Journal: Molecular Therapy Oncology

Article Title: Recombinant CALR polarizes and activates macrophages in tumors

doi: 10.1016/j.omton.2025.201121

Figure Lengend Snippet: Recombinant CALR reduces tumor growth and increases M1 macrophages (A) To measure the effect on tumor growth and immune cell activation, bacterial lysate was injected into tumor-bearing mice. Tumors were formed by subcutaneously injecting CT26 murine colon carcinoma cells into the flank of BALB/c mice. After 2 weeks, the mice were injected intratumorally with saline, bacterial control lysate, or lysate from CALR-expressing bacteria. One set of mice received injections at days 0, 3, and 6; were monitored for tumor growth; and their tumors were harvested at day 9 for analysis of immune cells. A second set of mice received only one injection at day 0, and tumors were harvested at day 3 for analysis. (B) Intratumoral injection of CALR lysate decreased tumor growth compared to saline controls ( p = 0.0089). Bacterial control lysate also reduced tumor growth compared to controls ( p = 0.0204). Volumes are reported relative to those on day 0. (C–F) On day 3, recombinant CALR did not affect (C) the number of leukocytes, (D) the number of M1 macrophages (per 10,000 cells analyzed), or the number of M1 macrophages expressing either (E) CD80 or (F) CD86 (per 10,000 cells analyzed) in tumors. (G) On day 9, injection with CALR lysate significantly increased the number of leukocytes in tumors compared to saline controls ( p = 0.0042). (H) On day 9, CALR lysate also significantly increased the number of M1 macrophages in tumors compared to bacterial controls ( p = 0.0480) and saline ( p = 0.0061). (I) CALR lysate significantly increased the number of M1 macrophages expressing CD80 (per 10,000 cells analyzed) compared to saline controls ( p = 0.0063). (J) CALR lysate also increased the number of M1 macrophages expressing CD86 (per 10,000 cells analyzed) compared to bacterial controls ( p = 0.0445) and saline ( p = 0.0077). Data are represented as mean ± SEM. The statistical comparisons in (B) are two-way ANOVA followed by Tukey’s method. The statistical comparisons in (C–J) are ANOVA followed by Tukey’s method. Asterisks indicate significance: ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: JAWSII murine dendritic cells and CT26 murine colon carcinoma cells were obtained from ATCC, confirmed by STR profiling by Charles River Research Animal Diagnostic Services, and passaged for fewer than 6 months.

Techniques: Recombinant, Activation Assay, Injection, Saline, Control, Expressing, Bacteria

Journal: Molecular Therapy Oncology

Article Title: Recombinant CALR polarizes and activates macrophages in tumors

doi: 10.1016/j.omton.2025.201121

Figure Lengend Snippet: Recombinant CALR increases helper T cell activity in tumors (A) The extent of T cell infiltration was determined in mice (see ) with CT26 tumors that were intratumorally injected (on days 0, 3, and 6) with saline (PBS), bacterial control lysate (BC), or lysate from CALR-expressing bacteria (CALR). On day 9, injection of CALR lysate significantly increased the number of T cells in tumors (per 10,000 cells analyzed) compared to saline controls ( p = 0.0495). (B) The percentage of helper T cells per 10,000 cells analyzed. (C) On day 9, injection with CALR lysate significantly increased the number of activated helper T cells (per 10,000 cells analyzed) in tumors compared to saline controls ( p = 0.0067). Data are represented as mean ± SEM. The statistical comparisons in (A–C) are ANOVA followed by Dunnett’s test. Asterisks indicate significance: ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: JAWSII murine dendritic cells and CT26 murine colon carcinoma cells were obtained from ATCC, confirmed by STR profiling by Charles River Research Animal Diagnostic Services, and passaged for fewer than 6 months.

Techniques: Recombinant, Activity Assay, Injection, Saline, Control, Expressing, Bacteria

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: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Ellagic Acid Alleviates Abnormal Fat Reduction by Activating the RXRβ–PPARγ Pathways in a CT26 Tumour‐Induced Cachexia Mouse Model

doi: 10.1002/jcsm.70176

Figure Lengend Snippet: EA restores reduced adipocyte function in CM‐stimulated white adipocytes. (A) The experimental scheme for the preparation of the CT26 CM is shown. (B) IL‐6 levels were measured in CT26 CM with ELISA kits ( n = 3). (C) Intracellular lipid droplets were stained with Oil Red O (magnification 400×, scale bar 75 = μm). (D) The quantification of intracellular lipid was detected at 500 nm in 3T3‐L1 cells differentiated into white adipocytes, and treated with 50% of CT26 CM or EA (3.1, 6.3 and 12.5 μM) ( n = 3). (E) The levels of intracellular and extracellular free fatty acids were measured in 3T3‐L1 cells differentiated into white adipocytes, and treated with 50% of CT26 CM or EA (6.3 and 12.5 μM) ( n = 3). (F, G) The expression of adipokines was analysed using a Mouse Adipokine Proteome Array kit. (H) The protein expression of IGFBP‐3 and lipocalin‐2 was analysed with ImageJ ( n = 2). All data are expressed as the mean ± SEM. Statistical significance was determined using a one‐way ANOVA with Tukey's post hoc test for multigroup comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001 or **** p < 0.0001 were considered statistically significant. CM, conditioned medium. DM (Wh), differentiation medium. EA, ellagic acid.

Article Snippet: Briefly, all mice were first randomized by body weight and divided into a non‐tumour‐bearing vehicle group, which received a subcutaneous injection of PBS, and a tumour‐induction group, which was injected with 5 × 10 5 CT26 colon cancer cells (CRL‐2638, ATCC, Rockville, MD, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Expressing

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Ellagic Acid Alleviates Abnormal Fat Reduction by Activating the RXRβ–PPARγ Pathways in a CT26 Tumour‐Induced Cachexia Mouse Model

doi: 10.1002/jcsm.70176

Figure Lengend Snippet: EA‐mediated RXRβ‐PPARγ axis activation mitigates the reduction of adipogenesis by CT26 CM. (A) Overview of the crystal structure of the complex between RXRB (PDB ID: 7A78) and EA, and expected intermolecular interactions are shown. (B) Relative mRNA expression of Rxrb was measured by RT‐PCR in 3T3‐L1 differentiated into white adipocytes with or without si Rxrb , and data are normalized to Gapdh ( n = 3). (C) Representative morphological images of 3T3‐L1 treated with siRxrb and/or EA are shown (magnification 400×, scale bar = 75 μm). (D) The lipid droplets were stained with BODIPY‐Green (magnification 1000×, scale bar = 25 μm). Lipid droplet sizes and area were measured using the ImageJ software ( n = 3). (E) Protein expression of PPARγ and ACC was measured by Western blot analysis. (F) Intensities of the protein bands were measured with ImageJ and normalized to β‐actin ( n = 6). All data are expressed as the mean ± SEM. Statistical significance was determined using a one‐way ANOVA with Tukey's post hoc test for multigroup comparisons and the non‐parametric Mann–Whitney U test for two‐group comparisons. * p < 0.05, ** p < 0.01 or *** p < 0.001 were considered statistically significant. CM, conditioned medium. DM (Wh), differentiation medium. EA, ellagic acid.

Article Snippet: Briefly, all mice were first randomized by body weight and divided into a non‐tumour‐bearing vehicle group, which received a subcutaneous injection of PBS, and a tumour‐induction group, which was injected with 5 × 10 5 CT26 colon cancer cells (CRL‐2638, ATCC, Rockville, MD, USA).

Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Software, Western Blot, MANN-WHITNEY

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Ellagic Acid Alleviates Abnormal Fat Reduction by Activating the RXRβ–PPARγ Pathways in a CT26 Tumour‐Induced Cachexia Mouse Model

doi: 10.1002/jcsm.70176

Figure Lengend Snippet: EA protects against body weight loss in CT26 tumour‐induced cachectic mice. (A) The experimental scheme of the in vivo study is shown. BALB/c mice were subcutaneously inoculated with 5 × 10 5 CT26 cells (CT26 group), except for the vehicle group. EA administration (10 mg/kg) via oral gavage was started 1 week after tumour cell injection (CT26 + EA group). 0.9% Normal saline (vehicle group and CT26 group) or EA (CT26 + EA group) was fed five times per week for 2 weeks. (B) The tumour‐free weight was calculated by subtracting the isolated tumour weight from the body weight ( n = 4). (C) The combined bilateral weight of the iWAT is shown ( n = 4). (D) The H&E‐stained image of the iWAT (magnification 400×, scale bar = 75 μm) is shown, and lipid droplet sizes were calculated using the ImageJ software. (E) The protein levels of C/EBPα, PPARγ, and pACC and ACC were analysed by Western blot analysis. Signal intensities of the protein bands were measured with ImageJ and normalized to β‐actin ( n = 4). (F) The paraffin‐embedded iWAT was stained with SREBP1 (green) and DAPI (blue) (magnification 1000×, scale bar = 25 μm), and representative images are shown. Fluorescence intensity of SREBP1 was quantified using the ImageJ software ( n = 4). Data are expressed as the mean ± SEM. Statistical significance was determined using a one‐way ANOVA with Tukey's post hoc test for multigroup comparisons. * p < 0.05, ** p < 0.01 or *** p < 0.001 were considered statistically significant. EA, ellagic acid. eWAT, epididymal white adipose tissue. iWAT, inguinal white adipose tissue.

Article Snippet: Briefly, all mice were first randomized by body weight and divided into a non‐tumour‐bearing vehicle group, which received a subcutaneous injection of PBS, and a tumour‐induction group, which was injected with 5 × 10 5 CT26 colon cancer cells (CRL‐2638, ATCC, Rockville, MD, USA).

Techniques: In Vivo, Injection, Saline, Isolation, Staining, Software, Western Blot, Fluorescence

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Ellagic Acid Alleviates Abnormal Fat Reduction by Activating the RXRβ–PPARγ Pathways in a CT26 Tumour‐Induced Cachexia Mouse Model

doi: 10.1002/jcsm.70176

Figure Lengend Snippet: EA increases the expression of RXRβ in the iWAT of the CT26 cachexia model. (A) The tumour‐free weight was measured ( n = 7), and the percentage of fat in the total body was measured with DEXA analysis ( n = 3). (B) The combined bilateral weight of the iWAT and eWAT is shown ( n = 7). (C) The combined bilateral weight of TA was measured, and grip strength was measured ( n = 6–7). (D) The paraffin‐embedded iWAT was stained with RXRβ (green) and DAPI (blue) (magnification 1000×, scale bar = 25 μm), and representative images are shown. The bottom panels show zoomed views of the boxed areas in the top panels. (E) Index of correlation (IC) between RXRβ and nuclear (DAPI) was measured with the Colocalization Colormap plugin using ImageJ ( n = 4). (F) Schematic of the experimental models and the mechanism of action for EA. Data are expressed as the mean ± SEM. Statistical significance was determined using a one‐way ANOVA with Tukey's post hoc test for multigroup comparisons. * p < 0.05, ** p < 0.01 or *** p < 0.001 were considered statistically significant. EA, ellagic acid. eWAT, epididymal white adipose tissue. iWAT, inguinal white adipose tissue.

Article Snippet: Briefly, all mice were first randomized by body weight and divided into a non‐tumour‐bearing vehicle group, which received a subcutaneous injection of PBS, and a tumour‐induction group, which was injected with 5 × 10 5 CT26 colon cancer cells (CRL‐2638, ATCC, Rockville, MD, USA).

Techniques: Expressing, Staining

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