Cancer-Associated-Fibroblast-Mediated Paracrine and Autocrine SDF-1/CXCR4 Signaling Promotes Stemness and Aggressiveness of Colorectal Cancers

Abstract

Colorectal cancer (CRC) is a leading cause of cancer mortality worldwide, and cancer-associated fibroblasts (CAFs) play a major role in the tumor microenvironment (TME), which facilitates the progression of CRC. It is critical to understand how CAFs promote the progression of CRC for the development of novel therapeutic approaches. The purpose of this study was to understand how CAF-derived stromal-derived factor-1 (SDF-1) and its interactions with the corresponding C-X-C motif chemokine receptor 4 (CXCR4) promote CRC progression. Our study focused on their roles in promoting tumor cell migration and invasion and their effects on the characteristics of cancer stem cells (CSCs), which ultimately impact patient outcomes. Here, using in vivo approaches and clinical histological samples, we analyzed the influence of secreted SDF-1 on CRC progression, especially in terms of tumor cell behavior and stemness. We demonstrated that CAF-secreted SDF-1 significantly enhanced CRC cell migration and invasion through paracrine signaling. In addition, the overexpression of SDF-1 in CRC cell lines HT29 and HCT-116 triggered these cells to generate autocrine SDF-1 signaling, which further enhanced their CSC characteristics, including those of migration, invasion, and spheroid formation. An immunohistochemical study showed a close relationship between SDF-1 and CXCR4 expression in CRC tissue, and this significantly affected patient outcomes. The administration of AMD3100, an inhibitor of CXCR4, reversed the entire phenomenon. Our results strongly suggest that targeting this signaling axis in CRC is a feasible approach to attenuating tumor progression, and it may, therefore, serve as an alternative treatment method to improve the prognosis of patients with CRC, especially those with advanced, recurrent, or metastatic CRC following standard therapy.

Keywords: C-X-C motif chemokine receptor 4 (CXCR4); cancer stem cells (CSCs); cancer-associated fibroblasts (CAFs); stromal-derived factor-1 (SDF-1); tumor microenvironment (TME).

Figure 1

Characterization and functional analysis of CAFs in CRC. (A) Flow cytometric analysis illustrating the identification of fibroblasts isolated from samples from CRC patients using the markers CD10 and GPR77, highlighting the heterogeneity within the CAF population. The experiment was conducted in triplicate to ensure the reliability and reproducibility of the results. (B) Morphological examination of CAFs through light microscopy, depicting their elongated spindle or stellate shapes with thin cytoplasmic extensions and prominent actin stress fibers. The adjacent panels show the immunofluorescent staining results for α-SMA and vimentin with a comparison of the expression levels in CAFs and NFs, demonstrating significantly higher expression in CAFs; magnification, ×400. (C) Gene expression microarray data showcasing significant changes in the expression profiles of the proteins, including chemokines and cytokine receptors, secreted by CAFs compared with NFs, with alterations marked as greater-than-five-fold increases. (D) Additional microarray results focusing on distinct clusters of upregulated genes related to tumor invasion and metastasis in CAFs. (E) qRT-PCR results displaying elevated mRNA levels of vimentin, fibroblast activation protein, α-SMA, SDF-1, and CXCR4 in CAFs extracted from CRC tissues; these were corroborated by corresponding increases in protein levels, reinforcing the pro-tumorigenic role of these markers in the tumor microenvironment. This experiment was conducted in triplicate to ensure the reliability and reproducibility of the results. (Scale bar: 50 μm). Data are shown as representative images and numerical data are represented as the mean ± SD of each group of cells from three separate experiments. *** p < 0.0001.

Figure 2

Analysis of SDF-1 and CXCR4 expression in CAFs and NFs, and their impacts on CRC cell motility. (A) ELISA results demonstrating the significant upregulation of SDF-1 protein secretion in CAFs compared with other groups, supporting the hypothesis that CAFs enhance SDF-1-induced migration in colorectal cancer. The bar graph shows the quantified protein levels with their standard deviations, illustrating marked differences between the groups. (B) qRT-PCR and Western blot analyses depicting the expression levels of SDF-1 and CXCR4 in NFs and CAFs. (C,D) EMT marker expression via Western blot in CRC cells following treatment with conditioned media. Increased levels of Twist-1, Snail, and Vimentin were observed, indicating EMT activation in response to SDF-1/CXCR4 signaling. (E) Migration and invasion were evaluated using Transwell assays to ascertain the influences of different fibroblast types on these processes. Each experiment was conducted in triplicate to ensure the reliability and reproducibility of the results; magnification, ×400. The mean ± SD of each group of cells from three separate experiments is given. * p < 0.05. ** p < 0.001.

Figure 3

Enhanced migratory and invasive capabilities of CRC cells mediated by SDF-1/CXCR4 signaling. (A) Phase-contrast micrograph showing the shape of SDF-1 expression levels in HT-29 and HCT-116 CRC cell lines following stable transfection compared to control cells; magnification, ×400. The data demonstrate significant SDF-1 upregulation in transfected cells, underscoring the effectiveness of the transfection process. (B) SDF-1 protein levels in the conditioned medium quantified by ELISA in three groups of HT-29 and HCT116 cells cultured separately for 72 h. (C) RT-PCR results detected for 48 h. (D) Western blot analysis showed SDF-1 and CXCR4 expression in HT-29 and HCT-116 cells. Compared with the vehicle, higher SDF-1 and CXCR4 expression were observed in rSDF-1 and transfected cells. (E) Transwell analysis showed that rSDF-1 and transfected cells demonstrated increased cell migration and invasiveness. AMD3100 inhibition of CXCR4 eliminated the migration and invasion induced by SDF-1; magnification, ×200. Data are represented within images and numerical data are represented as the mean ± SD for each group of cells from three separate experiments. * p < 0.05. (F) Western blot analysis showed that, in the presence of endogenous SDF-1, CXCR4, vimentin, Twist, and Snail were all upregulated, while E-cadherin was downregulated. Expression of CXCR4, vimentin, Twist, and Snail were downregulated in cells cultured via AMD3100 inhibition, while E-cad expression was upregulated. (G) Histological analysis shows organotypic tissue invasiveness of HT-29 and HCT-116 cells in SDF-1 expression groups (CAFs, rSDF-1, pMSCV SDF-1/NFs, and pMSCV SDF-1/CAFs), while NFs and pMSCV SDF-1/CAFs + AMD3100 demonstrated non-invasion. Data are represented as images and numerical data are represented as the mean ± SD for each group of cells from three separate experiments. * p < 0.05; ** p < 0.01.

Figure 4

Influence of SDF-1 on CSC characteristics in CRC cells mediated by cancer-associated fibroblasts (CAFs). (A) Analysis of sphere formation under different treatment conditions demonstrating the effect of SDF-1 on promoting spheroid phenotypes in CRC cells. This panel shows increased sphere formation in cultures treated with CAF-CM and rSDF-1 in comparison with the control groups. The graph quantifies the sphere formation efficiency, illustrating a significant enhancement in conditions stimulated by SDF-1. (B) Expression levels of the CSC markers CD44 and CD133 in CRC cells following exposure to SDF-1. The images of immunofluorescent staining highlight the upregulation of these markers in cells treated with SDF-1, which is indicative of enriched CSC properties. The quantitative analysis below these images shows the relative expression levels of CD44 and CD133, with bars representing statistical significance. (C) Detailed expression analysis of the transcription factor OCT4 and CSC markers CD44 and CD133 in CRC cells subjected to various treatments. The Western blot images and corresponding densitometric analysis illustrate that cells exposed to CAF-CM and SDF-1 transfection exhibit substantially higher levels of these proteins compared with the controls, confirming the role of SDF-1 in maintaining CSC characteristics. The graph adjacent to the blots quantifies protein expression, emphasizing the differential impact of SDF-1 on CSC marker expression.

Figure 4

Influence of SDF-1 on CSC characteristics in CRC cells mediated by cancer-associated fibroblasts (CAFs). (A) Analysis of sphere formation under different treatment conditions demonstrating the effect of SDF-1 on promoting spheroid phenotypes in CRC cells. This panel shows increased sphere formation in cultures treated with CAF-CM and rSDF-1 in comparison with the control groups. The graph quantifies the sphere formation efficiency, illustrating a significant enhancement in conditions stimulated by SDF-1. (B) Expression levels of the CSC markers CD44 and CD133 in CRC cells following exposure to SDF-1. The images of immunofluorescent staining highlight the upregulation of these markers in cells treated with SDF-1, which is indicative of enriched CSC properties. The quantitative analysis below these images shows the relative expression levels of CD44 and CD133, with bars representing statistical significance. (C) Detailed expression analysis of the transcription factor OCT4 and CSC markers CD44 and CD133 in CRC cells subjected to various treatments. The Western blot images and corresponding densitometric analysis illustrate that cells exposed to CAF-CM and SDF-1 transfection exhibit substantially higher levels of these proteins compared with the controls, confirming the role of SDF-1 in maintaining CSC characteristics. The graph adjacent to the blots quantifies protein expression, emphasizing the differential impact of SDF-1 on CSC marker expression.

Figure 5

Enhanced tumor growth and molecular characterization in SDF-1-expressing colon cancer xenografts. (A) A closer view of the cellular morphology in tumors, with images showcasing differences in cell density and organization between the SDF-1-expressing and control groups and further illustrating the impacts of SDF-1 on tumor architecture and cellular behavior. Quantitative analysis of tumor growth in nude mice xenografted with colon cancer cells. The graph displays a significant increase in tumor size in mice injected with cells overexpressing SDF-1 compared with the control group, demonstrating the role of SDF-1 in promoting tumor growth. Statistical significance with p-values of less than 0.05 is indicated. (B) Representative images of tumors harvested from the xenograft models, showing the visual difference in tumor size between the SDF-1-expressing group and the controls. (C) Immunohistochemical staining for SDF-1, CXCR4, CD44, and OCT4 within the tumor tissues. The images reveal higher expression levels of these markers in the SDF-1-expressing tumors compared with the controls, highlighting the molecular changes associated with SDF-1 expression. (D) Analysis of stemness-associated markers in tumor tissues with the quantification of Nanog expression levels. The elevated Nanog levels in SDF-1-expressing tumors suggest an increase in stem-like properties, corresponding to the aggressive nature of these tumors.

Figure 5

Enhanced tumor growth and molecular characterization in SDF-1-expressing colon cancer xenografts. (A) A closer view of the cellular morphology in tumors, with images showcasing differences in cell density and organization between the SDF-1-expressing and control groups and further illustrating the impacts of SDF-1 on tumor architecture and cellular behavior. Quantitative analysis of tumor growth in nude mice xenografted with colon cancer cells. The graph displays a significant increase in tumor size in mice injected with cells overexpressing SDF-1 compared with the control group, demonstrating the role of SDF-1 in promoting tumor growth. Statistical significance with p-values of less than 0.05 is indicated. (B) Representative images of tumors harvested from the xenograft models, showing the visual difference in tumor size between the SDF-1-expressing group and the controls. (C) Immunohistochemical staining for SDF-1, CXCR4, CD44, and OCT4 within the tumor tissues. The images reveal higher expression levels of these markers in the SDF-1-expressing tumors compared with the controls, highlighting the molecular changes associated with SDF-1 expression. (D) Analysis of stemness-associated markers in tumor tissues with the quantification of Nanog expression levels. The elevated Nanog levels in SDF-1-expressing tumors suggest an increase in stem-like properties, corresponding to the aggressive nature of these tumors.

Figure 6

Differential expression of the SDF-1/CXCR4 axis initially from CAF-induced paracrine SDF-1 and ultimately reinforcing SDF-1/CXCR4 signaling in CRC tissues in an autocrine manner, highlighting their roles in tumor progression and metastasis. (A,E) Immunohistochemical staining images showing the absent expression of SDF-1 and CXCR4 in normal colorectal tissues as negative controls. These panels establish the baseline expression levels for comparison with cancerous tissues. (B,F) High-magnification images displaying slight cytoplasmic and membrane staining of SDF-1 and CXCR4 in early CRC cell nests and moderate expression (C,G) in advanced CRC tissues, illustrating the active involvement of the expression of SDF-1 and CXCR4 signaling in CRC tissues and reinforcing the consistency of SDF-1 and CXCR4 expression in these aggressive cancer forms. Lastly, the images in (D,H) depict strong cytoplasmic positivity for SDF-1 and pronounced nuclear positivity for CXCR4 in CRC patients with liver metastasis. The arrows indicate patterns that demonstrate and reinforce how SDF-1/CXCR4 signaling is closely correlated with tumor behavior and metastatic potential, providing visual evidence of the significant role of the SDF-1/CXCR4 axis in promoting the aggressiveness of CRC. All images have a scale bar of 300 μm.

Figure 7

The progression of CRC aggressiveness through CAF-mediated SDF-1/CXCR4 signaling involving both paracrine and autocrine mechanisms; we designed a diagrammatic representation that highlights the following key stages: 1. Hemostasis/Pre-Neoplastic Stage: Illustration of the normal stroma with widely distributed NFs existing adjacent to normal or preneoplastic colonic cells, indicating minimal interaction and a stable dynamic balance between NFs and CRC cells. 2. Transformation into CRC: Depiction of the transition from preneoplastic colonic cells to CRC cells and the EMT with malignant behavior. This stage shows CAFs that have transformed from NFs accumulating around and closely interacting in the invasive fronts of CRC cell nests. The release of SDF-1 by CAFs and its binding to the CXCR4 receptor on CRC cells are highlighted. 3. Paracrine SDF-1/CXCR4 Signaling: Visualization of SDF-1 being secreted by CAFs in a paracrine manner, binding to CXCR4 receptors on CRC cells, and, thus, promoting increased aggressiveness in CRC cells. 4. Autocrine SDF-1/CXCR4 Signaling: Illustration of CRC cells expressing and releasing excessive SDF-1, which binds to their own CXCR4 receptors, creating a self-reinforcing autocrine cycle. This cycle enhances CSC characteristics, aggressiveness, and tumorigenicity, leading to a poor prognosis.