Fig 1.
Study treatment schema and antitumor activity in BBN-induced mause bladder cancer model.
(A) Schematic diagram illustrating the study work flow. Mice were given 0.05% BBN in drinking water, continuously for 16 weeks, and were then randomly divided into six groups (N = 15 per group). Starting with week 17, mice were treated once a week for 4 weeks with PBS, BCG, MMC, ADM, GEM, and DTX. One week after the last treatment, mice were euthanized and bladder, spleen, whole blood by cardiac puncture, and urine were harvested. (B) Illustration showing the procedure of intravesical instillation and occlusion of the urethra with a purse string suture. Chemotherapeutic agents are delivered by transurethral instillation through a catheter allowed to dwell in the bladder for 2 h. (C) Intravesical treatment with chemotherapeutic agents causes significant bladder weight loss compared to control (Mann-Whitney U test; * = P< 0.05). (D) Representative images of hematoxylin-eosin-stained bladder samples from each group. All chemotherapeutic agents have anti-tumor effect similar to BCG treatment in orthotopic model.
Fig 2.
Representative images of bladder samples from each treatment group stained with six immunological markers.
Tumoral infiltration of cells positive for CD4, CD8, CD56, CD204, Foxp3, and PD-L1 were noted. Expression level of each marker compared to control. CD4+ T cells, CD8+ T cells and NK cells were induced by BCG. Tregs and TAM were decreased in MMC and ADM.
Table 1.
The summary of immunohistochemical staining analysis and ELISAaray of serum.
Fig 3.
Representative images of human bladder cancer tissues from each treatment group stained with two immunological markers.
Tumoral infiltration of cells positive for CD204 and Foxp3 were noted. (A) M2 macropages are reduced after intravesical treatment with MMC and ADM compared to primary tumors (Mann-Whitney U test; * = P<0.05). (B) Tregs are reduced after intravesical treatment with MMC and ADM compared to primary tumors (Mann Whitney U test; * = P<0.05).
Fig 4.
Relative concentration of serum cytokines as determined by the ELISArray Kit.
(A) IL-17A is induced by BCG and MMC. (B) G-CSF is induced by BCG, MMC, ADM, and DTX.
Fig 5.
Time-course analysis of urinary IL-17A and G-CSF levels by ELISA.
(A, B) IL-17A displays a gradual increase with BCG and decrease with MMC. (C, D, E, F) G-CSF shows an increase with BCG, ADM, DTX, and especially MMC (Student’s t-test; * = P<0.05).
Fig 6.
Changes in the populations of blood cells caused by intravesical treatment.
(A) The number of neutrophils is increased in BCG, MMC, and ADM. (B) The number of lymphocytes is decreased in MMC. (C) The number of monocytes is increased in ADM (Mann-Whitney U test; * = P<0.05).
Fig 7.
Schematic summary of the study.
Intravesical treatment with chemotherapeutic agents shows anti-tumor activity through direct cytotoxicity and indirectly through modulation of anti-tumor immunity. Cytotoxic T cells recognize a cancer-specific antigen from apoptotic cancer cells and eliminate these cells. In addition, dendritic cells (DC) capture the antigen and present it to CD4+ T cells. Activated CD4+ T cells produce cytokines resulting in activation of helper T1 (Th1) cells and recruitment of cytotoxic T cells, NK cells, and macrophages through cytokines. IL-17A and G-CSF suppress protumoral immunity and enhance anti-tumor immunity (red allow: anti-tumor effect, black allow: general immunoresponse, blue allow: regulation by serum IL-17A and G-CSF, green allow: inhibition of immune-related cells, yellow allow: urine IL-17A and G-CSF as markers).