The objective of the present study was to investigate the association of B7-H3 expression and cluster of differentiation (CD)163+ tumor-associated macrophage (TAM) infiltration with clinicopathological parameters in urothelial cell carcinoma of the bladder (UCB), and to investigate their potential conjoint effects on progression of UCB. B7-H3 expression and CD163+ TAM infiltration in tumor specimens from 134 consecutive patients that underwent radical cystectomy for UCB were tested using immunohistochemistry, followed by statistical analysis. In these 134 patients, B7-H3 expression and CD163+ TAM infiltration in the bladder carcinoma tissues were significantly associated with an increased ratio of vascular invasion (P=0.009; P=0.012) and distant metastasis (P=0.015; P=0.038); however, they were not associated with gender, age, pathologic grade, tumor stage, recurrence or lymphatic metastasis. The results of χ2 test analysis indicated that CD163+ TAM infiltration and B7-H3 expression were positively correlated (χ2=20.714; P<0.001). Overall survival (OS) and progression-free survival (PFS) rates were significantly worsened by high B7-H3 expression (P=0.002; P=0.020). However, CD163+ TAM infiltration was not associated with OS or PFS rate. Notably, the OS and PFS rates in patients with high B7-H3 expression or high CD163+ TAM infiltration were significantly poorer than the patients with low B7-H3 expression (P<0.001; P<0.001) or low CD163+ TAM infiltration (P=0.022; P=0.017) in the subgroup of 115 patients with muscle-invasive bladder cancer. The results of the present study indicate that B7-H3 expression level could be used as an independent prognostic indicator following radical cystectomy for UCB and patients with high B7-H3 expression and high CD163+ TAM infiltration experience a poorer prognosis.
Bladder cancer is the second most common malignancy of the urinary system, with urothelial cell carcinoma of the bladder (UCB) being the most common type of bladder cancer (
The initiation and progression of tumors is closely associated with immune dysfunction in the tumor microenvironment. B7-H3 is a member of the B7 family of molecules, and serves an immunoregulatory function between tumor and immune cells (
Monocytes can enter tumor tissues through vessel walls, where they are able to differentiate into macrophages, designated tumor-associated macrophages (TAMs). TAMs are associated with tumor metastasis; conflicting studies regarding the positive or negative function of TAMs in survival outcomes exist for a number of human malignancies (
In the present study, which has 10 years of follow up data, the level of B7-H3 expression and the density of CD163+ TAM infiltration were investigated in postoperative tumor specimens from 134 patients undergoing radical cystectomy (RC) for UCB, and the associations with various clinicopathological features were analyzed. Survival analysis was performed to determine the prognostic significance of B7-H3 expression and the association of CD163+ TAMs with the postoperative survival of patients, and to evaluate the potential effects of these factors on the progression of UCB.
Data and tumor and adjacent non-tumor tissue samples were obtained during surgical resection from 134 consecutive patients (67.2±6.89 years old, 115 males and 19 females) who were followed-up following radical cystectomy (RC) for UCB in the Urology Department, Fourth Hospital of Hebei Medical University (Hebei, China), between March 2004 and October 2005, were analyzed. The criteria for RC were: Muscle-invasive bladder carcinoma, recurrent Ta/T1 stage non-muscle-invasive bladder carcinoma and carcinoma
The use of human samples and the research involving human participants in this study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University (Hebei, China). All participants provided written informed consent prior to participation in the present study.
Specimens were fixed using 10% formalin at room temperature for 4 h, followed by paraffin embedding. Samples were sliced into 5 µm thick sections. The latter were dewaxed in xylene at 60°C, and hydrated with serial dilutions of ethanol (100, 95, 90, 85 and 75%), heated in 1 mmol/l EDTA (pH 8.0) to 121°C, cooled to 90°C, and incubated for 5 min. The sections were submerged in 3% H2O2, followed by deionized water for 10 min to eliminate endogenous peroxidase activity, and blocked with goat serum at 37°C for 30 min (Origene Technologies, Inc., Beijing, China) for 30 min. Sections were incubated with primary mouse anti-human B7-H3 antibody (1:500; cat. no., ab134161;Abcam, Cambridge, UK) or mouse anti-human CD163 antibody (1:100; cat. no., GTX42364, EDHu-1; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 4°C overnight. The sections were incubated with horseradish peroxidase-conjugated goat anti-mouse Immunoglobulin G (1:500; cat. no., TA130004, Origene Technologies, Inc.) as the secondary antibody at room temperature for 30 min, and rendered with diaminobenzidine for 3–5 min. Subsequently, sections were counterstained with 5% hematoxylin at room temperature for 10 min, dehydrated in ethanol at room temperature (75 for 30 sec, 85 for 30 sec, 90 for 30 sec, 95 for 30 sec and 100% for 10 sec), cleared in 100% xylene for 30 sec, and mounted on coverslips. PBS was used to replace the primary antibody as the negative control.
A total of 5 random fields of view on each stained slide were viewed by light microscopy at magnification, ×400, with the positive rate of every 100 tumor cells counted each view, and the immunohistochemical results were analyzed according to the tumor cells positively-dyed percentage (percentage=number of positively dyed cells in each 100 cells counted) and strength evaluation. The scoring method of B7-H3 expression was based on the stained area and intensity of staining. The frequencies of positive cells were categorized into low, medium and high groups that represented <33%, 34–67% and >67% positively-stained cells, respectively (
The density of CD163+ TAM infiltration was based on the number of CD163+ cells per field(magnification, ×200). Quantification was performed as described previously (
Data were analyzed with SPSS software (version 19.0; IBM Corp., Armonk, NY, USA). Data are presented as the mean ± standard error of the mean. Associations betweenB7-H3 expression or CD163+ TAM infiltration and clinicopathological features were evaluated using χ2 tests and Fisher's exact tests. The correlation between B7-H3 staining and CD163+ TAM infiltration were analyzed with χ2 test. Associations of survival and tumor progression with B7-H3 expression or CD163+ TAM infiltration were estimated using the Kaplan-Meier method, and differences were assessed using log-rank tests. All tests were two sided and P<0.05 was considered to indicate a statistically significant difference.
Representative images of weak, moderate, and strong intensity of B7-H3 expression are presented in
Representative images of weak, moderate and strong expression CD163+ TAM infiltrations are presented in
Among the 75 UCB patients exhibiting a low density of CD163+ TAM infiltration, 35 (46.7%) exhibited a low level of B7-H3 expression. Of the 59 patients with a high density of CD163+ TAM infiltration, 53 cases (89.8%) also exhibited a high level of B7-H3 expression. The results of Spearman's rank correlation analysis indicated that CD163+ TAM infiltration and B7-H3 expression were positively correlated (rs=0.393, P<0.001,
Kaplan-Meier survival analyses revealed that the estimated cancer overall survival (OS) rates at 1, 5 and 10 years following RC for patients with high B7-H3 expression were 96.8±1.8, 23.9±4.9 and 13.6±6.6%, respectively, compared with 97.6±2.4, 40.2±7.8 and 27.8±8.2% for patients with low B7-H3 expression, respectively. The estimated progression free survival (PFS) rates at 1, 5 and 10 years following RC for patients with high B7-H3 expression were 68.5±4.8, 22.1±5.6 and 11.0±8.3%, respectively, compared with 90.1±4.7, 34.1±7.8 and 25.9±.0% for patients with low B7-H3 expression respectively. Thus, high B7-H3 expression was significantly associated with decreased OS (P=0.002) and PFS (P=0.020) in 134 patients with UCB (
In this cohort of 134 patients with UCB, a positive correlation was identified between the level of B7-H3 expression and CD163+ TAM infiltration, which were significantly associated with vascular invasion, distant metastasis and poor outcomes in the patients undergoing RC for UCB, indicating that B7-H3 may be involved in the progression of UCB with CD163+ TAM infiltration, and that B7-H3 may be a novel immunotherapeutic target for patients with UCB.
B7-H3, a member of the B7 family, has been demonstrated to mediate the proliferation of CD4+ and CD8+ T cells, and to enhance interferon-γ (IFN-γ) production (
Numerous growth factors, cytokines, chemokines and enzymes released by TAMs promote tumor progression and poor survival rates (
The rates of vascular invasion and distant metastasis were significantly elevated in the subgroup of patients with high B7-H3 expression or high CD163+ TAM infiltration. The results of χ2 test analysis revealed that there was a positive correlation between CD163+ TAM infiltration and B7-H3 expression, indicating that B7-H3 and TAMs may be involved in the initiation and malignant progression of UCB and jointly exhibit immunosuppressive effects on the tumor microenvironment. Pro-tumor TAM polarization facilitated by over-expression of B7-H3 expression maybe involved in the B7-H3/signal transducer and activation of transcription 3 signaling pathway (
As a novel co-regulatory molecule, the immunological function of B7-H3 remains controversial, which may reflect the potential presence of two receptors for B7-H3 with opposing co-regulatory functions on the proliferation of CD4+ T cells and induction of cytotoxic T cells (
The present study represents a valuable evaluation of the prognostic significance of B7-H3 and CD163+ TAM infiltration in UCB; however, the present study is not devoid of limitations. Although the identification of CD163+ TAM infiltration has been demonstrated to be associated with a poor prognosis in several types of cancer, including UCB (
To conclude, the expression levels of the co-stimulatory molecule B7-H3 and CD163+ TAM infiltration were investigated in tumor specimens following RC for UCB. Patients with high B7-H3 expression and high CD163+ TAM infiltration exhibited malignant progression and poor survival rates, particularly those with muscle-invasive bladder cancer. It was also identified that B7-H3 expression level maybe used as a valuable prognostic indicator following RC for UCB. B7-H3 and CD163+ TAM infiltration may function as prognostic indicators and, with additional study, may be a novel immunotherapeutic target for patients with UCB.
Not applicable.
No funding was received.
All data generated or analyzed during this study are included in this published article.
ZX and BS conceived and designed the experiments. ZX, LW, JT, HM and PL performed the experiments. LW, JT, HM and PL analyzed the data. ZX, LW, JT, HM, PL and BS contributed reagents, materials and analytical tools. ZX, LW and BS wrote the paper.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The use of human samples and the research involving human participants in this study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University (Hebei, China). All participants provided written informed consent prior to participation in the present study.
The patients, or legal guardians, have provided written informed consent for the publication of any associated data and accompanying images.
The authors declare that they have no competing interests.
Images of B7-H3 expression and CD163+ tumor-associated macrophage infiltration in urothelial cell carcinoma of the bladder tissues. Weak, moderate, and strong intensity of B7-H3 and CD163 expression are presented (magnification, ×200). Left panels, expression levels of B7-H3 protein. Right panels, expression of CD163 protein in the same samples as the adjacent section. CD163, cluster of differentiation 163.
Association of B7-H3 expression and CD163+ TAM infiltration with OS and PFS in patients with UCB. (A) OS rates with B7-H3 expression in cohort of 134 patients. Shorter OS times were observed in patients with high B7-H3 expression compared with low B7-H3 expression (P=0.002). (B) PFS rates with B7-H3 expression in cohort of 134 patients. Shorter PFS times were observed in patients with high B7-H3 expression than that in in low B7-H3 expression (P=0.020). (C) OS rates with B7-H3 expression in the subgroup of patients with muscle-invasive bladder cancer. Shorter OS times were observed in patients with high B7-H3 expression compare with low B7-H3 expression (P<0.001). (D) PFS rates with B7-H3 expression in the subgroup of patients with muscle-invasive bladder cancer. Shorter PFS times were observed in patients with high B7-H3 expression compared with low B7-H3 expression (P<0.001). (E) OS rates with CD163+ TAM infiltration in the subgroup of patients with muscle-invasive bladder cancer. Shorter OS times were observed in patients with high CD163+ TAM infiltration compared with low CD163+ TAM infiltration (P=0.022). (F) PFS rates with CD163+ TAM infiltration in the subgroup of patients with muscle-invasive bladder cancer patients. Shorter OS times were observed in patients with high CD163+ TAM infiltration compared with low CD163+ TAM infiltration (P=0.017). CD163, cluster of differentiation 163; OS, overall survival; PFS, progression-free survival; UCB, urothelial cell carcinoma of the bladder; TAM, tumor-associated macrophages.
Association between B7-H3 or CD163+ TAMs and clinicopathological features in 134 patients with UCB.
B7-H3 expression | CD163+ TAM infiltration | ||||||
---|---|---|---|---|---|---|---|
Feature | Patients, n% | Low, n (%) | High, n (%) | P-value | Low, n (%) | High, n (%) | P-value |
Age, years | 0.067 | 0.634 | |||||
<65 | 53 (39.6) | 21 (51.2) | 32 (34.4) | 31 (41.3) | 22 (37.3) | ||
≥65 | 81 (60.4) | 20 (48.8) | 61 (65.6) | 44 (58.7) | 37 (62.7) | ||
Gender | 0.330 | 0.855 | |||||
Male | 115 (85.8) | 37 (90.2) | 78 (83.9) | 64 (85.3) | 51 (86.4) | ||
Female | 19 (14.2) | 4 (9.8) | 15 (16.1) | 11 (14.7) | 8 (13.6) | ||
Tumor stage | 0.202 | 0.220 | |||||
Ta/CIS/T1 | 19 (14.1) | 8 (19.5) | 11 (11.8) | 12 (16.0) | 7 (11.9) | ||
T2 | 58 (43.3) | 21 (51.2) | 37 (39.8) | 36 (48.0) | 22 (37.3) | ||
T3 | 40 (29.9) | 9 (22.0) | 31 (33.3) | 21 (28.0) | 19 (32.2) | ||
T4 | 17 (12.7) | 3 (7.3) | 14 (15.1) | 6 (8.0) | 11 (18.6) | ||
Tumor grade | 0.201 | 0.177 | |||||
Low | 27 (20.1) | 11 (26.8) | 16 (17.2) | 12 (16.0) | 15 (25.4) | ||
High | 107 (79.9) | 30 (73.2) | 77 (82.8) | 63 (84.0) | 44 (74.6) | ||
Lymph node metastasis | 0.185 | 0.364 | |||||
Yes | 40 (29.9) | 9 (22.0) | 31 (33.3) | 20 (26.7) | 20 (33.9) | ||
No | 94 (70.1) | 32 (78.0) | 62 (66.7) | 55 (73.3) | 39 (66.1) | ||
Recurrence | 0.306 | 0.192 | |||||
Yes | 71 (53.0) | 19 (46.3) | 52 (55.9) | 36 (48.0) | 35 (59.3) | ||
No | 63 (47.0) | 22 (53.7) | 41 (44.1) | 39 (52.0) | 24 (40.7) | ||
Distant metastasis | 0.015 | 0.038 | |||||
Yes | 57 (42.5) | 11 (26.8) | 46 (49.5) | 26 (34.7) | 31 (52.5) | ||
No | 77 (57.5) | 30 (73.2) | 47 (50.5) | 49 (65.3) | 28 (47.5) | ||
Vascular invasion | 0.009 | 0.012 | |||||
Yes | 84 (62.7) | 19 (46.3) | 65 (69.9) | 40 (53.3) | 44 (74.6) | ||
No | 50 (37.3) | 22 (53.7) | 28 (30.1) | 35 (46.7) | 15 (25.4) |
CD, cluster of differentiation; TAM, tumor-associated macrophage.
Correlation between B7-H3 expression and CD163+ TAM infiltration in UCB patients.
B7-H3 expression | |||||
---|---|---|---|---|---|
CD163+ TAM infiltration | Patients, n | Low, n 41 | High, n 93 | χ2 | P-value |
Low | 75 | 35 | 40 | 20.714 | <0.001 |
High | 59 | 6 | 53 |
CD, cluster of differentiation; Tam, tumor-associated macrophage.
OS and PFS rates at different levels of B7-H3 expression and CD163+ TAM infiltration in patients with UCB.
A, All patients (n=134) | |||||||||
---|---|---|---|---|---|---|---|---|---|
OS, % (±SE) | PFS, % (±SE) | ||||||||
Feature | n | 1 year | 5 years | 10 years | P-value | 1 year | 5 years | 10 years | P-value |
B7-H3 expression | 0.002 | 0.020 | |||||||
High | 93 | 96.8 (1.8) | 23.9 (4.9) | 13.6 (6.6) | 68.5 (4.8) | 22.1 (5.6) | 11.0 (8.3) | ||
Low | 41 | 97.6 (2.4) | 40.2 (7.8) | 27.8 (8.2) | 90.1 (4.7) | 34.1 (7.8) | 25.9 (8.0) | ||
CD163+ TAM infiltration | 0.074 | 0.090 | |||||||
High | 59 | 98.3 (1.7) | 22.0 (5.8) | 16.5 (6.4) | 65.5 (6.2) | 23.7 (5.6) | 15.8 (7.5) | ||
Low | 75 | 97.3 (1.9) | 35.3 (5.8) | 22.1 (6.1) | 82.6 (4.4) | 32.2 (5.7) | 22.8 (6.2) | ||
B7-H3 expression | <0.001 | <0.001 | |||||||
High | 79 | 98.7 (1.3) | 14.9 (4.5) | 0 (0.0) | 71.8 (5.1) | 16.8 (4.6) | 0 (0.0) | ||
Low | 36 | 100 (0.0) | 58.1 (8.7) | 47.7 (9.9) | 94.4 (3.8) | 52.0 (9.2) | 43.4 (11.0) | ||
CD163+ TAM infiltration | 0.022 | 0.017 | |||||||
High | 54 | 100 (0.0) | 15.9 (5.5) | 8.0 (6.3) | 73.6 (6.1) | 14.9 (5.3) | 0 (0.0) | ||
Low | 61 | 98.4 (1.6) | 43.2 (6.7) | 26.4 (7.6) | 81.9 (4.9) | 35.8 (6.8) | 25.1 (8.1) |
OS, overall survival; PFS, progression-free survival; SE, standard error; CD163, cluster of differentiation; TAM, tumor-associated macrophage; UCB, urinary carcinoma of the bladder.