PD-L1upregulation is detected in MIBC
Westernblot was applied to investigate PD-L1 expression in the bladder cancer cell lines T24, SW780, UMUC-3 and the human immortalized bladder epithelial cell line SV-HUC-1. PD-L1 expression in invasive cell lines UMUC-3 and T24 was higher than SV-HUC-1 cells(Fig. 1A). UMUC-3 was chosen for follow-up experiment. The results from immunohistochemistry (IHC) analysis of 130 MIBC specimens demonstrated that PD-L1 expression was significantly increased in cancerous tissues in comparison with adjacent normal tissues(Fig. 1B). We detected positive expression of PD-L1 in 29.2%(38/130) of MIBC cases while in 6.3%(8/126) of adjacent normal tissues. Collectively, our data demonstrated that PD-L1 expression was increased in MIBC.
Association of PD-L1 expression with clinicopathologic parameters
Of 130 MIBC patients, positive and negative PD-L1 expression were 29.2% (38/130) and 70.8% (92/130), respectively(Table 1). The result from analysis of the relationship between PD-L1 expression and clinicopathological variables indicated increased PD-L1 expression was dramatically correlated with EMT(P=0.011, Fig. 2A) and basal subtype(P=0.001, Fig. 2B).We defined EMT when mesenchymal morphological changes and low E-cadherin expression were found at the invasive front of tumor. The upregulated PD-L1 expression is also dramatically related to regional lymph node metastases(P=0.01, Fig. 2C) and Oct4(P=0.038, Fig.2D). However, our data didn’t indicate intimate association of PD-L1 expression with other clinicopathologic parameters, including sex, age, differentiation, invasion depth as well as tumor size(Table 1).
Table 1
The relationship between PD-L1 expression and clinicopathological factors in muscle-invasive bladder cancer
Clinicopathological factors
|
PD-L1 expression
|
Positive (%)
|
Negative (%)
|
P
|
Age
|
|
|
0.384
|
≥65
|
23(60.5)
|
48(52.2)
|
|
<65
|
15(39.5)
|
44(47.8)
|
|
Sex
|
|
|
0.639
|
Male
|
28(73.7)
|
64(69.6)
|
|
Female
|
10(26.3)
|
28(30.4)
|
|
Size
|
|
|
0.402
|
≥3cm
|
18(47.4)
|
51(55.4)
|
|
<3cm
|
20(52.6)
|
41(42.6)
|
|
Invasion depth
|
|
|
0.711
|
T2
|
16(42.1)
|
42(45.7)
|
|
T3
|
22(57.9)
|
50(54.3)
|
|
Local nodal metastasis
|
|
|
0.010
|
N0
|
12(31.6)
|
52(56.5)
|
|
N1-3
|
26(68.4)
|
40(43.5)
|
|
Differentiation
|
|
|
0.341
|
G2
|
20(52.6)
|
40(43.5)
|
|
G3
|
18(47.4)
|
52(56.5)
|
|
Subtype
|
|
|
0.001
|
Basal
|
25(65.8)
|
31(33.7)
|
|
Luminal
|
13(34.2)
|
61(66.3)
|
|
EMT
|
|
|
0.011
|
Positive
|
27(71.1)
|
43(46.7)
|
|
Negative
|
11(28.9)
|
49(53.3)
|
|
Oct4
|
|
|
0.038
|
Positive
|
22(57.9)
|
35(38)
|
|
Negative
|
16(42.1)
|
57(62)
|
|
PD-L1 promotes tumor progression by conferring stemness and inducing EMT
Based on real-time RT-PCR and westernblot results, PD-L1 expression in UMUC-3 cell line was dramatically upregulated in plasmid transfection (overexpression, OE) group in comparison with OE control (OEC) group, at both mRNA and protein levels(Fig.3A). PD-L1 expression was significantly inhibited by the siRNA3(knockdown, KD) and siRNA1(KD1) compared to KD control(KDC)( Fig.3B). Cell proliferation assay indicated that PD-L1 knockdown significantly suppressed cell proliferation, while PD-L1 overexpression drastically increased the ability of cell proliferation (Fig. 3C). We then studied the influence of PD-L1expression on ability of migration and invasion. Cell invasion assay indicated that invasive ability was downregulated by 41% and upregulated by 56% by PD-L1 knockdown and overexpression, respectively(Fig. 3D). Cell migration assay showed that migration ability was decreased by 35% and increased by 56% by PD-L1 knockdown and overexpression, respectively(Fig. 3E). To investigate whether PD-L1 promotes the MIBC progression by enhancing EMT/CSC, EMT-related genes such as E-cadherin, N-cadherin, Snail, Fibronectin and Vimentin as well as renowned Yamanaka factors including Oct4, Klf4, Sox2 and C-Myc, which confer stemming and give rise to iPS cells, were detected by real-time RT-PCR. Our data showed that the expression of Yamanaka factors(Oct4 and C-myc) and mesenchymal markers(N-cadherin, Snail and Vimentin) was significantly decreased(P < 0.001), in contrast to remarkably increased expression of epithelial marker E-cadherin(P < 0.001). The upregulation of E-cadherin as well as downregulation of N-cadherin, Oct4 and C-myc at protein level was verified by westernblot (Fig. 4A). On the contrary, PD-L1 overexpression reversed the changes of stemness genes(Oct4 and C-myc) and EMT-related genes(E-cadherin, N-cadherin, Snail and Vimentin) upon PD-L1 knowdown. The opposite changes of Oct4, C-myc, E-cadherin and N-cadherin at protein level were also verified by westernblot (Fig. 4B).
PD-L1 expression indicates poor prognosis in MIBC
Kaplane-Meier analysis demonstrated that positive PD-L1 expression was associated with poorer OS (P< 0.001, Fig. 5A), so is EMT(P=0.002, Fig. 5B). In order to explore whether combined expression of PD-L1 and EMT presented with poorer prognosis, 130 MIBC patients were divided into four groups: one subgroup with both positive PD-L1 and EMT, either negative PD-L1 or negative EMT and both negative PD-L1 and EMT. Kaplane-Meier analysis indicated that OS of MIBC patients with positive PD-L1/positive EMT was poorer than that of other combinations (P< 0.001, Fig. 5C). Multivariate analysis demonstrated that PD-L1 expression was an independent prognostic factor (P=0.031), as was EMT(P= 0.026), local nodal metastasis(P= 0.015), subtype (P= 0.002).