Lenvatinib Induces Immunogenic Cell Death and Triggers Toll-Like Receptor-3/4 Ligands in Hepatocellular Carcinoma

Figures & data

Table 1 Antibodies Applied in Western Blot, Immunohistochemistry, Immunofluorescence and Flow Cytometry

Antibody	Sources	Article Number	Working Concentration
TLR3	ABclonal	A11778	1:1000 (WB)
TLR4	ABclonal	A5258	1:2000(WB)
PI3K	Cell Signaling Technology	17,366	1:1000(WB)
AKT	Cell Signaling Technology	4691	1:1000(WB)
pAKT	Cell Signaling Technology	4060	1:1000(WB)
Beta-actin	Cell Signaling Technology	8457	1:1000(WB)
Gapdh	abcam	ab8245	1:1000(WB)
Mouse IgG HRP	Cell Signaling Technology	7076	1:1000(WB)
Rabbit IgG HRP	Cell Signaling Technology	58,802	1:1000 (WB)
TLR3	ABclonal	A11778	1:1000 (IF)
Alexa Fluor Plus 594	ThermoFisher	A21203	1:500 (IF)
Calreticulin	ABclonal	A18013	1:1000(FC)
PD-L1	Biolegend	124,312	1:50

Figure 1 (A) The proportion of apoptotic cells in lenvatinib-treated cells was higher than that in the control group. (B) Extracellular ATP was significantly increased after treatment of lenvatinib and intracellular ATP was decreased or remained constant. (C) Lenvatinib significantly increased calreticulin expression on the surface of all hepatoma cells. (D) Elisa and Western blot analyses revealed higher HMGB1 in the culture medium of lenvatinib-treated hepatoma cells than that in untreated cells. *p < 0.05, **p < 0.01, *** p <0.001.

Figure 2 (A) GO and KEGG pathway analysis of increased genes after lenvatinib treatment. (B and C) Volcano plot showing differential expressed genes between lenvatinib-treated and untreated Huh7 cells and MHCC-97h cells. The TLR3 and TLR4 was listed in arrow heads.

Figure 3 After treatment with lenvatinib for 2 weeks, both the mRNA expression(A) and protein expression (B and C) of TLR3 was significantly increased. Moreover, the mRNA (D) and protein (E) expression of TLR4 were also upregulated. *p < 0.05, **p < 0.01.

Figure 4 Tumor size was significantly decreased after lenvatinib treatment (A and B). Western blot and IHC showed that protein expression of TLR3 (C and D) and TLR4 (E and F) were also increased in vivo. **p < 0.01.

Figure 5 (A) TCGA database revealed a strong correlation between TLR3/TLR4 and CD274. (B) PD-L1 expression was significantly increased after two weeks lenvatinib treatment and decreased after the inhibition of TLR4. (C) The inhibition of TLR3 had not significantly affected PD-L1 expression. *p < 0.05, **p < 0.01, *** p <0.001.

Figure 6 (A) The inhibition of TLR3 decreased the proportions of apoptotic cells. (B) CCK8 assay indicated that inhibition of TLR3 reinforced the proliferative capacity of MHCC-97H and Huh7 cells (B and C). *p < 0.05, **p < 0.01, *** p <0.001.

Table 2 Univariate and Multivariate Analysis of Overall Survival and Recurrence-Free Survival

Variables	Overall Survival			Recurrence-Free Survival
	Univariate Analysis	Multivariate Analysis		Univariate Analysis	Multivariate Analysis
	p value	p value	(95% CI)	p value	p value	(95% CI)
Sex (male)	0.227			0.904
Age (>60 years)	0.198			0.46
HbsAg(positive)	0.561			0.573
TB (>20.4 μmol/L)	0.173			0.23
γ-GT (≥60 U/L)	0.935			0.623
High differentiation	0.344			0.914
Albumin (<35 g/L)	0.008	0.011	0.506(0.398–0.899)	0.377
AFP (≥ 20 ng/mL)	<0.001	<0.001	2.424(1.560–3.764)	0.001	0.002	2.013(1.281–3.161)
Tumor size (>5 cm)	<0.001	<0.001	2.314(1.575–3.401)	0.003	<0.001	2.186(1.467–3.257)
Tumor number (>1)	0.542		NA	0.007	0.027	1.948(1.079–3.517)
Tumor capsule	<0.001	0.013	0.598(0.398–0.899)	0.01	0.077	0.682(0.447–1.042)
TLR3 positivity	0.01	0.009	0.690(0.460–1.033)	0.013	0.062	0.440(0.440–1.021)
TLR4 positivity	0.725			0.302

Abbreviations: CI, confidence interval; HbsAg, Hepatitis B surface antigen; TB, total bilirubin; AFP, alpha fetoprotein; TLR, toll-like receptor.

Table 3 Clinic Baseline and Correlation Analysis with TLR3 or TLR4

Variables		TLR3 Expression		p	TLR4 Expression		p
		Negative	Positivity		Negative	Positivity
Sex	Male	158	129	0.669	242	45	0.103
	Female	32	23		51	4
Age, year	<55	108	91	0.573	171	28	0.862
	≥55	82	61		98	17
HbsAg	Negative	22	23	0.334	38	7	0.801
	Positivity	168	129		255	42
TB, μmol/L	<20.4	169	142	0.152	265	46	0.438
	≥20.4	21	10		28	3
γ-GT, U/L	<40	121	91	0.47	184	28	0.450
	≥40	69	61		109	21
Albumin, U/L	<35	8	12	0.149	16	4	0.690
	≥35	182	140		245	77
AFP, ng/mL	<20	71	57	0.98	107	21	0.396
	≥20	119	95		186	28
Tumor size, cm	≤5	81	89	0.003	143	27	0.415
	>5	109	63		150	22
Tumor number	1	172	141	0.461	269	44	0.640
	>1	18	11		24	5
Tumor capsule	No	116	65	<0.001	158	23	0.365
	Yes	74	87		135	26
Tumor differentiation	I–II	137	120	0.146	223	34	0.314
	III–IV	53	32		70	15

Abbreviations: HbsAg, Hepatitis B surface antigen; TB, total bilirubin; AFP, alpha fetoprotein.

Figure 7 (A) Representative images of TLR3 in hepatocellular carcinoma (HCC). (B) Representative images of TLR4 in HCC. (C) Kaplan–Meier estimates overall survival (OS) and recurrence-free survival (RFS) according to TLR3 expression in HCC patients. TLR3-positive patients had a better prognosis than TLR3-negative patients. (D) Data from TCGA database showed higher TLR3 mRNA levels in HCC was positively associated with OS and RFS. (E) Kaplan–Meier estimates for OS and RFS according to TLR4 expression in HCC patients. TLR4 expression was not associated with prognosis. (F) Data from TCGA database showed higher TLR4 mRNA levels was not associated with prognosis in HCC.