A blockade of PD-L1 produced antitumor and antimetastatic effects in an orthotopic mouse pancreatic cancer model via the PI3K/Akt/mTOR signaling pathway

Figures & data

Table 1 Primers sequence for reverse transcription polymerase chain reaction

PTEN-F	CAGCCATCATCAAAGAGATCG
PTEN-R	TTGTTCCTGTATACGCCTTCAA
PI3K-F	AGGAGCGGTACAGCAAAGAA
PI3K-R	GCCGAACACCTTTTTGAGTC
Akt-F	TGAAAACCTTCTGTGGGACC
Akt-R	TGGTCCTGGTTGTAGAAGGG
mTOR-F	CTGGGACTCAAATGTGTGCAGTTC
mTOR-R	GAACAATAGGGTGAATGATCCGGG
MMP2-F	GCACTCTGGAGCGAGGATAC
MMP2-R	GCCCTCCTAAGCCAGTCTCT
MMP9-F	AAGGCAAACCCTGTGTGTTC
MMP9-R	GTGGTTCAGTTGTGGTGGTG
GAPDH-F	GGAAGGTGAAGGTCGGAGT
GAPDH-R	CCTGGAAGATGGTGATGGG

Abbreviations: PTEN, phosphatase and tensin homologue; PI3K, phosphati-dylinositol 3-kinase; mTOR, mammalian target of rapamycin; MMP2, Matrix metalloproteinases-2; MMP9, Matrix metalloproteinases-9; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; F, Forward; R, Reverse.

Figure 1 Effect of anti-PD-L1 antibody in an orthotopic pancreatic cancer mouse model.

Notes: (A) Average mouse body weight in the anti-PD-L1 group and control group. (B) The mean weight of pancreatic tumors after injection with anti-PD-L1 antibodies for 6 weeks. Data represent the mean ± standard deviation (n=5). **P<0.01. The data represent results from three separate experiments.
Abbreviation: PD-L1, programmed death-ligand 1.

Figure 2 Immunohistochemical analysis of PD-L1 expression in pancreatic cancer tissue and spontaneous liver metastases.

Notes: (A) Representative images of pancreatic tumor and spontaneous liver metastases. (B–C) Hematoxylin-eosin staining of pancreatic tumor tissue and spontaneous liver metastases at ×100 and ×400 magnification, respectively. (D) PD-L1 expression after injection with anti-PD-L1 antibody for 6 weeks in both pancreatic tumor tissue and spontaneous liver metastases at ×400 magnification.
Abbreviation: PD-L1, programmed death-ligand 1.

Figure 3 Effect of anti-PD-L1 antibody on expression of mRNA for components involved in the PI3K/Akt/mTOR pathway.

Notes: The levels of PTEN, PI3K, Akt, and mTOR mRNA expression in pancreatic tumor tissue (A) and spontaneous liver metastases (B) were examined by RT-PCR. The mean levels of PTEN, PI3K, Akt, and mTOR mRNA expression were shown in representative images (left) and quantitative analysis demonstrating the average of three separate experiments (right). Data represent the mean ± SD (n=5). **P<0.01.
Abbreviations: PD-L1, programmed death-ligand 1; PTEN, phosphatase and tensin homologue; PI3K, phosphatidylinositol 3-kinase; mTOR, mammalian target of rapamycin; RT-PCR, Reverse transcription polymerase chain reaction.

Figure 4 Effect of anti-PD-L1 antibody on proteins involved in the PI3K/Akt/mTOR pathway.

Notes: The levels of PTEN, PI3K, p-PI3K (Tyr⁴⁵⁸), Akt, p-Akt (Ser⁴⁷³), mTOR, and p-mTOR (Ser²⁴⁴⁸) protein expression in pancreatic tumor tissue (A) and spontaneous liver metastases (B) were detected by Western blotting. The mean levels of PTEN, PI3K, p-PI3K (Tyr⁴⁵⁸), Akt, p-Akt (Ser⁴⁷³), mTOR, and p-mTOR (Ser²⁴⁴⁸) protein expression were shown in representative images (left) and quantitative analysis demonstrating the average of three separate experiments (right). Data represent the mean ± standard deviation (n=5). **P<0.01.
Abbreviations: PD-L1, programmed death-ligand 1; PTEN, phosphatase and tensin homologue; p-Akt, phospho-Akt; PI3K, phosphatidylinositol 3-kinase; mTOR, mammalian target of rapamycin.

Figure 5 Inhibitory effects of anti-PD-L1 antibody on the invasion behavior of pancreatic tumor and spontaneous liver metastasis.

Notes: The levels of MMP2 and MMP9 mRNA expression in pancreatic tumor tissue (A) and spontaneous liver metastasis tissue (B) were examined by RT-PCR. The levels of MMP2 and MMP9 protein expression in pancreatic tumor tissue (C) and spontaneous liver metastasis tissue (D) were examined by Western blotting. The results were shown in representative images (left) and quantitative analysis demonstrating the average of three separate experiments (right). Data represent the mean ± standard deviation (n=5). *P<0.05, **P<0.01.
Abbreviations: MMP2, Matrix metalloproteinases-2; MMP9, Matrix metalloproteinases-9; PD-L1, programmed death-ligand 1; RT-PCR, reverse transcription polymerase chain reaction.

Figure S1 Immunohistochemical score of PD-L1 expression in pancreatic tumor and spontaneous liver metastases.

Notes: (A–D) Images represent pancreatic tumors with IHC scores. (E–H) Images represent liver tumors with IHC scores. Scores are reported as follows: 0 (A, E) represents no staining, 1+(B, F) represents weak staining, 2+(C, G) represents moderate staining, and 3+(D, H) represents intense stainin. Images of A–H are ×400 magnification.

Figure S2 ELISA analysis for the concentration of CA19-9 in serum after PANC 02 cells injection.

Notes: The CA19-9 concentrations in serum after PANC 02 cells injection for 7 days differed significantly from that of the normal level. Data represent the mean ± standard deviation (n=5). **P<0.01.

Abbreviations: CA19-9, carbohydrate antigen 19-9; PANC 02, pancreatic adeno-carcinoma cell line.

Figure S3 The mRNA expression of IFN-γ, granzyme B, and perforin in pancreatic tumor.

Notes: The expressions of IFN-γ, granzyme B, and perforin were significantly higher in the anti-PD-L1 group than in the controls. Data represent the mean ± standard deviation (n=5). **P<0.01.

Table S1 Primers sequence for reverse transcription polymerase chain reaction

IFN-γ-F	GTTACTGCCACGGCACAGTCATTG
IFN-γ-R	ACCATCCTTTTGCCAGTTCCTCCAG
Granzyme B-F	CCTGAAGGAGGCTGTGAAAGAATC
Granzyme B-R	CCCTGCACAAATCATGTTTAGTCC
Perforin-F	CTGCCACTCGGTCAGAATG
Perforin-R	CGGAGGGTAGTCACATCCAT

Abbreviations: IFN-γ, interferon- γ; F, Forward; R, Reverse.

Table S2 Results of mean H score from three independent pathologists

Tumor tissue		Control					Anti-PD-L1
		#1	#2	#3	#4	#5	#1	#2	#3	#4	#5
Pancreatic tumor	EXP.1	250	189	204	235	267	76	101	89	123	67
	EXP.2	265	243	189	173	–	132	108	65	89	34
	EXP.3	213	209	198	89	179	89	79	68	134	76
	P-valuea	<0.0001
Liver tumor	EXP.1	209	167	195	132	89	112	54	98	45	76
	EXP.2	198	138	119	165	72	76	32	128	21	19
	EXP.3	190	218	210	276	–	24	29	73	18	59
	P-valueb	<0.0001

Notes:

a The differences in H score between Control and Anti-PD-L1 groups in pancreatic tumors.

b The differences in H score between Control and Anti-PD-L1 groups in liver tumors. “–” denotes the mouse died before the end of the experiment.