murine colon carcinoma cell line ct26  (ATCC)

Journal: Science Advances

Article Title: PROTAC-based synthetic lethality strategy endogenously activates systemic STING to boost antitumor immunity

doi: 10.1126/sciadv.ado7448

Figure Lengend Snippet: ( A ) Schematic representation of PROTAC design targeting PARP1 and BRD4. ( B ) WB analysis of BRD4 and PARP1 protein levels in B16F10 cells treated with PRO P , PRO B , and PRO P+B for 12 hours. ( C ) WB analysis of BRD4 and PARP1 protein levels in B16F10 cells treated with PRO P+B for 24 hours at indicated concentrations. Quantification of BRD4 ( D ) and PARP1 ( E ) expression levels in B16F10 cells after incubating with PBS, PRO P , PRO B , and PRO P+B for 24 hours. a.u., arbitrary units. BRD4 ( F ) and PARP1 ( G ) expression detected by immunofluorescence in 4T1, H22, KPC, and CT26 cells after incubating with PRO P+B for 24 hours. The signals of BRD4 and PARP1 were generated by Alexa Fluor 488– and Alexa Fluor 647–conjugated secondary antibodies, respectively. ( H ) Heatmap visualization of target protein expression levels after incubating with PRO P+B for 24 hours. ( I ) Schematic diagram of PROTAC preventing DNA damage repair. ( J ) WB analysis of γ-H2AX protein levels in B16F10 cells treated with PRO P , PRO B , and PRO P+B for 24 hours. ( K ) Comet assay for B16F10 cells under diverse treatments. ( L ) Immunofluorescence of γ-H2AX staining for DNA fragmentation visualization after incubation of PRO P+B for 24 hours in 4T1, H22, KPC, and CT26 cells. The signals of γ-H2AX were generated by Alexa Fluor 488–conjugated secondary antibodies. ( M ) Comet assay for 4T1, H22, KPC, and CT26 cells after PRO P+B treatments. ( N ) DNA damage was expressed by the ratio of comet tail on the basis of the comet assay in (M). Results are presented as the means ± SD.

Article Snippet: B16F10, 4T1, H22, KPC, and CT26 cell lines were obtained from American Type Culture Collection.

Techniques: Expressing, Immunofluorescence, Generated, Single Cell Gel Electrophoresis, Staining, Incubation

Journal: Science Advances

Article Title: PROTAC-based synthetic lethality strategy endogenously activates systemic STING to boost antitumor immunity

doi: 10.1126/sciadv.ado7448

Figure Lengend Snippet: ( A ) dsDNA expression levels detected by immunofluorescence in B16F10 cells after incubating with indicated treatments (PBS, PRO P , PRO B , and PRO P+B ) for 24 hours. The signals of dsDNA were generated by Alexa Fluor 488–conjugated secondary antibodies. Quantification of dsDNA in the cytoplasm of the B16F10 cells ( B ) and 4T1, H22, KPC, and CT26 cells ( C ). n = 3 biologically independent samples. ( D ) Schematic diagram of STING pathway activation upon recognition of cytosolic dsDNA. ELISA revealed cGAMP production in B16F10 cells ( E ) and 4T1, H22, KPC, and CT26 cells ( G ) 24 hours after indicated treatments (PBS, PRO P , PRO B , and PRO P+B ). ( F ) ELISA revealed cGAMP production in B16F10 cells after incubation of PRO P+B with or without DNase I addition. ( H ) WB analysis of cGAS, p-STING, and p-IRF3 protein levels in B16F10 cells treated with PBS, PRO P , PRO B , and PRO P+B for 24 hours. ELISA revealed IFN-β production in B16F10 cells ( I ) and 4T1, H22, KPC, and CT26 cells ( J ) 24 hours after indicated treatments (PBS, PRO P , PRO B , and PRO P+B ). ( K ) PD-L1 expression levels detected by immunofluorescence in B16F10 cells after incubating with indicated treatments (PBS, PRO P , PRO B , and PRO P+B ) for 24 hours. The signals of PD-L1 were generated by Alexa Fluor 488–conjugated secondary antibodies. ( L ) Flow cytometry analysis of PD-L1 expression in 4T1, H22, KPC, and CT26 cells treated with indicated treatments (PBS, PRO P , PRO B , and PRO P+B ) for 24 hours. (K) is the quantification of ( M ). Results are presented as the means ± SD.

Article Snippet: B16F10, 4T1, H22, KPC, and CT26 cell lines were obtained from American Type Culture Collection.

Techniques: Expressing, Immunofluorescence, Generated, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry

Journal: Science Advances

Article Title: PROTAC-based synthetic lethality strategy endogenously activates systemic STING to boost antitumor immunity

doi: 10.1126/sciadv.ado7448

Figure Lengend Snippet: ( A ) Schematic diagram of STING pathway activation and maturation of DCs. ( B ) Representative flow cytometric analysis image and relative quantifications of mature DC markers CD80/CD86 in BMDCs co-incubated with B16F10 cells treated with PBS, PRO P , PRO B , and PRO P+B for 24 hours. ( C ) Schematic diagram of STING pathway activation and maturation of DCs. Production of cGAMP in DC2.4 cells ( D ) and THP1 cells ( E ), which were co-incubated with B16F10, 4T1, H22, KPC, and CT26 cells after the tumor cells were treated with PBS, PRO P , PRO B , and PRO P+B , respectively, measured by ELISA assay. ( F ) WB analysis of cGAS, p-STING, and p-IRF3 protein levels in DC2.4 cells that were co-incubated with B16F10, 4T1, H22, KPC, and CT26 cells after the tumor cells were treated with PBS, PRO P , PRO B , and PRO P+B , respectively. Results are presented as the means ± SD.

Article Snippet: B16F10, 4T1, H22, KPC, and CT26 cell lines were obtained from American Type Culture Collection.

Techniques: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: PROTAC-based synthetic lethality strategy endogenously activates systemic STING to boost antitumor immunity

doi: 10.1126/sciadv.ado7448

Figure Lengend Snippet: ( A ) IC 50 (median inhibitory concentration) and CI values of different cell lines after incubation with LNP@PRO P , LNP@PRO B , and LNP@PRO P+B . ( B and C ) Representative pictures of clonogenic assay in B16F10, 4T1, H22, KPC, and CT26 cells treated with PBS (G1), LNP@PRO P (G2), LNP@PRO B (G3), or LNP@PRO P+B (G4) for 24 hours. ( D ) Colony-forming efficiency of B16F10, 4T1, H22, KPC, and CT26 cells. ( E ) Schematic illustration of the timeline for B16F10 tumor inoculation and treatments in a C57BL/6 mouse model. Mice with ~100-mm 3 subcutaneous tumors were intravenously (iv) administered with LNP@PRO P , LNP@PR B , and LNP@PRO P+B five times at a PRO B equivalent dose of 10 mg kg −1 . sc, subcutaneously. ( F ) Tumor growth of B16F10 tumor–bearing mice treated with LNP@PRO P , LNP@PRO B , or LNP@PRO P+B . n = 7 biologically independent samples. ( G ) Survival of B16F10 tumor–bearing mice treated with LNP@PRO P , LNP@PRO B , or LNP@PRO P+B . ( H ) Immunofluorescence staining of BRD4, PARP1, PD-L1, and γ-H2AX expression levels in B16F10 tumor after indicated treatment. ( I ) WB analysis of cGAS, p-STING, and p-IRF3 protein levels in B16F10 tumor after indicated treatment. ( J ) Schematic illustration of the timeline for 4T1/CT26 tumor inoculation and treatments in a BALB/C mouse model. Tumor growth ( K ) and survival ( L ) of 4T1 tumor–bearing mice treated with LNP@PRO P , LNP@PRO B , or LNP@PRO P+B . n = 7 biologically independent samples. Tumor growth ( M ) and survival ( N ) of CT26 tumor–bearing mice treated with PBS (G1), LNP@PRO P (G2), LNP@PRO B (G3), or LNP@PRO P+B (G4). n = 7 biologically independent samples. Results are presented as the means ± SEM. ( O ) Schematic illustration of the timeline for B16F10 tumor inoculation and treatments in a NCG mouse model. ( P ) Tumor growth of B16F10 tumor–bearing mice treated with PBS or LNP@PRO P+B . n = 6 biologically independent samples. ( Q ) Photographs of the excised tumors. ( R ) Tumor weights of different groups after treatments. ( S ) Tumor inhibition of different groups after treatments. Results are presented as the means ± SD.

Article Snippet: B16F10, 4T1, H22, KPC, and CT26 cell lines were obtained from American Type Culture Collection.

Techniques: Concentration Assay, Incubation, Clonogenic Assay, Immunofluorescence, Staining, Expressing, Inhibition

Journal: iScience

Article Title: Fluorescence tracking Treg movement identifies anti-CCR8 and radiation as a therapeutic combination

doi: 10.1016/j.isci.2025.114572

Figure Lengend Snippet: Combination RT and anti-CCR8 reduces tumor burden and improve survival in distant non-irradiated tumors BALB/c mice were injected with CT26 on both flanks. Tumor-bearing mice were treated with a “pre” treatment strategy. Mice were injected i.p. with anti-CCR8 on days 7, 10, and 14 and treated with 12 Gy RT to the right flank tumor (treated) on day14 post-tumor inoculation. The left tumor (untreated) did not receive RT. Tumor growth and survival were assessed. A total of 8 mice were analyzed per group. Data are represented as mean ± SD. Statistics for survival plots were performed using a Mantel-Cox test between groups. ns = not significant, ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: The CT26 murine colorectal carcinoma cell line was purchased from ATCC (CRL-2638).

Techniques: Irradiation, Injection

Journal: Cell Reports Medicine

Article Title: DF6215, an α-optimized IL-2-Fc fusion, expands immune effectors and drives robust preclinical anti-tumor activity

doi: 10.1016/j.xcrm.2025.102518

Figure Lengend Snippet: DF6215 mediates robust lymphocyte expansion and modulates the tumor microenvironment, resulting in potent anti-tumor activity (A) Kinetics of Ki-67 + immune subset counts in the blood after human (h)IgG1 isotype control or DF6215 administration (0.23 mg/kg in light blue and 1.35 mg/kg in dark blue) in naive BALB/c mice ( n = 3/group). Data represent the mean ± SEM. (B) Efficacy of DF6215 in the mouse CT26 tumor model. Individual tumor volumes, per caliper measurements, are shown with treatment days and frequencies as indicated (vertical dotted lines, QW schedule). The number of complete responders (CRs) in each DF6215 treatment group is noted within each image. n = 10/group. Kaplan-Meier survival curves are shown, with the median survival indicated in brackets (log rank Mantel-Cox test: ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001). Top: mice were enrolled into treatment groups on day 11, when tumors averaged 218 mm 3 in size, and once weekly treatment began. Bottom: mice were enrolled into treatment groups on day 12, when tumors averaged 181 mm 3 in size, and once weekly treatment began. See also . (C and D) BALB/c mice ( n = 10/group) were engrafted with CT26 and treated i.p. with a single dose of DF6215 (0.675 mg/kg) or hIgG1 isotype after mice were enrolled into treatment groups when tumors averaged ∼218 mm 3 in size. (C) (Left) Absolute cell count quantification of indicated immune subsets in the tumor microenvironment 7 days post-treatment. Data shown are the mean ± SEM. (Right) Immune subset counts in tumors 96 h post-treatment. Significance for DF6215 relative to isotype by unpaired t test was noted as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, and ns, not significant. (D) Cytokines in blood as assessed by a Luminex proinflammatory panel 24 h post-treatment. Significance for DF6215 relative to isotype by one-way ANOVA with Tukey’s test is noted as ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, and ns, not significant.

Article Snippet: The mouse melanoma cell line B16F10 and colon carcinoma cell line CT26 were obtained from ATCC.

Techniques: Activity Assay, Control, Cell Characterization, Luminex

Journal: Cell Reports Medicine

Article Title: DF6215, an α-optimized IL-2-Fc fusion, expands immune effectors and drives robust preclinical anti-tumor activity

doi: 10.1016/j.xcrm.2025.102518

Figure Lengend Snippet: DF6215 treatment augments anti-tumor activity compared to a non-⍺ IL-2-Fc (A) Efficacy of DF6215 monotherapy vs. non-⍺ IL-2 Fc monotherapy administered i.p. in the mouse CT26 tumor model. Individual tumor volumes are shown with treatment days and frequencies as indicated (vertical dotted lines). The number of complete responders (CRs) in each treatment group is noted in each image. N = 10/group. Kaplan-Meier survival curves are shown, with the median survival indicated in brackets (∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05; log rank Mantel-Cox test). Some of the DF6215 monotherapy data presented in to illustrate the dose response are included here to allow direct comparison with the non-α-binding IL-2-Fc, which was included as a comparator in the same experiment. See also . (B–D) BALB/c mice ( n = 10/group) were engrafted with CT26 and treated i.p. with a single dose of hIgG1 isotype, DF6215 (0.675 mg/kg), or non-⍺ IL-2-Fc (0.675 mg/kg). (B) The effector cell ratios of cells in the tumor microenvironment 96 h post-treatment (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; one-way ANOVA with Tukey’s test). (C) Frequencies of CD69 + NK and CD8 + T cells in the tumor microenvironment 48 h post-treatment. (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; one-way ANOVA with Tukey’s test). (D) Frequencies of granzyme B + NK cells and CD8 + T cells in the tumor microenvironment 48 h post-treatment (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; one-way ANOVA with Tukey’s test).

Article Snippet: The mouse melanoma cell line B16F10 and colon carcinoma cell line CT26 were obtained from ATCC.

Techniques: Activity Assay, Comparison, Binding Assay

Journal: Biomacromolecules

Article Title: Lopinavir Derivative as Potent P‑gp Inhibitor Enables Delivery through HPMA Copolymer Conjugates and Overcoming Tumor Chemoresistance to Conventional Cytostatic Drugs

doi: 10.1021/acs.biomac.5c02097

Figure Lengend Snippet: LD and P-LD cause potent sensitization of P-gp-expressing cancer cells to the cytostatic activity of free and HPMA copolymer–bound conventional cytostatic drugs, respectively. Sensitization of P388/MDR, CT26, and SCC7 cells to the cytostatic activity of DOX or DTX in the presence of LD at various constant concentrations (A) or to that of P-DOX or P-DTXD at various constant concentrations of P-LD (B) after 72 h of incubation. [ 3 H]-thymidine incorporation assay was employed herein. Concentrations shown in experiments with polymer conjugates represent free drug equivalents. Proliferation of cells exposed to the test drugs relative to those exposed to the same concentration of only LD or P-LD have been plotted. IC 50 values for each cytostatic drug in the absence or presence of LD or P-LD are presented in brackets. Each data point represents the mean ± SD of the tetraplicate samples. Each experiment was conducted at least twice, and similar results were obtained.

Article Snippet: The CT26 mouse colon adenocarcinoma cell line (catalog no. CRL-2638, RRID: CVCL_7256) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated in RPMI-1640 medium (Sigma-Aldrich, Czech Republic) supplemented with heat activated fetal bovine serum (10%), 100 U/mL of penicillin-streptomycin solution, 1 mM sodium pyruvate, 4.5 g/L of glucose, and 10 mM HEPES.

Techniques: Expressing, Activity Assay, Incubation, Thymidine Incorporation Assay, Polymer, Concentration Assay

Journal: Biomacromolecules

Article Title: Lopinavir Derivative as Potent P‑gp Inhibitor Enables Delivery through HPMA Copolymer Conjugates and Overcoming Tumor Chemoresistance to Conventional Cytostatic Drugs

doi: 10.1021/acs.biomac.5c02097

Figure Lengend Snippet: LD and P-LD considerably potentiate the induction of apoptosis in cancer cells expressing P-gp by conventional cytostatic drugs and their polymer-bound counterparts, respectively. Induction of apoptosis in P388/MDR (A, G) and CT26 (B, C, H, and I) cells was determined by annexin V–Dy647/Hoechst 33258 staining followed by flow cytometry analysis. The cells were incubated with 10 or 1 μM DOX (A and B, respectively) or 120 μM DTX (C) alone or in combination with the indicated concentrations of LD for 48 h. The cells were incubated with 80 or 8 μM (G and H, respectively) of P-DOX or 150 μM P-DTXD (I) alone or in combination with the indicated concentrations of P-LD for 48 h. Untreated cells (control) and cells treated with the corresponding concentrations of LD or P-LD alone were included as controls. Representative dot plots from one of at least two independent experiments, each with triplicate samples, which yielded similar results are shown. Caspase-3 activity was determined for titrated concentrations of DOX in P388/MDR (D) and CT26 (E) cells and titrated concentrations of DTX in CT26 cells (F) alone or in combination with 2 μM LD after 48 h of incubation via a fluorescence-based assay for detecting the activity of activated caspase-3. Caspase-3 activity was determined for titrated concentrations of P-DOX in P388/MDR (J) and CT26 (K) cells and for titrated concentrations of P-DTXD in CT26 cells (L) alone or combined with 4 μM P-LD after 48 h of incubation. For the control group, the cells were incubated with only culture medium. Concentrations are represented as drug equivalents in experiments involving polymer conjugates. Each bar showing the amount of released AMC represents the mean ± SD of triplicate samples. Experiments were conducted at least twice and yielded similar results. Statistically significant differences between compared compounds evaluated via unpaired two-tailed Student’s t -test are represented by *, **, and *** denoting P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively.

Article Snippet: The CT26 mouse colon adenocarcinoma cell line (catalog no. CRL-2638, RRID: CVCL_7256) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated in RPMI-1640 medium (Sigma-Aldrich, Czech Republic) supplemented with heat activated fetal bovine serum (10%), 100 U/mL of penicillin-streptomycin solution, 1 mM sodium pyruvate, 4.5 g/L of glucose, and 10 mM HEPES.

Techniques: Expressing, Polymer, Staining, Flow Cytometry, Incubation, Control, Activity Assay, Fluorescence, Two Tailed Test

Journal: Biomacromolecules

Article Title: Lopinavir Derivative as Potent P‑gp Inhibitor Enables Delivery through HPMA Copolymer Conjugates and Overcoming Tumor Chemoresistance to Conventional Cytostatic Drugs

doi: 10.1021/acs.biomac.5c02097

Figure Lengend Snippet: P-LD improves the antitumor efficacy of P-DOX in mouse tumor models of induced and intrinsic MDR without increasing toxicity. The combination of P-LD with P-DOX significantly inhibited tumor growth and improved survival in mice bearing P388/MDR-derived tumors. DBA/2 mice ( n = 8) were subcutaneously (s.c.) administered 2.5 × 10 5 P388/MDR cells on day 0. P-LD (100 mg LD/kg per dose, i.p.) and P-DOX (25 mg DOX/kg per dose, i.v.) were administered on days 6, 9, and 12. Toxicity (A), tumor growth (B), and survival of the experimental mice (C) were recorded. The combination of P-LD with P-DOX significantly inhibited tumor growth and improved survival in mice bearing CT26-derived tumors. BALB/c mice ( n = 8; n = 9 for the combination treatment group) were administered 2 × 10 5 CT26 cells on day 0. P-LD (120 mg LD/kg per dose, i.p.), P-DOX (30 mg DOX/kg per dose, i.v.), and DOX (5 mg/kg per dose, i.v.) were administered on days 7, 10, and 13. Toxicity (D), tumor growth (E), and survival of the experimental mice (F) were recorded. P-DOX was administered 1 h following the administration of P-LD in all of the experiments. Mice in the control group were injected with the same volume (250 μL) of phosphate buffered saline. Unpaired two-tailed Student’s t -test and Mantle–Cox log-rank test were employed for analyzing the statistical significance of the data, which has been indicated by *, **, and *** (denoting P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively). MS denotes the mean survival in days. Experiments were done twice with comparable results.

Article Snippet: The CT26 mouse colon adenocarcinoma cell line (catalog no. CRL-2638, RRID: CVCL_7256) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated in RPMI-1640 medium (Sigma-Aldrich, Czech Republic) supplemented with heat activated fetal bovine serum (10%), 100 U/mL of penicillin-streptomycin solution, 1 mM sodium pyruvate, 4.5 g/L of glucose, and 10 mM HEPES.

Techniques: Derivative Assay, Control, Injection, Saline, Two Tailed Test