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R&D Systems anti ccl2 mouse monoclonal antibody
Anti Ccl2 Mouse Monoclonal Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+mouse+ccl2+antibody/pm41832208-257-44-49?v=R%26D+Systems
Average 94 stars, based on 51 article reviews

anti ccl2 mouse monoclonal antibody - by Bioz Stars, 2026-08

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FAs from Adipose Tissues Drive HIF‐1α‐Mediated CCL2 Secretion in Cancer Cells. A) Structural and schematic illustration of PDMS‐3D culture chips. Total 5 × 10 5 cells were seeded onto the chips. For monoculture, only cancer cells were seeded, whereas for coculture, cancer cells and adipocyte‐derived stem cells (ADSCs) were seeded at a 10:1 ratio. Conditioned media (CM) were collected after 48 h from monocultured 3D culture chips (CM: C only) and cocultured 3D culture chips (CM: C+A). B) HIF‐1α protein levels were analyzed by western blotting following 24 h of treatment with each CM. To deplete lipids, the coculture‐derived CM were incubated with activated charcoal for 8 h following collection, as indicated. To restore lipids, 2% lipid mixture (LM) was added to the charcoal stripped coculture‐derived CM. Mean ± SD (n = 3); * , p < 0.05. C) Western blotting was performed to assess HIF‐1α protein levels after 24‐h treatment with 2% LM to cancer cells. Mean ± SD (n = 3); * , p < 0.05. D) A cytokine array was performed with the CM from monocultured cancer cells (CM: C only) and the CM from cancer cells cocultured with ADSCs (CM: C+A). The levels of cytokines regulated by HIF‐1α were compared between monoculture‐derived CM (CM: C only) and coculture‐derived CM (CM: C+A), and the relative increases in coculture‐derived CM (CM: C+A) were visualized in a graph. The mouse breast cancer cells (4T1) were cocultured with mouse ADSCs and human breast cancer cells (MDA‐MB‐231) were cocultured with human ADSCs. E) GFP‐tagged stable cancer cell lines were monocultured or cocultured with ADSCs. The mouse cancer cell line (4T1) was cocultured with mouse ADSCs and human cancer cell lines (MDA‐MB‐231, MCF7, and PC3) were cocultured with human ADSCs. The cocultured cancer cells were sorted as indicated in Figure (Supporting Information). The CCL2 mRNA levels between cocultured cancer cells and monocultured cancer cells were compared by qRT‐PCR. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001. F) Cancer cells were transfected with si‐Con or si‐HIF‐1α and treated with PBS or 2% LM for 48 h. The CCL2 mRNA levels were measured by qRT‐PCR. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001. G) Immunofluorescence (IF) analysis was performed using the indicated antibodies following transfection with si‐Con or si‐HIF‐1α and treatment with PBS or 2% LM for 48 h. Nuclei were stained with DAPI, and F‐actin was stained with Alexa Fluor 488–phalloidin. Scale bar = 20 µm. Mean ± SD (n = 3); ** , p < 0.001; *** , p < 0.0001. H) Diagram of the FA/HIF‐1α/CCL2 axis in cancer.

Journal: Advanced Science

Article Title: Cancer Manipulates Adjacent Adipose Tissue to Exploit Fatty Acids via HIF‐1α/CCL2/PPARα Axis: A Metabolic Circuit to Support Tumor Progression

doi: 10.1002/advs.202515186

Figure Lengend Snippet: FAs from Adipose Tissues Drive HIF‐1α‐Mediated CCL2 Secretion in Cancer Cells. A) Structural and schematic illustration of PDMS‐3D culture chips. Total 5 × 10 5 cells were seeded onto the chips. For monoculture, only cancer cells were seeded, whereas for coculture, cancer cells and adipocyte‐derived stem cells (ADSCs) were seeded at a 10:1 ratio. Conditioned media (CM) were collected after 48 h from monocultured 3D culture chips (CM: C only) and cocultured 3D culture chips (CM: C+A). B) HIF‐1α protein levels were analyzed by western blotting following 24 h of treatment with each CM. To deplete lipids, the coculture‐derived CM were incubated with activated charcoal for 8 h following collection, as indicated. To restore lipids, 2% lipid mixture (LM) was added to the charcoal stripped coculture‐derived CM. Mean ± SD (n = 3); * , p < 0.05. C) Western blotting was performed to assess HIF‐1α protein levels after 24‐h treatment with 2% LM to cancer cells. Mean ± SD (n = 3); * , p < 0.05. D) A cytokine array was performed with the CM from monocultured cancer cells (CM: C only) and the CM from cancer cells cocultured with ADSCs (CM: C+A). The levels of cytokines regulated by HIF‐1α were compared between monoculture‐derived CM (CM: C only) and coculture‐derived CM (CM: C+A), and the relative increases in coculture‐derived CM (CM: C+A) were visualized in a graph. The mouse breast cancer cells (4T1) were cocultured with mouse ADSCs and human breast cancer cells (MDA‐MB‐231) were cocultured with human ADSCs. E) GFP‐tagged stable cancer cell lines were monocultured or cocultured with ADSCs. The mouse cancer cell line (4T1) was cocultured with mouse ADSCs and human cancer cell lines (MDA‐MB‐231, MCF7, and PC3) were cocultured with human ADSCs. The cocultured cancer cells were sorted as indicated in Figure (Supporting Information). The CCL2 mRNA levels between cocultured cancer cells and monocultured cancer cells were compared by qRT‐PCR. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001. F) Cancer cells were transfected with si‐Con or si‐HIF‐1α and treated with PBS or 2% LM for 48 h. The CCL2 mRNA levels were measured by qRT‐PCR. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001. G) Immunofluorescence (IF) analysis was performed using the indicated antibodies following transfection with si‐Con or si‐HIF‐1α and treatment with PBS or 2% LM for 48 h. Nuclei were stained with DAPI, and F‐actin was stained with Alexa Fluor 488–phalloidin. Scale bar = 20 µm. Mean ± SD (n = 3); ** , p < 0.001; *** , p < 0.0001. H) Diagram of the FA/HIF‐1α/CCL2 axis in cancer.

Article Snippet: A monoclonal antibody neutralizing murine CCL2 (nAb‐CCL2) (InVivoMAb anti‐mouse/human/rat CCL2, clone number: 2H5, #BE0185) was purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Derivative Assay, Western Blot, Incubation, Quantitative RT-PCR, Transfection, Immunofluorescence, Staining

CCL2‐Induced PPARα Accumulation Drives Lipolysis and FA Release. A,B) Using the indicated FA quantification kit, we measured the FA concentrations in CM collected from the experiment shown in Figure ,E (Supporting Information). Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001. C) In the GEO dataset comprising tumor‐adjacent adipose tissues from 34 breast cancer patients ( GSE153316 ), gene expression profiles were compared between normal‐weight and obese groups. The groups were classified as follows: Normal (20–24.9 kg m −2 , n=21), Obese (≥25 kg m −2 , n=13). D) ADSCs were treated with CCL2 for 2 h with the indicated concentrations and the protein levels were analyzed by western blotting with indicated antibodies. Mean ± SD (n = 3); * , p < 0.05. E) ADSCs were treated with CCL2 (20 ng mL −1 ) and nAb‐CCL2 (1 µg mL −1 ) for 2 h. Proteins were analyzed by western blotting with indicated antibodies. Mean ± SD (n = 3); * , p < 0.05. F) ADSCs were transfected with PPRE‐Luc plasmid and treated with CCL2 (20 ng mL −1 ) and nAb‐CCL2 (1 µg mL −1 ) for 24 h. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001. G) Lipid accumulation was estimated by Nile red staining. ADSCs were treated with CCL2 (20 ng mL −1 ), nAb‐CCL2 (1 µg mL −1 ), or GW6471 (PPARα inhibitor, 1 µ m ) for 24 h. After staining with Nile Red and DAPI, the cells were visualized by fluorescence microscopy. Scale bar = 100 µm H) FACS analysis of Nile Red stained cells. The graph (right) indicate the mean fluorescence intensity. Mean ± SD (n = 3) *** , p < 0.0001. I) ADSCs were treated with CCL2 (20 ng mL −1 ), nAb‐CCL2 (1 µg mL −1 ), or GW6471 (1 µ m ) for 24 h as indicated. The mRNA levels were quantified by qRT‐PCR. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001. J) FA concentrations in CM collected after 24 h of treatment with CCL2 (20 ng mL −1 ), nAb‐CCL2 (1 µg mL −1 ), or GW6471 (1 µ m ) to ADSCs were measured using the indicated FA quantification kit. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001. K) Diagram of the CCL2/PPARα/FA axis in adipose lipolysis.

Journal: Advanced Science

Article Title: Cancer Manipulates Adjacent Adipose Tissue to Exploit Fatty Acids via HIF‐1α/CCL2/PPARα Axis: A Metabolic Circuit to Support Tumor Progression

doi: 10.1002/advs.202515186

Figure Lengend Snippet: CCL2‐Induced PPARα Accumulation Drives Lipolysis and FA Release. A,B) Using the indicated FA quantification kit, we measured the FA concentrations in CM collected from the experiment shown in Figure ,E (Supporting Information). Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001. C) In the GEO dataset comprising tumor‐adjacent adipose tissues from 34 breast cancer patients ( GSE153316 ), gene expression profiles were compared between normal‐weight and obese groups. The groups were classified as follows: Normal (20–24.9 kg m −2 , n=21), Obese (≥25 kg m −2 , n=13). D) ADSCs were treated with CCL2 for 2 h with the indicated concentrations and the protein levels were analyzed by western blotting with indicated antibodies. Mean ± SD (n = 3); * , p < 0.05. E) ADSCs were treated with CCL2 (20 ng mL −1 ) and nAb‐CCL2 (1 µg mL −1 ) for 2 h. Proteins were analyzed by western blotting with indicated antibodies. Mean ± SD (n = 3); * , p < 0.05. F) ADSCs were transfected with PPRE‐Luc plasmid and treated with CCL2 (20 ng mL −1 ) and nAb‐CCL2 (1 µg mL −1 ) for 24 h. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001. G) Lipid accumulation was estimated by Nile red staining. ADSCs were treated with CCL2 (20 ng mL −1 ), nAb‐CCL2 (1 µg mL −1 ), or GW6471 (PPARα inhibitor, 1 µ m ) for 24 h. After staining with Nile Red and DAPI, the cells were visualized by fluorescence microscopy. Scale bar = 100 µm H) FACS analysis of Nile Red stained cells. The graph (right) indicate the mean fluorescence intensity. Mean ± SD (n = 3) *** , p < 0.0001. I) ADSCs were treated with CCL2 (20 ng mL −1 ), nAb‐CCL2 (1 µg mL −1 ), or GW6471 (1 µ m ) for 24 h as indicated. The mRNA levels were quantified by qRT‐PCR. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001. J) FA concentrations in CM collected after 24 h of treatment with CCL2 (20 ng mL −1 ), nAb‐CCL2 (1 µg mL −1 ), or GW6471 (1 µ m ) to ADSCs were measured using the indicated FA quantification kit. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001. K) Diagram of the CCL2/PPARα/FA axis in adipose lipolysis.

Techniques: Gene Expression, Western Blot, Transfection, Plasmid Preparation, Staining, Fluorescence, Microscopy, Quantitative RT-PCR

CCL2 Increases PPARα Stability via p‐ERK Pathway. A) The PPARα mRNA level was quantified by qRT‐PCR following treatment of human ADSC with CCL2 (20 ng mL −1 ) for the indicated time points. Mean ± SD (n = 3); n.s., not significant. B) ADSCs were pre‐treated with or without CCL2 for 4 h and incubated with cycloheximide (CHX) (100 µ m ) for the indicated time points. Cell lysates were subjected to western blot using the indicated antibodies (left). Band intensities on the blots were analyzed using ImageJ and plotted (right). Mean ± SD (n = 3); * , p < 0.05. C) HA‐Ub and PPARα plasmid were transfected into ADSCs, and CCL2 (20 ng mL −1 ) was treated for 4 h and incubated with MG132 (10 µ m ) for 2 h. Cell lysates were subjected to immunoprecipitation (IP) using HA‐affinity beads, and the precipitated proteins were analyzed by western blotting. D) CCL2 (20 ng mL −1 ) was treated to ADSCs for the indicated time points. The protein levels were analyzed by western blotting with indicated antibodies. E) CCL2 (20 ng mL −1 ) and nAb‐CCL2 (1 µg mL −1 ) were treated for 30 min and the protein levels were analyzed by western blotting with indicated antibodies. Mean ± SD (n = 3); * , p < 0.05. F) ADSCs were pre‐treated with PD98059 (20 µ m ) for 4 h, followed by CCL2 (20 ng mL −1 ) treatment for 2 h. Western blotting was performed on cell lysates using the indicated antibodies. Mean ± SD (n = 3); * , p < 0.05. G) ADSCs were pre‐treated with PD98059 (20 µ m ) for 4 h, followed by treatment with CCL2 (20 ng mL −1 ) for 4 h and MG132 (10 µM) for 2 h. Cell lysates were subjected to IP using a HUWE1 antibody, and the precipitated proteins were analyzed by western blotting with indicated antibodies. H) ADSCs were transfected with siCon or siHUWE1 and subsequently treated with CCL2 (20 ng mL −1 ) for 2 h. The protein levels were analyzed by western blotting with indicated antibodies. I) Schematic illustration of PPARα regulation via the CCL2/p‐ERK/p‐PPARα axis.

Journal: Advanced Science

Article Title: Cancer Manipulates Adjacent Adipose Tissue to Exploit Fatty Acids via HIF‐1α/CCL2/PPARα Axis: A Metabolic Circuit to Support Tumor Progression

doi: 10.1002/advs.202515186

Figure Lengend Snippet: CCL2 Increases PPARα Stability via p‐ERK Pathway. A) The PPARα mRNA level was quantified by qRT‐PCR following treatment of human ADSC with CCL2 (20 ng mL −1 ) for the indicated time points. Mean ± SD (n = 3); n.s., not significant. B) ADSCs were pre‐treated with or without CCL2 for 4 h and incubated with cycloheximide (CHX) (100 µ m ) for the indicated time points. Cell lysates were subjected to western blot using the indicated antibodies (left). Band intensities on the blots were analyzed using ImageJ and plotted (right). Mean ± SD (n = 3); * , p < 0.05. C) HA‐Ub and PPARα plasmid were transfected into ADSCs, and CCL2 (20 ng mL −1 ) was treated for 4 h and incubated with MG132 (10 µ m ) for 2 h. Cell lysates were subjected to immunoprecipitation (IP) using HA‐affinity beads, and the precipitated proteins were analyzed by western blotting. D) CCL2 (20 ng mL −1 ) was treated to ADSCs for the indicated time points. The protein levels were analyzed by western blotting with indicated antibodies. E) CCL2 (20 ng mL −1 ) and nAb‐CCL2 (1 µg mL −1 ) were treated for 30 min and the protein levels were analyzed by western blotting with indicated antibodies. Mean ± SD (n = 3); * , p < 0.05. F) ADSCs were pre‐treated with PD98059 (20 µ m ) for 4 h, followed by CCL2 (20 ng mL −1 ) treatment for 2 h. Western blotting was performed on cell lysates using the indicated antibodies. Mean ± SD (n = 3); * , p < 0.05. G) ADSCs were pre‐treated with PD98059 (20 µ m ) for 4 h, followed by treatment with CCL2 (20 ng mL −1 ) for 4 h and MG132 (10 µM) for 2 h. Cell lysates were subjected to IP using a HUWE1 antibody, and the precipitated proteins were analyzed by western blotting with indicated antibodies. H) ADSCs were transfected with siCon or siHUWE1 and subsequently treated with CCL2 (20 ng mL −1 ) for 2 h. The protein levels were analyzed by western blotting with indicated antibodies. I) Schematic illustration of PPARα regulation via the CCL2/p‐ERK/p‐PPARα axis.

Techniques: Quantitative RT-PCR, Incubation, Western Blot, Plasmid Preparation, Transfection, Immunoprecipitation

Blocking CCL2/CCR2/PPARα Axis Reduces Adipose‐lipolysis and FA Release, Suppressing Cancer Progression. A) Schematic diagram for Figure of mono‐ or co‐culture of cancer cells with transfected ADSCs in 3D culture system. B) Single‐size images of spheroids on 3D culture chips on the 5th day. Cancer cells were monocultured or cocultured with ADSCs transfected with siCon, siCCR2, or siPPARα at a 10:1 ratio. The spheroid roundness was calculated as described. Scale bar = 200 µm. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001. C) Using a FA quantification kit, we measured the FA concentration in CM obtained from the experiment shown in Figure . Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001. D) CM collected from the experiment shown in Figure were added to the lower chamber of a transwell system to evaluate its effect on cancer cell migration. After 48 h, the number of migrated cells was determined by counting. Scale bar = 200 µm. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001. E) Single‐size images of cancer cells on 3D culture chips on the 5th day. CM obtained from the chips in Figure were applied to 3D culture chips seeded with equal numbers of cancer cells. The treated CM were replaced with newly collected CM every 48 h. The average diameter was measured, and the spheroid roundness was calculated as described. Scale bar = 200 µm. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001. F) CM obtained from Figure were applied to 3D culture chips seeded with equal numbers of cancer cells. On day 5, spheroids were collected, sectioned, and mounted on slides. Immunofluorescence (IF) was performed on each slide using the indicated Ki67 antibody and DAPI to assess Ki67 expression levels. Scale bar = 200 µm. Mean ± SD (n = 3); * , p < 0.05. G) Cancer cells were monocultured or indirectly cocultured with ADSCs at a 5:1 ratio. Cancer cells were seeded in the bottom wells of a 12‐well transwell plate, and ADSCs transfected with siCon, siCCR2, or siPPARα were cultured in the upper inserts to assess the proliferative effect of indirect coculture. The number of cancer cells in the lower chamber was measured by cell counting following three days of indirect coculture. Mean ± SD (n = 3); * , p < 0.05.

Journal: Advanced Science

Article Title: Cancer Manipulates Adjacent Adipose Tissue to Exploit Fatty Acids via HIF‐1α/CCL2/PPARα Axis: A Metabolic Circuit to Support Tumor Progression

doi: 10.1002/advs.202515186

Figure Lengend Snippet: Blocking CCL2/CCR2/PPARα Axis Reduces Adipose‐lipolysis and FA Release, Suppressing Cancer Progression. A) Schematic diagram for Figure of mono‐ or co‐culture of cancer cells with transfected ADSCs in 3D culture system. B) Single‐size images of spheroids on 3D culture chips on the 5th day. Cancer cells were monocultured or cocultured with ADSCs transfected with siCon, siCCR2, or siPPARα at a 10:1 ratio. The spheroid roundness was calculated as described. Scale bar = 200 µm. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001. C) Using a FA quantification kit, we measured the FA concentration in CM obtained from the experiment shown in Figure . Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001. D) CM collected from the experiment shown in Figure were added to the lower chamber of a transwell system to evaluate its effect on cancer cell migration. After 48 h, the number of migrated cells was determined by counting. Scale bar = 200 µm. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001. E) Single‐size images of cancer cells on 3D culture chips on the 5th day. CM obtained from the chips in Figure were applied to 3D culture chips seeded with equal numbers of cancer cells. The treated CM were replaced with newly collected CM every 48 h. The average diameter was measured, and the spheroid roundness was calculated as described. Scale bar = 200 µm. Mean ± SD (n = 3); * , p < 0.05; ** , p < 0.001. F) CM obtained from Figure were applied to 3D culture chips seeded with equal numbers of cancer cells. On day 5, spheroids were collected, sectioned, and mounted on slides. Immunofluorescence (IF) was performed on each slide using the indicated Ki67 antibody and DAPI to assess Ki67 expression levels. Scale bar = 200 µm. Mean ± SD (n = 3); * , p < 0.05. G) Cancer cells were monocultured or indirectly cocultured with ADSCs at a 5:1 ratio. Cancer cells were seeded in the bottom wells of a 12‐well transwell plate, and ADSCs transfected with siCon, siCCR2, or siPPARα were cultured in the upper inserts to assess the proliferative effect of indirect coculture. The number of cancer cells in the lower chamber was measured by cell counting following three days of indirect coculture. Mean ± SD (n = 3); * , p < 0.05.

Techniques: Blocking Assay, Co-Culture Assay, Transfection, Concentration Assay, Migration, Immunofluorescence, Expressing, Cell Culture, Cell Counting

CCL2 Inhibition Decreases Adipose Lipolysis, Attenuating Cancer Progression In Vivo. A) Schematic diagram of the in vivo model. Starting from 4 weeks of age, all the mice were fed either a CD or HFD. At week 5 of feeding, 4T1‐Luc cells were orthotopically injected into the fourth mammary fat pad of female NOD/SCID mice. Starting one week after cancer cell transplantation, nAb‐CCL2 was injected intratumorally every other (n=6 independent animals in each group). B) Bioluminescence images of mice obtained every 7 days after transplantation using the Xenogen IVIS Lumina Spectrum. Color scale bars represent luminescence intensity ranging from low (purple) to high (red). Total flux (photons/sec/cm 2 /sr) was measured and the bioluminescence intensities were plotted. Mean ± SD (n = 6 independent animals in each group); * , p < 0.05 (HFD_IgG compared with HFD_nAb‐CCL2); n.s., not significant (CD_IgG compared with CD_nAb‐CCL2); ## , P < 0.001 (HFD_IgG compared with CD_IgG). C) Representative images of N.A. and T.A. with tumor obtained from IgG injected CD group (CD_IgG), nAb‐CCL2 injected CD group (CD_nAb), IgG injected HFD group (HFD_IgG), and nAb‐CCL2 injected HFD group (HFD_nAb). Each tumor was collected along with the adjacent T.A. The outlines of each tumor were indicated by a yellow dashed line. Scale bar = 10 mm. Tumor and fat volumes (mm 3 ) were calculated using the formula: (width × length 2 )/2. Mean ± SD (n =6 in each group); * , p < 0.05; ** , p < 0.001; *** , P < 0.0001; n.s., not significant. D) PPARα protein levels in adipose tissues were quantified for each group. Adipose tissue lysates were subjected to western blotting using the indicated antibodies. Band intensities on the blots were analyzed using ImageJ. Mean ± SD (n =6 in each group); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001; n.s., not significant. E) qRT‐PCR was performed on adipose tissues to assess the expression of lipolysis‐related genes that are downstream targets of PPARα. Mean ± SD (n=6 in each group); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001; n.s., not significant. F) H&E staining and IHC of tumor sections using the indicated antibodies and DAB staining. Scale bar = 100 µm. (n=6 in each group). G) qRT‐PCR was performed on tumor tissues to assess mRNA level of CCL2. Mean ± SD (n =6 in each group); * , p < 0.05. H) qRT‐PCR was performed on tumor tissues to assess the expression of proliferation‐related genes, including HIF‐1α downstream targets, in tumor tissues. Mean ± SD (n=6 in each group); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001; n.s., not significant.

Journal: Advanced Science

Article Title: Cancer Manipulates Adjacent Adipose Tissue to Exploit Fatty Acids via HIF‐1α/CCL2/PPARα Axis: A Metabolic Circuit to Support Tumor Progression

doi: 10.1002/advs.202515186

Figure Lengend Snippet: CCL2 Inhibition Decreases Adipose Lipolysis, Attenuating Cancer Progression In Vivo. A) Schematic diagram of the in vivo model. Starting from 4 weeks of age, all the mice were fed either a CD or HFD. At week 5 of feeding, 4T1‐Luc cells were orthotopically injected into the fourth mammary fat pad of female NOD/SCID mice. Starting one week after cancer cell transplantation, nAb‐CCL2 was injected intratumorally every other (n=6 independent animals in each group). B) Bioluminescence images of mice obtained every 7 days after transplantation using the Xenogen IVIS Lumina Spectrum. Color scale bars represent luminescence intensity ranging from low (purple) to high (red). Total flux (photons/sec/cm 2 /sr) was measured and the bioluminescence intensities were plotted. Mean ± SD (n = 6 independent animals in each group); * , p < 0.05 (HFD_IgG compared with HFD_nAb‐CCL2); n.s., not significant (CD_IgG compared with CD_nAb‐CCL2); ## , P < 0.001 (HFD_IgG compared with CD_IgG). C) Representative images of N.A. and T.A. with tumor obtained from IgG injected CD group (CD_IgG), nAb‐CCL2 injected CD group (CD_nAb), IgG injected HFD group (HFD_IgG), and nAb‐CCL2 injected HFD group (HFD_nAb). Each tumor was collected along with the adjacent T.A. The outlines of each tumor were indicated by a yellow dashed line. Scale bar = 10 mm. Tumor and fat volumes (mm 3 ) were calculated using the formula: (width × length 2 )/2. Mean ± SD (n =6 in each group); * , p < 0.05; ** , p < 0.001; *** , P < 0.0001; n.s., not significant. D) PPARα protein levels in adipose tissues were quantified for each group. Adipose tissue lysates were subjected to western blotting using the indicated antibodies. Band intensities on the blots were analyzed using ImageJ. Mean ± SD (n =6 in each group); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001; n.s., not significant. E) qRT‐PCR was performed on adipose tissues to assess the expression of lipolysis‐related genes that are downstream targets of PPARα. Mean ± SD (n=6 in each group); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001; n.s., not significant. F) H&E staining and IHC of tumor sections using the indicated antibodies and DAB staining. Scale bar = 100 µm. (n=6 in each group). G) qRT‐PCR was performed on tumor tissues to assess mRNA level of CCL2. Mean ± SD (n =6 in each group); * , p < 0.05. H) qRT‐PCR was performed on tumor tissues to assess the expression of proliferation‐related genes, including HIF‐1α downstream targets, in tumor tissues. Mean ± SD (n=6 in each group); * , p < 0.05; ** , p < 0.001; *** , p < 0.0001; n.s., not significant.

Techniques: Inhibition, In Vivo, Injection, Transplantation Assay, Western Blot, Quantitative RT-PCR, Expressing, Staining

Activation of the HIF‐1α/CCL2/PPARα Axis in Obese Breast Cancer Patients with Tumor Progression and Adipolysis. A) H&E staining and IHC of tumor sections using the indicated antibodies and DAB staining. Total 26 breast cancer patients were stratified into normal, overweight, and obese groups based on their BMI as follows: Normal (20‐22.9 kg m −2 , n=12; black), Overweight (23‐24.9 kg m −2 , n=5; orange), Obese (≥25 kg m −2 , n=9; red). Quantification of the stained areas for HIF‐1α, CCL2, and Ki67 was performed using ImageJ, and the results were visualized as graphs. Scale bar = 100 µm. Mean ± SD; * , P < 0.05; ** , P < 0.001. B) H&E staining and IHC of adipose tissue sections using the indicated antibodies and DAB staining. Among the breast cancer patients shown in Figure , 12 individuals with identifiable adipose tissues adjacent to normal breast tissue or tumor tissue were stratified into two BMI‐based groups: Normal (20–22.9 kg m − 2 , n = 6; dark yellow and yellow) and Overweight & Obese (≥23 kg m − 2 , n = 6; blue and light blue). Quantification of CCR2, ATGL, and PPARα staining per adipocyte was performed using ImageJ. Cytoplasmic staining areas were measured for CCR2 and ATGL, whereas nuclear staining intensity was measured for PPARα. The results are presented as graphs. Scale bar = 50 µm. Mean ± SD; * , p < 0.05; ** , p < 0.001; *** , p < 0.0001.

Journal: Advanced Science

Article Title: Cancer Manipulates Adjacent Adipose Tissue to Exploit Fatty Acids via HIF‐1α/CCL2/PPARα Axis: A Metabolic Circuit to Support Tumor Progression

doi: 10.1002/advs.202515186

Figure Lengend Snippet: Activation of the HIF‐1α/CCL2/PPARα Axis in Obese Breast Cancer Patients with Tumor Progression and Adipolysis. A) H&E staining and IHC of tumor sections using the indicated antibodies and DAB staining. Total 26 breast cancer patients were stratified into normal, overweight, and obese groups based on their BMI as follows: Normal (20‐22.9 kg m −2 , n=12; black), Overweight (23‐24.9 kg m −2 , n=5; orange), Obese (≥25 kg m −2 , n=9; red). Quantification of the stained areas for HIF‐1α, CCL2, and Ki67 was performed using ImageJ, and the results were visualized as graphs. Scale bar = 100 µm. Mean ± SD; * , P < 0.05; ** , P < 0.001. B) H&E staining and IHC of adipose tissue sections using the indicated antibodies and DAB staining. Among the breast cancer patients shown in Figure , 12 individuals with identifiable adipose tissues adjacent to normal breast tissue or tumor tissue were stratified into two BMI‐based groups: Normal (20–22.9 kg m − 2 , n = 6; dark yellow and yellow) and Overweight & Obese (≥23 kg m − 2 , n = 6; blue and light blue). Quantification of CCR2, ATGL, and PPARα staining per adipocyte was performed using ImageJ. Cytoplasmic staining areas were measured for CCR2 and ATGL, whereas nuclear staining intensity was measured for PPARα. The results are presented as graphs. Scale bar = 50 µm. Mean ± SD; * , p < 0.05; ** , p < 0.001; *** , p < 0.0001.

Techniques: Activation Assay, Staining

A Diagram of the obesity‐driven tumor progression through tumor–adipose metabolic crosstalk. FA supply activates HIF‐1α in cancer cells, promoting CCL2 secretion. This, in turn, leads to PPARα accumulation and enhances PPARα‐mediated lipolysis in adjacent adipose tissue. Consequently, FAs are continuously released from tumor‐associated adipose tissue, further activating HIF‐1α and amplifying CCL2 production, thereby accelerating tumor progression. This vicious cycle is illustrated in the schematic diagram. TME; Tumor microenvironment, FA; fatty acid.

Journal: Advanced Science

Article Title: Cancer Manipulates Adjacent Adipose Tissue to Exploit Fatty Acids via HIF‐1α/CCL2/PPARα Axis: A Metabolic Circuit to Support Tumor Progression

doi: 10.1002/advs.202515186

Figure Lengend Snippet: A Diagram of the obesity‐driven tumor progression through tumor–adipose metabolic crosstalk. FA supply activates HIF‐1α in cancer cells, promoting CCL2 secretion. This, in turn, leads to PPARα accumulation and enhances PPARα‐mediated lipolysis in adjacent adipose tissue. Consequently, FAs are continuously released from tumor‐associated adipose tissue, further activating HIF‐1α and amplifying CCL2 production, thereby accelerating tumor progression. This vicious cycle is illustrated in the schematic diagram. TME; Tumor microenvironment, FA; fatty acid.

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