Tumor microenvironment B cells increase bladder cancer metastasis via modulation of the IL-8/androgen receptor (AR)/MMPs signals

Abstract

While B cells in the tumor microenvironment may play important roles in cancer progression, their impacts on the bladder cancer (BCa) metastasis remain unclear. Here we found from human clinical BCa samples that BCa tissues could recruit more B cells than the surrounding normal bladder tissues and the in vitro co-culture assay also demonstrated that B cells could be recruited more easily towards BCa cells compared to normal bladder cells. Chamber invasion and 3D invasion assays showed the recruited B cells could then significantly increase the BCa cell invasion. Mechanism dissection found that recruited B cells could increase IL-8/androgen receptor (AR) signals in BCa cells that could then promote the expression of metastasis genes including MMP1 and MMP13. Blocking the IL-8/AR/MMPs signals either by anti-IL-8 neutralizing antibody, AR-siRNA, or MMPs inhibitors all partially reversed the infiltrating B cells capacity to increase the BCa cell invasion. The in vivo data from orthotopically xenografted BCa mouse model also confirmed that infiltrating B cells could increase BCa cell invasion via increasing AR signals. Together, these results demonstrate the key roles of B cells within the bladder tumor microenvironment that increase the BCa metastasis and may help us to develop the potential therapies via targeting these newly identified IL-8/AR/MMPs signals to better battle the BCa progression.

Keywords: B cell; androgen receptor; bladder cancer; tumor metastasis; tumor microenvironment.

Figure 1. Bladder cancer tissues/cells can better recruit B cells than non-malignant tissues/urothelial cells

a. More B cells infiltration was noted in BCa tissues compared to adjacent normal bladder area tissues. IHC staining of human bladder tissues was conducted using anti-CD20 antibody (N = 24). b. Cartoon shows the transwell B cells recruitment assay. Conditioned media (CM) of BCa cells or SVHUC cells was plated into the lower chambers of the transwells. 1 × 10⁵ B cells were plated onto the upper chambers with 5 μm pore polycarbonate membranes. The B cells migrated into the lower chambers were collected after 6 hrs and counted. Data are presented as mean ± SD. *P < 0.05 by student's t-test.

Figure 2. B cells can promote BCa cells migration and invasion

a. We co-cultured TCCUSP, T24 and J82 cells with B cells for 3 days. The 1 × 10⁵ co-cultured BCa cells were seeded into the upper chambers (with 8 μm size pore) to perform migration assays, 1 × 10⁵ BCa cells without co-culture with B cells were used as controls. After 24 hrs, 0.1% crystal violet blue staining results show BCa cells co-cultured with B cells had a higher invasive capacity as compared to control cells. b. BCa cells were subjected to invasion assays using 8 μm size pore chambers coated with matrigel. Image shows BCa cells co-cultured with B cells have a higher ability for migration than BCa cells alone (*P < 0.05). c. 3D invasion assay showed that more protrusions structures formed in co-cultured J82 cells than in J82 cells alone. The right panels in A, B, C are the quantification data of left panels. (*P < 0.05).

Figure 3. B cells could promote BCa cell invasion via up-regulation of AR/MMP1/MMP13 signaling

a–b. qRT-PCR (A) and Western blot (B) results showed AR expressions were increased in TCCSUP, T24 and J82 cells after co-culture with B cells. c. Validation of AR siRNA knocking down efficiency in TCCSUP and J82 using western blot. d. Knocking down AR in BCa cells can reverse the effects of B cells on BCa cell invasion ability. e. qRT-PCR and Western blot results show MMP1 and MMP13 expression were increased in TCCSUP and J82 cells after co-culture with B cells. f. qRT-PCR (left panels)and Western blot (right panels) results show MMP1 and MMP13 expressions were increased in TCCSUP and J82 cells after overexpressing AR (oeAR). g. Invasion assays show B cells promotion of TCCSUP and J82 cells invasion can be partly reversed by MMP1 or MMP13 inhibitor. *P < 0.05 by student's t-test. NS: not significant.

Figure 3. B cells could promote BCa cell invasion via up-regulation of AR/MMP1/MMP13 signaling

a–b. qRT-PCR (A) and Western blot (B) results showed AR expressions were increased in TCCSUP, T24 and J82 cells after co-culture with B cells. c. Validation of AR siRNA knocking down efficiency in TCCSUP and J82 using western blot. d. Knocking down AR in BCa cells can reverse the effects of B cells on BCa cell invasion ability. e. qRT-PCR and Western blot results show MMP1 and MMP13 expression were increased in TCCSUP and J82 cells after co-culture with B cells. f. qRT-PCR (left panels)and Western blot (right panels) results show MMP1 and MMP13 expressions were increased in TCCSUP and J82 cells after overexpressing AR (oeAR). g. Invasion assays show B cells promotion of TCCSUP and J82 cells invasion can be partly reversed by MMP1 or MMP13 inhibitor. *P < 0.05 by student's t-test. NS: not significant.

Figure 4. AR transcriptionally up-regulates MMP1 and MMP13 by binding to their promoters

a. Predicted localization of AREs in MMP1 (top lane) and MMP13 promoter region (bottom lane). b. J82-oeAR/vector cells were transfected with MMP1/MMP13-pGL3 luciferase reporter plasmid and pCMVtk vector was used as an internal control for transfection efficiency. After 36 hrs transfection, luciferase activities in triplicate samples were measured. (*P < 0.05) c. ChIP analysis of AR binding to the MMP1/MMP13 promoters.

Figure 5. Mechanisms why infiltrating B cells could increase BCa cells AR expression with increased invasion

a. qRT-PCR results showed IL-8 expressions were significantly increased in both B cells and BCa cells after co-culture Ramos (TCCSup) or Ramos (J82) stands for Ramos cells after co-culture with TCCSUP cells or J82 cells; TCCSUP (Ramos) or J82 (Ramos) stands for TCCSUP or J82 cells after co-culture with Ramos cells. b. B cells promotion of BCa cells invasion can be partly reversed by anti-IL-8 neutralizing antibody. c. qRT-PCR (upper panels) and Western blot (lower panels) results showed that blocking IL-8 using anti-IL-8 neutralizing antibody can partially reverse the B cells-mediated AR, MMP1 and MMP13 up-regulation in BCa cells. d. qRT-PCR results showed rhIL-8 can up-regulate AR, MMP1 and MMP13 expression in BCa cells. e. rhIL-8 can promote BCa cells invasion. (*P < 0.05).

Figure 5. Mechanisms why infiltrating B cells could increase BCa cells AR expression with increased invasion

a. qRT-PCR results showed IL-8 expressions were significantly increased in both B cells and BCa cells after co-culture Ramos (TCCSup) or Ramos (J82) stands for Ramos cells after co-culture with TCCSUP cells or J82 cells; TCCSUP (Ramos) or J82 (Ramos) stands for TCCSUP or J82 cells after co-culture with Ramos cells. b. B cells promotion of BCa cells invasion can be partly reversed by anti-IL-8 neutralizing antibody. c. qRT-PCR (upper panels) and Western blot (lower panels) results showed that blocking IL-8 using anti-IL-8 neutralizing antibody can partially reverse the B cells-mediated AR, MMP1 and MMP13 up-regulation in BCa cells. d. qRT-PCR results showed rhIL-8 can up-regulate AR, MMP1 and MMP13 expression in BCa cells. e. rhIL-8 can promote BCa cells invasion. (*P < 0.05).

Figure 6. B cells promote BCa metastasis using In vivo orthotopic BCa model

a. J82 cells were mixed with Ramos B cells and orthotopically implanted into bladders of nude mice. 6 weeks after implantation, the bladder tumors growth and metastasis were monitored by IVIS images. b. The images show bladder tumors (black arrows) and metastatic lymph nodes foci (red arrows). c. Quantification data for tumor metastases in mouse BCa model (*P < 0.05). d. Quantification data for tumor metastatic foci (*P < 0.05). e. IHC staining for AR, MMP1 and MMP13 in orthotopic bladder tumor tissues (400X).