Interleukin-34 Enhances the Tumor Promoting Function of Colorectal Cancer-Associated Fibroblasts

Abstract

The stromal compartment of colorectal cancer (CRC) is marked by the presence of large numbers of fibroblasts, termed cancer-associated fibroblasts (CAFs), which promote CRC growth and progression through the synthesis of various molecules targeting the neoplastic cells. Interleukin (IL)-34, a cytokine over-produced by CRC cells, stimulates CRC cell growth. Since IL-34 also regulates the function of inflammatory fibroblasts, we hypothesized that it could regulate the tumor promoting function of colorectal CAFs. By immunostaining and real-time PCR, we initially showed that IL-34 was highly produced by CAFs and to lesser extent by normal fibroblasts isolated from non-tumoral colonic mucosa of CRC patients. CAFs and normal fibroblasts expressed the functional receptors of IL-34. IL-34 induced normal fibroblasts to express α-SMA, vimentin and fibroblast activation protein and enhanced fibroblast growth, thus generating a cellular phenotype resembling that of CAFs. Consistently, knockdown of IL-34 in CAFs with an antisense oligonucleotide (AS) decreased expression of such markers and inhibited cell proliferation. Co-culture of CRC cells with IL-34 AS-treated CAFs supernatants resulted in less cancer cell proliferation and migration. Among CAF-derived molecules known to promote CRC cell growth/migration, only netrin-1 and basic-fibroblast growth factor were induced by IL-34. Data suggest a role for IL-34 in the control of colorectal CAF function.

Keywords: b-FGF; colon cancer; cytokines; netrin-1; stromal cells.

Figure 1

Cancer-associated fibroblasts express interleukin-34 (IL-34). (A) Representative images of immunofluorescence staining of fibroblasts isolated from tumoral (cancer-associated fibroblasts—CAFs) and non-tumoral (normal fibroblasts—NFs) area of one patient with colon cancer and analyzed for the expression of CD31 (green), cytocheratin (green), vimentin (green), and DAPI (blue). (Scale bars: 50 µm). One of three representative experiments, in which cells of three patients were used, is shown. (B) NFs and CAFs were evaluated for the expression of IL-34 by real-time PCR; levels were normalized to β-Actin and data were expressed as mean ± SEM of five patients. (C) Representative images of immunofluorescence staining of CAFs and NFs taken from tumoral and non-tumoral area, respectively, of one patient with colon cancer and one patient ulcerative colitis-associated colon cancer (CAC) and analyzed for the expression of IL-34 (green), and DAPI (blue). The example is representative of six separate experiments in which cells of three patients with colon cancer and three patients with CAC were analyzed. The scale bars are 75 µm.

Figure 2

Both normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) express IL-34 receptors. A–B: NFs and CAFs were evaluated for the expression of macrophage colony-stimulating factor receptor (M-CSFR-1) (A) and PTP-ζ (B) RNA transcripts by real-time PCR; levels were normalized to β-Actin and data were expressed as mean ± SEM of six patients. (C) Representative images of immunofluorescence staining of NFs and CAFs taken from tumoral and non-tumoral area, respectively, of one patient with colon cancer and analyzed for the expression of M-CSFR-1 (green), PTP-ζ (green), and DAPI (blue). The example is representative of three separate experiments in which cells of three patients with colon cancer were analyzed. The scale bars are 75 µm.

Figure 3

IL-34 stimulates normal fibroblasts (NFs) to acquire a tumoral phenotype. (A–C). Serum-starved NFs were either left unstimulated (UNST) or treated with IL-34 (25–100 ng/mL) for 6 h, and then α-SMA (A), FAP (B), Vimentin (C) RNA transcripts were evaluated by real-time PCR; levels were normalized to β-Actin and data were expressed as mean ± SEM of six experiments in which cells of six patients were analyzed. (D) CAFs were transfected with either negative control antisense oligonucleotide (NCAS) or IL-34 antisense oligonucleotide (IL-34 AS) for 48 h. IL-34 and β-Actin were then analyzed by Western blotting. Uncropped Western Blot images are available in Figure S2. (E–G). CAFs were transfected with negative control antisense oligonucleotide (NCAS) or IL-34 antisense oligonucleotide (IL-34 AS) for 24 h and α-SMA (E), FAP (F), Vimentin (G) RNA transcripts were analyzed by real-time PCR; levels were normalized to β-Actin, and data were expressed as mean ± SEM of six experiments in which cells of six patients were analyzed.

Figure 4

IL-34 enhances fibroblast proliferation. (A) Serum-starved normal fibroblasts (NFs) were stimulated with IL-34 (50 ng/mL) or basic-fibroblast growth factor (b-FGF) (20 ng/mL) for 48 h. Cell proliferation was evaluated by flow cytometry, and proliferation index was calculated with Modfit LT. Data indicate mean ± SEM of six independent experiments in which cells of six patients were analyzed. Right insets: representative histograms showing the expression of carboxyfluorescein diacetate succinimidyl ester (CFSE) in NFs analyzed by flow cytometry. (B) CAFs were transfected with negative control antisense oligonucleotide (NCAS) or IL-34 antisense oligonucleotide (IL-34 AS) for 48 h. Cell proliferation was evaluated by flow cytometry, and proliferation index was calculated with Modfit LT. Data indicate mean ± SEM of six independent experiments in which cells of six patients were analyzed. Right insets: representative histograms showing the expression of CFSE in CAFs analyzed by flow cytometry. UNST= unstimulated.

Figure 5

IL-34 produced by fibroblasts decreases DLD-1 cell proliferation and migration. (A) DLD-1 cells were incubated with supernatants of CAFs previously transfected with either negative control antisense oligonucleotide (NCAS) or IL-34 antisense oligonucleotide (IL-34 AS) for 24 h. DLD-1 proliferation was evaluated after 48 h by flow cytometry and proliferation index was calculated with Modfit LT. Data indicate mean ± SEM of six independent experiments in which cells of six patients were analyzed. Right insets: representative histograms showing the expression of CFSE in DLD-1 analyzed by flow cytometry. (B) Representative images of “pseudo” wound in monolayer of DLD-1 treated with supernatants of CAFs previously transfected with either negative control antisense oligonucleotide (NCAS) or IL-34 antisense oligonucleotide (IL-34 AS) for 24 h. Cells were photographed at the time of scratch (T0) and examined for cell migration after 24 h from the specific stimulation. Right panel shows the % of “pseudo” wound area in a monolayer of DLD-1 cells at the specific time point (T) with respect to area in T0 (defined as 100%), and the data are expressed as mean ± SEM of six independent experiments in which cells of six patients were analyzed.

Figure 6

IL-34 induces Netrin-1 and b-FGF RNA expression. (A) Serum-starved normal fibroblasts (NFs) were treated with IL-34 (50 ng/mL) for 6 h and Igf1; Igf2, Tgf-α, Hgf, Netrin-1 and b-FGF RNA transcripts were analyzed by real-time PCR; levels were normalized to β-Actin and data were expressed as mean ± SEM of six experiments in which cells of six patients were analyzed. (B) CAFs were transfected with either negative control antisense oligonucleotide (NCAS) or IL-34 antisense oligonucleotide (IL-34 AS) for 24 h and Igf1; Igf2, Tgf-α, Hgf, Netrin-1 and b-FGF RNA transcripts were analyzed by real-time PCR; levels were normalized to β-Actin and data were expressed as mean ± SEM of six experiments in which cells of six patients were analyzed. UNST = unstimulated.

Figure 7

IL-34 induces Netrin-1 and b-FGF protein expression. (A) Serum-starved normal fibroblasts (NFs) were treated with IL-34 (50 ng/mL) for 24 h. Netrin-1 and β-Actin were evaluated by Western blotting. One of three independent experiments, in which cells of three patients were analyzed, is shown. Right panel shows the quantitative analysis of Netrin-1/β-Actin ratio. Values are expressed in arbitrary units (a.u.) and indicate mean ± SEM. Uncropped Western Blots images are available in Figure S5. (B) Serum-starved NFs were treated with IL-34 (50 ng/mL) for 48 h and b-FGF was evaluated in cell-free supernatants by ELISA. Values are expressed in arbitrary units (a.u.) and indicate mean ± SEM of six experiments in which cells of six patients were analyzed. C-D. CAFs were transfected with either negative control antisense oligonucleotide (NCAS) or IL-34 antisense oligonucleotide (IL-34 AS) for 24 h, and Netrin-1 and β-Actin (C) or b-FGF and β-Actin (Uncropped Western Blots images are available in Figure S6) (D) were evaluated by Western blotting. Right panels show the quantitative analysis of Netrin-1/β-Actin (C) or b-FGF /β-Actin (D) ratio, and values are expressed in arbitrary units (a.u.) and indicate mean ± SEM (Uncropped Western Blots images are available in Figure S7). One of three independent experiments, in which cells of three patients were analyzed, is shown. UNST = unstimulated.