Journal: Frontiers in Immunology

Article Title: STAT6 inhibition of M2 macrophages suppresses tumor growth by modulating the tumor microenvironment in colon cancer model

doi: 10.3389/fimmu.2026.1733991

Figure Lengend Snippet: TAMpep-IP suppresses tumor growth in the colon cancer model. (A) BALB/c mice were subcutaneously inoculated with CT26 colon carcinoma cells (3 × 10 5 cells per mouse). Starting on day 7 post-inoculation, TAMpep-IP (400 nmol/kg) was administered subcutaneously every three days for a total of seven doses. (B) Representative images of tumors excised at the experimental endpoint (day 25) showed visibly reduced tumor size in the TAMpep-IP–treated group compared to control. (C) Tumor volumes were measured every 3 days following tumor implantation. Mice treated with TAMpep-IP exhibited significantly reduced tumor growth relative to the control group (control: n = 6; TAMpep-IP: n = 6). (D) Tumor proliferation was evaluated by immunohistochemical staining of Ki-67 in tumor sections. Quantitative analysis showed a significantly lower proportion of Ki-67 + proliferating cells in TAMpep-IP–treated tumors. Representative immunohistochemistry images were acquired at ×100 magnification. Scale bar = 1000 μm. All data are presented as mean ± SEM. *p<0.05, ***p<0.001.

Article Snippet: The murine colon carcinoma cell line CT26 (KCLB 80009; Korean Cell Line Bank, Seoul, Korea) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (D5671; Welgene, Gyeongsangbuk, Korea) supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 1% penicillin–streptomycin.

Techniques: Control, Tumor Implantation, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: STAT6 inhibition of M2 macrophages suppresses tumor growth by modulating the tumor microenvironment in colon cancer model

doi: 10.3389/fimmu.2026.1733991

Figure Lengend Snippet: TAMpep-IP reduces M2 macrophages in tumor tissues of colon cancer model. (A) Tumor-infiltrating immune cells were isolated from CT26 tumors in control and TAMpep-IP–treated mice. Flow cytometry was used to identify CD206 + F4/80 + macrophages within the CD45 + CD11b + population. TAMpep-IP significantly decreased the proportion of M2-like tumor-associated macrophages. (B) Quantitative RT-PCR analysis of tumor tissues revealed that TGF-β mRNA expression, a key M2-associated cytokine, was significantly reduced in TAMpep-IP–treated tumors compared to controls. (C) Western blot analysis of tumor showed a marked decrease in CD206 protein levels following TAMpep-IP, indicating effective suppression of M2 macrophage markers. (D) CD206 + macrophages were further visualized by immunohistochemical staining of tumor sections. ImageJ-based quantification confirmed a significant reduction in CD206 + area in TAMpep-IP–treated tumors. Representative immunohistochemistry images were acquired at ×100 magnification. Scale bar = 1000 μm. All data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The murine colon carcinoma cell line CT26 (KCLB 80009; Korean Cell Line Bank, Seoul, Korea) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (D5671; Welgene, Gyeongsangbuk, Korea) supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 1% penicillin–streptomycin.

Techniques: Isolation, Control, Flow Cytometry, Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: STAT6 inhibition of M2 macrophages suppresses tumor growth by modulating the tumor microenvironment in colon cancer model

doi: 10.3389/fimmu.2026.1733991

Figure Lengend Snippet: TAMpep-IP enhances inflammatory cytokine expression and activated CD8 + T cells in tumor tissues of colon cancer model. (A) Flow cytometry was used to evaluate CD8 + T cell function in CT26 tumor tissues from control and TAMpep-IP-treated mice. TAMpep-IP significantly increased the proportion of activated Granzyme B + CD8 + T cells, while reducing the frequency of exhausted Tim-3 + CD8 + T cells, indicating enhanced cytotoxic T cell activity. (B, C) Confocal immunofluorescence analysis was performed on tumor sections stained with DAPI (nuclei), anti-CD8 (green), anti-Granzyme B (red), and anti-PD-1 (red). Activated CD8 + T cells were identified by co-localization of CD8 and Granzyme B, whereas exhausted CD8 + T cells were identified by co-localization of CD8 and PD-1. Quantification revealed a significant increase in intertumoral CD8 + Granzyme B + T cells and a concomitant decrease in CD8 + PD-1 + exhausted T cells following TAMpep-IP. Representative confocal images were acquired using a 40× objective lens. Scale bar = 20 μm. (D) Quantitative RT-PCR analysis of CT26 tumor tissues showed significantly elevated mRNA levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-12 in TAMpep-IP–treated mice compared to controls, indicating induction of a pro-inflammatory tumor microenvironment. All data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p < 0.0001.

Article Snippet: The murine colon carcinoma cell line CT26 (KCLB 80009; Korean Cell Line Bank, Seoul, Korea) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (D5671; Welgene, Gyeongsangbuk, Korea) supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 1% penicillin–streptomycin.

Techniques: Expressing, Flow Cytometry, Cell Function Assay, Control, Activity Assay, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Long-chain Polyunsaturated Fatty Acids Mitigate In Vitro Skeletal Muscle Wasting Induced by Colorectal Carcinoma Cells via a 15-LOX-dependent Pathway

doi: 10.64898/2026.05.22.726995

Figure Lengend Snippet: (A) : Day 3 post-differentiation C2C12 myotubes were treated with fresh DM, CT26 CM, DM containing 50 µM NDGA, or 10 µM BLX-3887 for 72 hours. C2C12 myotubes were fixed and stained for sarcomeric myosin (MF20c, green) and myogenin (F5Dc, red). Nuclei was counter stained by DAPI (blue). (B) : Myotube diameter for C2C12 myotubes was quantified as described in Methods. Groups labeled with different letters are significantly different from one another, while groups sharing a common letter are not significantly different.

Article Snippet: The murine skeletal muscle cell line C2C12 (ATCC, CRL-1772) and colorectal carcinoma cell line CT26 (ATCC, CRL-2638) were cultured separately in growth media (GM) consisting of Dulbecco’s modified Eagle medium (DMEM, Gibco, 11995-065) containing 10% fetal bovine serum (FBS, Corning, 35-010-CV) and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL, Gibco, 15140-122) at 37 °C in cell incubator with 5% CO 2 .

Techniques: Staining, Labeling

Journal: bioRxiv

Article Title: Long-chain Polyunsaturated Fatty Acids Mitigate In Vitro Skeletal Muscle Wasting Induced by Colorectal Carcinoma Cells via a 15-LOX-dependent Pathway

doi: 10.64898/2026.05.22.726995

Figure Lengend Snippet: Day 3 post-differentiation C2C12 myotubes were treated with CT26 conditioned media (CM) with or without the pan-LOX inhibitor nordihydroguaiaretic acid (NDGA) (50 µM) or the 15-LOX specific inhibitor BLX3887 (10 µM). Then, individual LC-PUFAs including ARA, EPA, DHA, and DPA were spiked into the wells to reach a final concentration of 25 µM. After 72 hours, C2C12 myotubes were fixed in 4% paraformaldehyde (PFA) in preparation for immunocytochemistry analysis. (A) C2C12 myotubes were stained for sarcomeric myosin (MF20c, green) and myogenin (F5Dc, red). Nuclei was counter stained by DAPI (blue). (B-C) Myotube diameter for C2C12 myotubes with or without NDGA (B) or BLX-3887 (C) was quantified as described in Methods. Scale bar is 200 μm. *P < 0.05 for difference of PUFA treatments vs. vehicle, and #P < 0.05 for difference between respective LC-PUFA treatments with or without LOX inhibitors. (D-E) C2C12 myoblasts were differentiated for 3 days and then exposed to CT26 CM and individual PUFAs including ARA, EPA, DPA, and DHA for 3 hours. RNA was extracted from mature myotubes. mRNA expression fold change of Il6 (D) and Ccl2 (E) in response to CT26 CM and PUFA treatments was shown. *P < 0.05 for effects of PUFA treatments, and #P < 0.05 for difference between DM and CT26 CM.

Article Snippet: The murine skeletal muscle cell line C2C12 (ATCC, CRL-1772) and colorectal carcinoma cell line CT26 (ATCC, CRL-2638) were cultured separately in growth media (GM) consisting of Dulbecco’s modified Eagle medium (DMEM, Gibco, 11995-065) containing 10% fetal bovine serum (FBS, Corning, 35-010-CV) and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL, Gibco, 15140-122) at 37 °C in cell incubator with 5% CO 2 .

Techniques: Concentration Assay, Immunocytochemistry, Staining, Expressing

Journal: bioRxiv

Article Title: Long-chain Polyunsaturated Fatty Acids Mitigate In Vitro Skeletal Muscle Wasting Induced by Colorectal Carcinoma Cells via a 15-LOX-dependent Pathway

doi: 10.64898/2026.05.22.726995

Figure Lengend Snippet: Co-cultures of mature C2C12 myotubes and CT26 carcinoma cells were treated with individual LC-PUFAs including ARA, EPA, DPA, and DHA (25 µM). After 72 hours, conditioned media from the lower compartments was collected and C2C12 myotubes were fixed and stained for sarcomeric myosin (MF20c, green) and myogenin (F5Dc, red). Nuclei was counter stained by DAPI (blue) (A) . Myotube diameter for C2C12 myotubes in the base plates was quantified as described in Methods (B) . Scale bar is 200 μm. Groups labeled with different letters are significantly different from one another, while groups sharing a common letter are not significantly different. (C- G) Conditioned media from C2C12-CT26 co-culture was analyzed by targeted Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) to profile lipid metabolites in response to PUFA treatments. (C) : A heatmap of the top 50 most differentially regulated lipid mediators detected in conditioned culture media samples from C2C12 myotubes. (D-G) : Concentration of representative lipid mediator metabolites downstream of arachidonic acid (ARA) (e.g., PGE 2 , 15-HETE, and LXA 4 ) (D) , EPA (e.g., 15-HEPE, RvE1) (E), DPA (e.g., RvD5 n-3 DPA ) (F) , and DHA (e.g., 14-HDoHE, MaR2, 17-HDoHE, and RvD1) (G) . Bars show the mean ± SEM of media from 3 wells (biological replicates). P-values were determined by two-tailed unpaired t-tests. ∗p < 0.05, **p<0.01, ***p<0.001, and ****p<0.0001 vs. C2C12 myotubes without PUFAs or CT26 inserts.

Article Snippet: The murine skeletal muscle cell line C2C12 (ATCC, CRL-1772) and colorectal carcinoma cell line CT26 (ATCC, CRL-2638) were cultured separately in growth media (GM) consisting of Dulbecco’s modified Eagle medium (DMEM, Gibco, 11995-065) containing 10% fetal bovine serum (FBS, Corning, 35-010-CV) and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL, Gibco, 15140-122) at 37 °C in cell incubator with 5% CO 2 .

Techniques: Staining, Labeling, Co-Culture Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Long-chain Polyunsaturated Fatty Acids Mitigate In Vitro Skeletal Muscle Wasting Induced by Colorectal Carcinoma Cells via a 15-LOX-dependent Pathway

doi: 10.64898/2026.05.22.726995

Figure Lengend Snippet: Concentration of lipid mediator metabolites downstream of arachidonic acid (ARA) (e.g., 5-HETE, 6-keto-PGF 1α , PGD 2 , and PGF 2α ) (A) , EPA (e.g., 18-HEPE, RvE2) (B), DPA (e.g., MaR1 n-3 DPA , PD1 n- 3 DPA ) (C) , and DHA (e.g., 4-HDoHE, 7-HDoHE, PD1, PDX, RvD2, RvD3, RvD5, RvD6, AT-RvD3, AT-RvD6) (D) Bars show the mean ± SEM of media from 3 wells (biological replicates). P-values were determined by two- tailed unpaired t-tests. ∗p < 0.05, **p<0.01, ***p<0.001, and ****p<0.0001 vs. C2C12 myotubes without PUFAs or CT26 inserts.

Article Snippet: The murine skeletal muscle cell line C2C12 (ATCC, CRL-1772) and colorectal carcinoma cell line CT26 (ATCC, CRL-2638) were cultured separately in growth media (GM) consisting of Dulbecco’s modified Eagle medium (DMEM, Gibco, 11995-065) containing 10% fetal bovine serum (FBS, Corning, 35-010-CV) and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL, Gibco, 15140-122) at 37 °C in cell incubator with 5% CO 2 .

Techniques: Concentration Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Long-chain Polyunsaturated Fatty Acids Mitigate In Vitro Skeletal Muscle Wasting Induced by Colorectal Carcinoma Cells via a 15-LOX-dependent Pathway

doi: 10.64898/2026.05.22.726995

Figure Lengend Snippet: (A) : Day 3 post-differentiation C2C12 myotubes were treated with 100 ng/mL TNFα (top) or CT26 CM (bottom) in the presence or absence of 100 nM of individual mature SPMs including RvD1, RvD5, MaR1, PD1, RvE1, LXA 4 , and RvD2 n-3 DPA for 72 hours. Resulting C2C12 myotubes were fixed and stained for sarcomeric myosin (MF20c, green) and myogenin (F5Dc, red). Nuclei was counter stained by DAPI (blue). (B- C) Myotube diameter for C2C12 myotubes receiving TNFα (B) or CT26 CM (C) was quantified as described in Methods. Scale bar is 200 μm. *P < 0.05 for difference compared to C2C12 myotubes receiving TNFα or CT26 CM alone. (D-E) C2C12 myoblasts were differentiated for 3 days and exposed to TNFα (100 ng/mL) with or without individual SPMs including RvD1, RvD5, MaR1, PD1, RvE1, LXA 4 , and RvD2 n-3 DPA (100nM) for 3 hours, after which RNA was extracted from mature myotubes. mRNA expression was determined by RT-qPCR and fold change of Il6 (D) and Fbxo32 (E) in response to TNFα exposure and SPM treatments was shown. *P < 0.05 for difference compared to C2C12 myotubes receiving TNFα.

Article Snippet: The murine skeletal muscle cell line C2C12 (ATCC, CRL-1772) and colorectal carcinoma cell line CT26 (ATCC, CRL-2638) were cultured separately in growth media (GM) consisting of Dulbecco’s modified Eagle medium (DMEM, Gibco, 11995-065) containing 10% fetal bovine serum (FBS, Corning, 35-010-CV) and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL, Gibco, 15140-122) at 37 °C in cell incubator with 5% CO 2 .

Techniques: Staining, Expressing, Quantitative RT-PCR