Tumor-associated macrophages promote bladder tumor growth through PI3K/AKT signal induced by collagen

Abstract

The tumor microenvironment is associated with various tumor progressions, including cancer metastasis, immunosuppression, and tumor sustained growth. Tumor-associated macrophages (TAMs) are considered an indispensable component of the tumor microenvironment, participating in the progression of tumor microenvironment remodeling and creating various compounds to regulate tumor activities. This study aims to observe enriched TAMs in tumor tissues during bladder cancer development, which markedly facilitated the proliferation of bladder cancer cells and promoted tumor growth in vivo. We determined that TAMs regulate tumor sustained growth by secreting type I collagen, which can activate the prosurvival integrin α2β1/PI3K/AKT signaling pathway. Furthermore, traditional chemotherapeutic drugs combined with integrin α2β1 inhibitor showed intensive anticancer effects, revealing an innovative approach in clinical bladder cancer treatment.

Keywords: PI3K/AKT; bladder cancer; collagen; integrin α2β1; tumor-associated macrophages.

Figure 1

Tumor‐associated macrophages (TAMs) promote bladder cancer cell growth and tumor progression. A, Immunohistochemistry of CD68 in bladder tumor tissues from patients in stage T0 and T3. Scale bar, 50 μm. The arrows indicate the macrophages in tumor sites. B, Percentage of macrophages in the immune cell subpopulation of patients (stage T0 and T3) bladder tumor tissues. C, Relative cell number of MB49 cells cocultured with mouse peritoneal macrophages, TAMs, or PBS for 72 h. D, Relative numbers of T24 cells cocultured with macrophages from patients’ paracarcinoma tissues, TAMs, or PBS for 72 h. E, Relative colony numbers of MB49 cells pretreated with mouse peritoneal macrophages, TAMs, or PBS for 72 h. F, Relative colony numbers of T24 cells pretreated with macrophages from patients’ paracarcinoma tissues, TAMs, or PBS for 72 h. G, Bladder tumor weights of mice treated with PBS or TAMs. Of note, 10⁶ MB49 cells were intravesically instilled into C57 mice bladders. On days 4 and 8, mice were instilled with 2 × 10⁵ TAMs by bladder irrigation. H, Histological H&E staining of the bladders of C57 mice in PBS or TAM. Scale bar, 500 μm. Error bars, mean ± SEM; *P < .05; **P < .01

Figure 2

Tumor‐associated macrophages (TAMs) facilitate bladder cancer cell growth by secreting collagen. A, Relative cell numbers and colony numbers of MB49 cells treated with TAM cultured medium (CM) or not (PBS) for 72 h. B, Relative cell numbers and colony numbers of T24 cells treated with CM or PBS for 72 h. C, Relative gene expression of collagen, interleukin (IL)‐10, transforming growth factor (TGF)‐β, vascular endothelial growth factor (VEGF), chemokine (C‐C motif) ligand (CCL)2, CCL17, and CCL22 in mice peritoneal macrophages and TAMs. D, Relative type I collagen quantity in peritoneal macrophage cultured medium or TAM cultured medium detected by ELISA. E, Relative gene expression of integrin β1, IL‐10 receptor (R), TGF‐βR, VEGFR, CCR2, and CCR4 in MB49 cells treated with TAMs or not. F, Immunofluorescence of integrin β1 of MB49 cells treated with TAM cultured medium or not for 72 h. Scale bar, 10 μm. G, Relative cell numbers and colony numbers of MB49 cells treated with collagen (0.1 μg/mL) or PBS for 72 h. H, Relative cell numbers and colony numbers of T24 cells treated with collagen (0.1 μg/mL) or PBS for 72 h. I, Bladder tumor weights of mice treated with PBS, collagen, or collagenase. Of note, 106 MB49 cells were intravesically instilled into C57 mice bladders. On days 4 and 8, mice were instilled with 1 μg collagen or 0.1 μg collagenase by bladder irrigation. J, Immunohistochemistry of type I collagen in bladder cancer tissues from patients in stage T0 or stage T3. Scale bar, 50 μm. Error bars, mean ± SEM. *P < .05; **P < .01. n.s, not statistically significant

Figure 3

Collagen induces tumor progression through integrin α2β1. A, Relative gene expression of integrin α2, α1, α10, and α10 in collagen‐treated MB49 cells (0.1 μg/mL) for 72 h or not (PBS). B, Western blot analysis of integrin α2β1 and actin in MB49 cells treated with collagen (0.1 μg/mL) for 72 h. C, Relative colony numbers and cell numbers of MB49 cells treated with collagen (0.1 μg/mL) or collagen (0.1 μg/mL) combined with E7820 (0.05 μg/mL) for 72 h. D, Relative colony numbers and cell numbers of T24 cells treated with collagen (0.1 μg/mL) or collagen (0.1 μg/mL) combined with E7820 (0.05 μg/mL) for 72 h. E, Bladder tumor weights of mice treated with collagen (1 μg) or collagen (1 μg) combined with E7820 (50 μg) on days 4 and 8. Error bars, mean ± SEM. *P < .05; **P < .01. n.s, not statistically significant

Figure 4

Collagen‐induced tumor progression by activation of the PI3K/AKT signaling pathway. A, Immunofluorescence of p‐PI3K in MB49 cells treated with collagen (0.1 μg/mL), collagen (0.1 μg/mL) combined with E7820 (0.05 μg/mL), or PBS for 72 h. Scale bar, 10 μm. B, Immunofluorescence of p‐AKT in MB49 cells treated with collagen (0.1 μg/mL), collagen (0.1 μg/mL) combined with E7820 (0.05 μg/mL), or PBS for 72 h. Scale bar, 10 μm. C, Relative numbers of MB49 cells treated with collagen (0.1 μg/mL), or collagen (0.1 μg/mL) combined with LY294002 (0.5 μmol/L) or MK‐2206 (10 nmol/L) for 72 h. D, Relative colony numbers of MB49 cells treated with collagen (0.1 μg/mL), or collagen (0.1 μg/mL) combined with LY294002 (0.5 μmol/L) or MK‐2206 (10 nmol/L) for 72 h. E, Relative numbers of T24 cells treated with collagen (0.1 μg/mL), or collagen (0.1 μg/mL) combined with LY294002 (0.5 μmol/L) or MK‐2206 (10 nmol/L) for 72 h. F, Relative colony numbers of T24 cells treated with collagen (0.1 μg/mL), or collagen (0.1 μg/mL) combined with LY294002 (0.5 μmol/L) or MK‐2206 (10 nmol/L) for 72 h. G, Bladder tumor weights of mice treated with collagen (1 μg), or collagen (1 μg) combined with LY294002 (50 μg) or MK‐2206 (2 μg) on days 4 and 8. Error bars, mean ± SEM. *P < .05; **P < .01. n.s, not statistically significant

Figure 5

Combination of integrin α2β1 and chemotherapeutic agents enhanced the anticancer effects in bladder cancer treatment. A, Incidence of blood urine on day 12 in mice treated with PBS, doxorubicin (DOX; 0.1 mg), or DOX (0.1 mg) combined with E7820 (50 μg) on days 4 and 8. B, Bladder tumor weights in mice on day 12 treated with PBS, DOX (0.1 mg), or DOX (0.1 mg) combined with E7820 (50 μg) on days 4 and 8. C, Survival time of mice with bladder tumors treated with PBS, DOX (0.1 mg), or DOX (0.1 mg) combined with E7820 (50 μg) on days 4 and 8. D, Bladder tumor weight in mice on day 12 treated with PBS, mitomycin C (MMC; 0.1 mg), or MMC (0.1 mg) combined with E7820 (50 μg) on days 4 and 8. E, Bladder tumor weights in mice on day 12 treated with PBS, hydroxycamptothecine (HCPT; 0.05 mg), or HCPT (0.05 mg) combined with E7820 (50 μg) on days 4 and 8. Error bars, mean ± SEM. *P < .05; **P < .01. n.s, not statistically significant