Targeted gene silencing of CCL2 inhibits triple negative breast cancer progression by blocking cancer stem cell renewal and M2 macrophage recruitment

Abstract

Triple negative breast cancers are an aggressive subtype of breast cancer, characterized by the lack of estrogen receptor, progesterone receptor and Her2 expression. Triple negative breast cancers are non-responsive to conventional anti-hormonal and Her2 targeted therapies, making it necessary to identify new molecular targets for therapy. The chemokine CCL2 is overexpressed in invasive breast cancers, and regulates breast cancer progression through multiple mechanisms. With few approaches to target CCL2 activity, its value as a therapeutic target is unclear. In these studies, we developed a novel gene silencing approach that involves complexing siRNAs to TAT cell penetrating peptides (Ca-TAT) through non-covalent calcium cross-linking. Ca-TAT/siRNA complexes penetrated 3D collagen cultures of breast cancer cells and inhibited CCL2 expression more effectively than conventional antibody neutralization. Ca-TAT/siRNA complexes targeting CCL2 were delivered to mice bearing MDA-MB-231 breast tumor xenografts. In vivo CCL2 gene silencing inhibited primary tumor growth and metastasis, associated with a reduction in cancer stem cell renewal and recruitment of M2 macrophages. These studies are the first to demonstrate that targeting CCL2 expression in vivo may be a viable therapeutic approach to treating triple negative breast cancer.

Keywords: CCL2; TAT cell penetrating peptide; breast cancer; cancer stem cell; macrophage.

Figure 1. Ca-TAT peptides complexed to CCL2 siRNAs significantly reduce CCL2 protein expression in breast cancer cells

A. MDA-MB-231 or B. DCIS.com cells cultured in 2D were transfected with Ca-TAT peptides complexed to control (Con) or CCL2 siRNAs (huCCL2si1 or huCCL2si2). 48 hours post transfection, conditioned media was measured by CCL2 ELISA. Par= Parental. C-D. MDA-MB-231cells were cultured in 3D collagen, and were transfected with Ca-TAT peptides complexed to siRNAs (C) or treated with 10 μg/ml anti-CCL2 or IgG (D). CCL2 expression was measured in conditioned medium by ELISA, 24 hours and 48 hours post-treatment. Statistical analysis was performed by One way ANOVA followed by Bonferonni post-hoc analysis. Statistical significance was determined by p-value less than 0.05. *p<0.05, **p<0.01, n.s=not significant. Mean+SEM is shown.

Figure 2. Ca-TAT delivery of CCL2 siRNAs inhibits growth and enhances cell death of primary MDA-MB-231 tumor xenografts

A. Experimental plan for treatment of MDA-MB-231 tumor bearing mice with Ca-TAT/siRNA complexes. 21 days post transplantation, tumors received injections every three days with Ca-TAT peptides complexed to 10 micrograms control or CCL2 siRNAs (huCCL2si1, huCCL2si2) for a total of three injections, n=6 per group. Animals were euthanized 30 days post-transplantation for tissue harvest and analysis. B. Tumor and surrounding normal mammary tissues were analyzed for CCL2 expression by flow cytometry C. Representative tumors with quantified tumor mass. D. Representative H&E stain of primary tumor xenografts. Necrotic areas were identified on H&E stains at 5 different depths of the tumor and quantified by Image J analysis. Necrotic areas are outlined. Scale bar= 200 microns. Statistical analysis was performed by One way ANOVA followed by Bonferonni post-hoc comparisons with Control siRNA group. Statistical significance was determined by p-value less than 0.05. **p<0.01, ***p<0.001, n.s= not significant. Mean+SEM is shown.

Figure 3. CCL2 gene silencing inhibits primary and secondary invasion of MDA-MB-231 breast tumors

A. Top panels: Low magnification (4X) H&E stain with muscle tissue (M) and tumor tissue (T). Scale bar=1000 microns. Middle panels: Higher magnification (10x) H&E stain with muscle tissue and tumor tissue. Scale bar=400 microns. Bottom panels: CO-IF stain of Calsquestrin (green) and CK5 (red) in primary tumor xenografts. Overlays of Calsequestrin, CK5 and DAPI stain are shown. Scale bar= 400 microns. B. Metastatic lesions throughout the lung tissue were visually identified by hematoxylin staining as round shaped nodules using an inverted microscope and quantified. Representative images are shown. Metastatic nodules are outlined. Scale bar=80 microns. C. H&E stain of lung tissues. Metastatic lesions are circled. Scale bar=40 microns. Statistical analysis was performed by One way ANOVA followed by Bonferonni post-hoc comparisons with Control siRNA group (Con). Statistical significance was determined by p-value less than 0.05. **p<0.01. Mean+SEM is shown. N=6 animals per group.

Figure 4. CCL2 gene silencing enhances necrosis and autophagy in MDA-MB-231 breast tumor xenografts

MDA-MB-231 breast tumor xenografts were injected with Ca-TAT peptides complexed to control siRNAs (Con), or CCL2 siRNAs (huCCL2si1 or huCCL2si2), and immunostained for expression of A. PCNA, B. Cleaved caspase-3, C. HMGB1, or D. LC3B. Scale=100 microns. Statistical analysis was performed by One Way ANOVA followed by Bonferonni post-hoc analysis. Statistical significance was determined by p-value less than 0.05. *p<0.05, ***p<0.001, n.s=not significant. Mean+SEM is shown. N=6 animals per group.

Figure 5. CCL2 gene silencing in breast cancer cells by Ca-TAT/siRNA complexes inhibits cancer stem cell renewal

A-B. MDA-MB-231 breast tumor xenografts (N=6 animals per group) were treated with control (Con) or Ca-TAT complexed to CCL2 siRNAs (huCCL2si1, huCCL2si2), and were analyzed for CD24 and CD44 expression by flow cytometry (A), or ALDH1 expression by immunohistochemistry stain (B). ALDH1 expression was quantified by Image J; arbitrary units are shown. Scale bar=100 microns. C. MDA-MB-231 cells in culture were transfected with Ca-TAT/siRNA complexes and examined for mammosphere formation. Representative images are shown at passage 4. Scale bar=200 microns. Statistical analysis was performed by One Way ANOVA followed by Bonferonni post-hoc comparisons with Control group. Statistical significance was determined by p-value less than 0.05. *p<0.05, **p<0.01. Mean+SEM is shown.

Figure 6. CCL2 gene silencing in MDA-MB-231 breast tumor xenografts inhibits M2 macrophage recruitment but not tumor angiogenesis

MDA-MB-231 breast tumor xenografts were examined for A. CD11b expression by flow cytometry, B. Arginase I, or C. Von Willebrand Factor 8 (VWF8) expression, by immunohistochemistry. Scale bar=100 microns. Expression levels were quantified by Image J; arbitrary units are shown. Statistical analysis was performed by One Way ANOVA followed by Bonferonni post-hoc comparisons with Control group. Statistical significance was determined by p-value less than 0.05. *p<0.05, **p<0.01, n.s= not significant. Mean+SEM is shown. N=6 animals per group

Figure 7. CCL2 gene silencing inhibits macrophage recruitment to MDA-MB-231 breast cancer 3D cultures

A. Diagram of MMD function. Dual wall design allows user to control size of flow-through opening by twisting the inner and outer chambers in and out of alignment. B. Experimental design: MDA-MB-231 breast cancer cells were embedded in collagen within the MMDs, which were placed in 6 well dishes. mCherry labeled Raw264.7 macrophages were plated outside the devices, and assayed for migration into the MMD over 24-48 hours. C. Representative images of Raw 264.7 mcherry macrophage infiltration into collagen alone or collagen embedded with breast cancer cells after 48 hours. Fluorescence images overlayed with phase contrast images are shown. Scale bar=100 microns. Arrow points to unlabeled MDA-MB-231 breast cancer cells. D. MDA-MB-231 cells were cultured in 3D collagen in the MMDs, transfected with Ca-TAT/siRNA complexes, and analyzed for macrophage infiltration into the MMD. Macrophage infiltration was quantified by Image J; arbitrary units shown. Statistical analysis was performed by One way ANOVA followed by Bonferonni post-hoc analysis. Statistical significance was determined by p-value less than 0.05. *p<0.05, **p<0.01, ***p<0.0001, n.s=not sigificant. Mean+SEM is shown.