The present study sought to estimate the applicability of apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1), vascular endothelial growth factor A (VEGFA) expression and CD163+ tumor-associated macrophage (TAM) ratio as prognostic factors in bladder cancer (BCa). A total of 127 patients with bladder urothelial cancer who underwent radical cystectomy at Daping Hospital were recruited between January 2013 and January 2017, including 45 cases of non-muscle invasive BCa (NMIBC) and 82 of MIBC. Immunohistochemical detection of APE1, VEGFA and CD163, as well as multiple immunofluorescence staining for APE1, VEGFA, CD163 and CD34, were performed on tissue samples. For APE1 and VEGFA, the staining was graded based on intensity (0–3), while CD163 was graded (0–3) based on the percentage of positively stained cells. The prognostic value of APE1, VEGF and CD163 was assessed using Kaplan-Meier and Cox regression analysis. The results suggested that in BCa, high APE1 expression was associated with high VEGFA expression and more infiltration of CD163+ TAM. Furthermore, high expression of APE1 was associated with lymphovascular invasion of BCa, as well as reduced survival time. This indicates that APE1 may be associated with CD163+ TAM infiltration in BCa, with VEGFA as a possible influencing factor.
Bladder cancer (BCa) is one of the most common types of urological cancer, resulting in ~150,000 deaths worldwide every year (
Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) protein, also known as redox factor-1 (Ref-1), was originally identified as a multifunctional protein involved in DNA base excision repair (BER) and redox signaling. Previous studies have indicated that APE1 expression is not only significantly higher in multiple cancer types, including liver cancer, ovary cancer and osteosarcoma, but also highly associated with prognosis (
Previously, Sun and Nelson (
Therefore, the present study sought to evaluate the association among the expression of APE1 and vascular endothelial growth factor A (VEGFA) and the infiltration of M2 macrophages, as detected via the M2 marker CD163, as well as their prognostic significance in BCa. To the best of our knowledge, the present study was the first to implicate APE1 as a key factor in the DNA damage response with polarization of TAM, laying a foundation for elucidating the mechanism of the recurrence of BCa and exploring the efficacy of immunotherapy for this neoplasm.
A total of 167 patients with BCa subjected to radical cystectomy at Daping Hospital between January 2013 and January 2017 were recruited without any restriction by age or sex. Data collection was closed by May 2019. Those patients who did not have a reported event of death at the time of closure were included for calculation of the median follow-up time. Patient follow-up was usually performed every 3 months in the first year post-surgery and then every 6 months. The cause of death was determined by chart review from patients' medical records or from the death certificate. The follow-up time was 4–78 months and the median follow-up time was 48 months. Cases with non-urothelial carcinoma types and patients who died of causes not associated with cancer were excluded, and cases without precise follow-up were also excluded, resulting in 127 remaining cases. All subjects were from a Han Chinese population and were subjected to radical cystectomy at Daping Hospital (Chongqing, China) with the diagnosis made based on pathology. The general characteristics of the patients are summarized in
Tumor tissues were fixed overnight in 4% paraformaldehyde, dehydrated, embedded in paraffin and sectioned into 8–10 mm (RM2235; Leica Microsystems). Experienced pathologists blinded to the clinical outcomes reviewed these pathological specimens. The tumors were staged according to the 8th AJCC TNM staging classification (
Multiple immunofluorescence staining was performed on paraffin-embedded tissues. Sections of 4 µm thickness were cut from selected BCa tissues. The slides were deparaffinized in xylene, rehydrated and washed with tap water prior to boiling in Tris-EDTA buffer (pH 9, 643901; Klinipath) for epitope retrieval/microwave treatment. Endogenous peroxidase was blocked using Antibody Diluent/Block (cat. no. 72424205; Perkin Elmer) and incubated at room temperature. Protein blocking was performed using Antibody Diluent/Block at 37°C for 1 h. The slides were incubated with primary antibodies to CD163 (1:150 dilution; cat. no. ZM0428; Zsbio), APE1 (1:2,000 dilution; cat. no. ab194; Abcam) and VEGFA (1:100 dilution; cat. no. ab52917; Abcam) for 1 h at 37°C, and then incubated with antibody to CD34 (cat. no. kit-0004; MXB) at 4°C overnight. Next, incubation with Opal Polymer HRP Ms+Rb (cat. no. 2414515; Perkin Elmer) was performed at 37°C for 10 min. Tyramide signal amplification (TSA) visualization was performed with the Opal eight-color IHC kit (cat. no. NEL797B001KT; Perkin Elmer), containing fluorophores DAPI, Opal 690 (CD34), Opal 650 (VEGFA), Opal 570 (APE1) and Opal 520 (CD163), as well as a TSA Coumarin system (cat. no. NEL703001KT; Perkin Elmer).
Slides were scanned using the Perkin Elmer Vectra (Vectra 3.0.5; Perkin Elmer). Multispectral images were unmixed using spectral libraries built from images of single stained tissues for each reagent using the inform Advanced Image Analysis software (inForm 2.3.0; Perkin Elmer). A selection of 5–10 representative original multispectral images was used to train the inForm software (tissue segmentation, cell segmentation, phenotyping tool and positivity score). All the settings applied to the training images were saved within an algorithm to allow the inForm software (PerkinElmer; v2.3.0) batch analysis of multiple original multispectral images of the same tissue (
Data analysis was performed using SPSS (version 22; IBM, Corp.) and GraphPad Prism 7 (GraphPad Software, Inc.). Cumulative survival probabilities were estimated using the Kaplan-Meier method and differences between survival rates were tested for significance using the log-rank test. Univariate and multivariate Cox regression analyses were calculated using SPSS. Hazard ratios (HRs) and 95% confidence intervals (95% CI) were used to present the associations of dependent and independent variables with the risk of mortality. The χ2 test was employed to assess differences between two groups. All tests were two-sided with a 95% CI and P<0.05 was considered to indicate statistical significance. Receiver operating characteristic (ROC) analysis was conducted for further evidence. Correlation of APE1, VEGFA and CD163 expression in muscle invasive bladder cancer tissues with multiple immunofluorescence staining was analyzed with Pearson correlation.
IHC was used to estimate the expression of APE1 and VEGFA, as well as the ratio of CD163+ TAMs, and the association between each of them with the clinicopathological characteristics was determined. APE1 was mainly localized in the nuclei with a certain amount residing in the cytoplasm, whereas VEGFA appeared in the cytoplasm only (
The correlation between the expression of APE1 and VEGFA and the proportion of CD163+ M2 macrophages was then assessed. The results suggested that high expression of APE1 in tumor cells was positively correlated with the grade regarding CD163+ TAMs (r=0.196, P=0.027), as well as with VEGFA expression (r=0.208, P=0.018). Furthermore, VEGFA expression was strongly correlated with the grade regarding CD163+ TAMs (r=0.408, P<0.001;
KM analysis revealed that high APE1 expression and a high CD163+ TAM ratio were associated with a shorter OS (
Multiple nomograms and models have been constructed in the past to predict the outcomes of Bca (
The presence of LVI, defined as the presence of tumor cells in the lymphatic vessel and in vascular walls, has been reported to be of prognostic value in patients with Bca. Evidence suggests that LVI is a characteristic of biologically and clinically aggressive BCa and may be of prognostic value. In the present study, 34 out of 127 patients were identified to have LVI, 32 of which had MIBC and 2 had NMIBC (
Aromatic amines are well-known risk factors for BCa (
Previous studies have indicated that high expression of APE1 in lung cancer and osteosarcoma is associated with upregulated VEGF through hypoxia-inducible factor-1α activation and retardation of APE1 results in a significant drop in VEGF expression (
VEGF and its receptors have profound effects on the early development and differentiation of both vascular endothelial and hematopoietic progenitors. Kloepper
Macrophage infiltration is associated with significantly better cancer-specific survival in patients with CD163+ TAM-associated tumors. A meta-analysis of 1,400 cases of BCa including 13 studies further indicated that only CD163+ TAMs, not CD68+ TAMs, were closely associated with OS, recurrence-free survival and progression-free survival (
Recently, Hudson
In the univariate linear regression analysis performed in the present study, VEGFA was indicated to have no association with OS but it was a confounding factor in the multivariate analysis. This is possibly due to several reasons: i) The number of cases was too low to accurately present the association; ii) VEGFA may not be a decisive factor on OS in BCa, but may still be a contributing factor due to its association with other meaningful factors, including APE1 and CD163 expression. Further studies on this mechanism will be required to draw a conclusion.
The present study demonstrated that high APE1 expression is associated with the presence of LVI in BCa. APE1 expression was also determined to be positively associated with VEGFA expression and increased infiltration of CD163+ TAM. LVI, high APE1 expression and a high ratio of CD163+ TAM in BCa may lead to a reduced survival time of patients. The results may add valuable insight to optimize models predicting BCa prognosis, consequently guiding perioperative treatments that benefit patients the most. The present study also indicated that there may be a mechanism by which high APE1 expression contributes to an increase of M2-type TAMs by definition as CD163+, possibly involving regulation of VEGFA expression through its redox function. However, the present results are limited in that only patients subjected to radical cystectomy were recruited, and studies including patients with broader recruitment criteria (not only restricting to patients who underwent cystectomy but, for example, also those who underwent transurethral resection) will provide a better analysis. In addition, only paraffin-embedded tissues were assessed and there was a lack of fresh tissues, which may limit the conclusions of the present study. Further prospective studies with standardized protocols should be performed to fully assess the impact of LVI, APE1 and VEGFA expression, as well as the CD163+ TAM ratio, on the outcomes for patients with BCa.
Not applicable.
This study was supported by the National Natural Science Foundation of China (grant nos. 81772704 and 81972398 to JJ) and the Clinical Medical Research Personnel Training Program of Army Medical University (grant no. 2018XLC3076 to LW).
The datasets used and analyzed during the present study are available from the corresponding author upon reasonable request.
LAW contributed to the conception and design, acquisition of data, analysis and interpretation of the data and IHC of the tissue and was a major contributor in writing the manuscript. BY was a major contributor to the analysis and interpretation of the data. TT contributed to the acquisition of data. YY, JX and LL contributed to the IHC analysis of the tissues. DZ contributed to the writing and critical revision of the manuscript, as well as towards the conception of the study. HX is an experienced pathologist and contributed to reviewing and analyzing the results of IHC and multiple immunofluorescence. LW contributed to the retrieval of funding for the study and revision of the manuscript. JJ contributed to the conception and design, writing and revision of the manuscript and retrieval of funding for the study. All authors read and approved the final manuscript.
This study involved the use of patient tissues and data. Approval was acquired from the Ethics Committee of the Research Institute of Surgery and Daping Hospital, Army Medical University (Chongqing, China), serial number 2018 No.25. Written informed consent was obtained from a patient representative who is involved in this study for the usage of the lesion tissue for research purposes was acquired at the time-point of surgery.
Not applicable.
The authors declare that they have no competing interests.
(A) Representative immunohistochemistry results for APE1, VEGFA and CD163 indicated that APE1 staining was mainly located in the nuclei and partly in the cytoplasm and VEGFA was present in the cytoplasm of tumor cells, while CD163 was expressed on the infiltrating macrophages (magnification, ×200). The staining was rated as 0, 1+, 2+ or 3+ based on intensity for APE1 and VEGFA and based on the percentage of positive cells for CD163. (B) Heatmap presentation of the gradings of APE1, VEGFA and CD163 among the 127 patients. VEGFA, vascular endothelial growth factor A; APE1, apurinic/apyrimidinic endodeoxyribonuclease 1.
Multiple immunofluorescence staining for APE1, VEGFA, CD163 and CD34 in bladder cancers. (A) Representative sample with high expression of APE1 and VEGFA in which the intratumor CD163-labeled M2 macrophages are easily distinguishable. (B) Example of a sample with high expression of APE1 and VEGFA in which the CD163-labeled M2 macrophages are easily distinguishable in the tumor stroma. (C) Representative sample with low expression of APE1 and VEGFA, in which the CD163-labeled M2 macrophages are less distinguishable. APE1-positive staining in tumor epithelial cells displayed as orange and VEGFA-positive staining in tumor epithelial cells was cyan, while CD163-positive infiltrating M2 macrophages were green and CD34-positive vascular endothelial cells displayed as magenta (magnification, ×200). VEGFA, vascular endothelial growth factor A; APE1, apurinic/apyrimidinic endodeoxyribonuclease 1.
Factors influencing the survival of patients with bladder cancer. (A-C) Kaplan-Meier survival curves; (A) High expression of APE1 was associated with shorter OS; (B) VEGFA expression was not significantly associated with OS; (C) a high ratio of CD163+ tumor-associated macrophages was associated with shorter OS. (D) ROC curves of APE1, VEGFA and CD163 were assessed with T stage and N stage for best prediction efficiency. VEGFA, vascular endothelial growth factor A; APE1, apurinic/apyrimidinic endodeoxyribonuclease 1; OS, overall survival; ROC, receiver operating characteristic; m, months.
Univariate and multivariate COX analysis of the prognosis of IHC marker for OS.
Univariate | Multivariate | |||||
---|---|---|---|---|---|---|
Variable | HR | 95% CI | P-value | HR | 95% CI | P-value |
Age |
1.492 | 0.829–2.687 | 0.182 | |||
Sex |
1.047 | 0.413–2.654 | 0.923 | |||
Smoking |
0.913 | 0.479–1.741 | 0.783 | |||
LVI |
2.974 | 1.642–5.387 | <0.001 | |||
T-stage |
8.279 | 2.948–23.255 | <0.001 | 12.857 | 3.883–42.575 | <0.001 |
N-stage |
4.265 | 2.262–8.045 | <0.001 | |||
Grade |
2.201 | 0.530–9.135 | 0.277 | |||
APE1 |
2.797 | 1.486–5.267 | 0.001 | 3.644 | 1.863–7.129 | <0.001 |
VEGFA |
0.860 | 0.479–1.544 | 0.613 | 0.410 | 0.217–0.773 | 0.006 |
CD163 |
2.425 | 1.300–4.521 | 0.005 | 2.883 | 1.480–5.617 | 0.002 |
Age, ≤65 vs. >65 years
sex, male vs. female
smoking: positve smoking history vs. negative smoking history
LVI, lymphovasular invasion vs. no lymphovascular invasion
T-stage, Tis and T1 vs. T2, T3 and T4
N-stage, N0 vs. N1 and N2
grade, high grade vs. low grade
APE1, high APE1 expression vs. low APE1 expression
VEGFA, high VEGFA expression vs. low VEGFA expression
CD163, high CD163 ratio vs. low CD163 ratio. Analysis were performed utilizing logistic regression using the Cox proportional hazards model on SPSS 17.0. Area under the curve is shown in