet: A20, 4T1, MC38, and CT26 mouse tumor cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques:

Journal: bioRxiv

Article Title: Indomethacin exerts both cyclooxygenase inhibition-dependent and independent mechanisms to enhance chemo-immunotherapy in mice

doi: 10.64898/2026.01.07.698231

Figure Lengend Snippet: (A) FTY720 administration reduces T cells in circulation but does not alter T cell frequency in secondary lymphoid organs. CT26-bearing BALB/c mice were treated as described in . A cohort of mice were euthanized after receiving 4 doses of FTY720 or solvent. The frequencies of CD4+ and CD8+ T cells in blood, draining lymph node and spleen were determined by flow cytometry. Representative dot plots are shown. Numbers in plots denote percent of CD4+ and CD8+ T cells. (B) FTY720 administration diminishes the efficacy of CTX+Indo in MC38 tumor model. Following the same experimental procedures depicted in , C57BL/6 mice with established MC38 tumors (60-90mm 2 ) were treated as indicated. Tumor growth curves are shown with mice numbers in each group indicated. Mouse survival is summarized in the Kaplan-Meier plot (C). Statistics: Log-rank (Mantel-Cox) test. *, p < 0.05; ns, not significant.

Article Snippet: A20, 4T1, MC38, and CT26 mouse tumor cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Solvent, Flow Cytometry

Journal: bioRxiv

Article Title: Indomethacin exerts both cyclooxygenase inhibition-dependent and independent mechanisms to enhance chemo-immunotherapy in mice

doi: 10.64898/2026.01.07.698231

Figure Lengend Snippet: BALB/c mice bearing established CT26 tumors (60-90mm 2 ) were randomly assigned to three groups to receive no treatment, CTX alone, or CTX+Indo. 7 days after CTX, tumors were harvested, processed and cryopreserved for scRNA-seq analysis. Mice responsive to CTX+Indo treatment (responders) were identified using the bilateral tumor model shown in . Only tumors from responders were included in the scRNA-seq analysis. (A) t-SNE plots showing annotated cell populations based on gene expression profiles in the indicated tumor samples. (B) Bar graph summarizing the percentage distribution of tumor cells and immune cells across the analyzed samples. (C) Bar graph summarizing the percentage distribution of different immune cell subsets across the analyzed samples. (D) Heatmap showing the top signaling pathways in tumor cells affected by the indicated treatment conditions. Columns represent samples, and rows represent average pathway AUC scores generated by AUCell package.

Article Snippet: A20, 4T1, MC38, and CT26 mouse tumor cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Gene Expression, Protein-Protein interactions, Generated

Journal: bioRxiv

Article Title: Indomethacin exerts both cyclooxygenase inhibition-dependent and independent mechanisms to enhance chemo-immunotherapy in mice

doi: 10.64898/2026.01.07.698231

Figure Lengend Snippet: (A) scRNA-seq feature plots showing the expression patterns of selected genes under the indicated treatment conditions. Data are derived from the scRNA-seq analysis described in . (B) Schematic illustrating the timeline of tumor sample collection for flow cytometric analysis. BALB/c mice bearing established CT26 tumors (60-90mm 2 ) were randomly assigned to three groups to receive no treatment, CTX alone, or CTX+Indo. 7 days after CTX, tumors were harvested and processed into single cell suspensions for flow cytometric analysis. A bilateral tumor model was used for the CTX+Indo treatment group. Mice responsive (R) or non-responsive (NR) to CTX+Indo treatment were determined based on the outcome of the indicator tumors by day 20, and the corresponding tumor samples analyzed at day 7 were retrospectively categorized. (C) Bar graph summarizing the percentages of CD8+ TILs within lymphocytes under the indicated conditions, shown as mean ± SEM from at least 3 samples per condition. Differences between groups did not reach statistical significance by one-way ANOVA. (D) Representative dot plots showing IFNγ production by CD8+ TILs measured by cytokine intracellular stain after 4-hour stimulation with PMA/inomycin. Numbers denote the percentage of IFNγ⁺ cells within the gated (red rectangles) CD8⁺ T cell population. An unstimulated sample was included to aid in setting the gating strategy. The percentages of IFNγ⁺ CD8⁺ TILs (mean ± SEM) are summarized in (E), and the mean fluorescence intensities (MFIs) of IFNγ in CD8⁺ TILs (mean ± SEM) are summarized in (F). (G) Representative FACS data showing the expression of PD1 in CD8 + TILs under the indicated conditions. The percentages of PD1⁺ CD8⁺ TILs (mean ± SEM) are summarized in (H), and the MFIs of PD1 in CD8⁺ TILs (mean ± SEM) are summarized in (I). (J) CTX+Indo augments the efficacy of aPD1 treatment in the CT26 tumor model. The schema depicts the timeline of the experimental procedures. Mice with established CT26 tumors (60-90mm 2 ) were randomly assigned to groups to receive the indicated treatments. Tumor growth curves are shown, with the number of mice per group indicated. Mouse survival is summarized in the Kaplan-Meier plot (K). Statistics: (C, E, F, H, I) one-way ANOVA; (K) Log-rank (Mantel-Cox) test. **, p < 0.01; ***, p < 0.001; ns, not significant.

Article Snippet: A20, 4T1, MC38, and CT26 mouse tumor cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Derivative Assay, Staining, Fluorescence

Journal: bioRxiv

Article Title: Indomethacin exerts both cyclooxygenase inhibition-dependent and independent mechanisms to enhance chemo-immunotherapy in mice

doi: 10.64898/2026.01.07.698231

Figure Lengend Snippet: Indo enhances chemo-immunotherapy through both COX-dependent and COX-independent mechanisms. (A) Western blot analysis showing COX-2 expression in the indicated mouse tumor cell lines. β-actin was used as a loading control. (B) PGE₂ concentrations in tumor cell culture supernatants. Tumor cells were seeded in 6-well plates (0.5 × 10⁶ cells per well), and supernatants were collected at confluence. PGE₂ levels were quantified by LC-MS. Data are shown as mean ± SEM of triplicate samples. (C) Quantification of PGE₂ in tumor tissues. BALB/c mice were implanted s.c. with CT26 or A20 tumor cells. Tumor tissues were resected at the sizes of 100-200 mm 2 and processed for PGE₂ quantification by LC-MS. Results were normalized to tumor tissue weight and shown as mean ± SEM of triplicate samples. (D) CTX+Indo augments the efficacy of aPD1 treatment in the A20 tumor model. The schema depicts the timeline of the experimental procedures. Mice with established A20 tumors (80-100mm 2 ) were randomly assigned to groups to receive the indicated treatments. Tumor growth curves are shown, with the number of mice per group indicated. Data shown are pooled from two independent experiments. Mouse survival is summarized in the Kaplan-Meier plot (E). (F) CT26.COXKO cells do not produce measurable PGE₂. Supernatants from CT26.COXKO cell culture were collected for PGE₂ quantification by LC-MS. Cell cultures from untreated or Indo-treated wild-type CT26 cells were included as controls. Results are shown as mean ± SEM of triplicate samples. (G) PGE₂ is not detectable in CT26.COXKO tumor tissues regardless of treatment. BALB/c mice with established wild-type CT26 or CT26.COXKO tumors (60-90mm 2 ) were randomly assigned to three groups to receive no treatment, CTX alone, or CTX+Indo. 7 days after CTX, tumors were harvested and processed for PGE₂ quantification by LC-MS. Results were normalized to tumor tissue weight and shown as mean ± SEM of triplicate samples. (H) Waterfall plots summarizing the responses of CT26.COXKO tumor response to the indicated treatments. As depicted in the schema, mice with established CT26.COXKO tumors (60-90mm 2 ) were randomly assigned to groups to receive the indicated treatments. Tumor size changes from treatment initiation to endpoint were normalized to the initial tumor sizes for each mouse and used to generate the waterfall plots. (I) Indo reduces the level of RAS-GTP in CT26 cells. CT26 cells were treated with DMSO or Indo (10uM) for 16 hours before being harvested for RAS pull-down assays. Similar assays were conducted for CT26.COXKO cells (J) and A20 cells (K). Representative Western blots for RAS-GTP and total RAS are shown. Normalized RAS-GTP values were calculated as the ratio of RAS-GTP to total RAS in Indo-treated samples divided by the corresponding ratio in DMSO-treated samples. Data are summarized as mean ± SEM from at least three biological replicates per condition. Statistics: (B, F, G, I, J, K), one-way ANOVA; (C), student t- test; (E) Log-rank (Mantel-Cox) test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Article Snippet: A20, 4T1, MC38, and CT26 mouse tumor cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Control, Cell Culture, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Indomethacin exerts both cyclooxygenase inhibition-dependent and independent mechanisms to enhance chemo-immunotherapy in mice

doi: 10.64898/2026.01.07.698231

Figure Lengend Snippet: A COX-deficient CT26 cell line was generated by targeted CRISPR/Cas9-mediated gene editing of both Ptgs1 and Ptgs2 genes. PCR products spanning the sgRNA target site were amplified and analyzed using long-read sequencing. Images shown are representative IGV browser views of sequencing read alignments illustrating deletions and sequence variants at the CRISPR-edited region. Unmodified CT26 cells were used as a sequencing control.

Article Snippet: A20, 4T1, MC38, and CT26 mouse tumor cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Generated, CRISPR, Amplification, Sequencing, Control