Lysyl oxidase activates cancer stromal cells and promotes gastric cancer progression: quantum dot-based identification of biomarkers in cancer stromal cells

Figures & data

Figure 1 Expression of LOX in GC.

Notes: (A1 and A2) LOX expressed in cytoplasm of cancer cells (blue arrows) and stromal cells (red arrows) revealed by IHC. (B1 and B2) LOX was stained orange and expressed in cytoplasm of cancer cells (blue arrows) and stromal cells (red arrows) revealed by QDs-585-conjugated antibody nanoprobe. (C) LOX expressed in nucleus of cancer cells (green arrows) revealed by IHC. (D) LOX expressed in nucleus of tumor cells (green arrows) revealed by QDs-585 probe-based single molecular imaging. Magnification: 200× (A1, B1, C and D), 400× (A2 and B2), 100× (C and D). Scale bar: 50 μm for (A1 and B1), 20 μm for (A2 and B2) and 100 μm for (C and D).

Abbreviations: LOX, lysyl oxidase; GC, gastric cancer; IHC, immunohistochemistry; QD, quantum dot.

Figure 2 Comparison of conventional fluorescent dyes and QD-based molecular probe.

Notes: (A1) The expression of LOX in GC cells revealed by Cy3 fluorescent imaging (excited by 570 nm emission fluorescence, yellow arrows). Cell nuclei are indicated by green arrows. (A2) Cell nucleus revealed by DAPI (green arrows). Cytoplasms are indicated by yellow arrows. (A3) Merged imaging of A1 and A2; LOX expressed in GC cells (yellow arrows) and stromal cells (green arrows). (B1) The expression of LOX in GC revealed by DyLight 488 in GC cells (yellow arrows) and stromal cells (red arrows) (merged image). (B2 and B3) The LOX was stained red and expressed in GC cells (yellow arrows) and stromal cells (red arrows), which was revealed by QDs-655 imaging probes. (C1–C3) The LOX was stained yellow and expressed in GC cells (yellow arrows) and stromal cells (red arrows), which was revealed by QDs-585 imaging probes. Magnification: 200× (A1–A3, B1, B2 and C2), 400× (B3 and C3) and 100× (C1). Scale bar: 50 μm for (A1–A3, B1, B2 and C2), 20 μm for (B3 and C3) and 100 μm for (C1).

Abbreviations: QD, quantum dot; LOX, lysyl oxidase; GC, gastric cancer; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3 QD probe-based molecular imaging methods.

Notes: QD probe-based single molecular imaging. (A1) Type IV collagen was stained yellow by QDs-585-conjugated antibody nanoprobe (yellow arrow); the area that had been degraded was revealed (red arrow). (A2) Infiltrating macrophages were stained green by QDs-525-conjugated nanoprobe (green arrow). (A3) MMP-9 was stained red by QDs-655-conjugated nanoprobe (red arrow). (A4) MMP-9 is mainly expressed in the cytoplasm of cancer cells (red arrow) but not nuclear cells (blue arrow). QD probe-based double molecular imaging. (B1 and B2) Type collagen (yellow arrows) and MMP-9 (red arrows) are labeled simultaneously in GC tissues with nanoprobes QDs-585 and QDs-655, respectively. (B3 and B4) Type collagen (green arrows) and LOX (yellow arrows) are labeled simultaneously in GC tissues with nanoprobes QDs-525 and QDs-585, respectively. (C1–C4) Infiltrating macrophages (green arrows), LOX (yellow arrows) and neovessels (red arrows) are labeled simultaneously in GC tissues with nanoprobes QDs-525, QDs-585 and QDs-655, respectively. In C3, the expression of LOX was significantly higher than that in C2. Major vessels were microvessels. Magnification: 200× (A1, B1, B3, C2 and C3), 400× (A2, A3, B2, B4 and C4), 100× (C1) and 1,000× (A4). Scale bar: 50 μm for (A1, B1, B3, C2 and C3), 20 μm for (A2, A3, B2, B4 and C4), 100 μm for (C1) and 10 μm for (A4).

Abbreviations: QD, quantum dot; MMP-9, matrix metalloproteinase 9; GC, gastric cancer; LOX, lysyl oxidase.

Figure 4 Multiplexed spectrum analysis of QD probe-based multiple molecular imaging.

Notes: (A1, B1, C1, D1, E1 and F1) The conventional fluorescent images of QD probe-based multiple molecular imaging. Infiltrating macrophages (green arrows), LOX (yellow arrows) and neovessels (red arrows) are labeled simultaneously in GC tissues with nanoprobes QDs-525, QDs-585 and QDs-655, respectively. (A2, B2, C2, D2, E2 and F2) Unmixed composite multiple spectrum image of QD probe-based multiple molecular imaging. Infiltrating macrophages (green arrows), LOX (yellow arrows) and neovessels (red arrows) are labeled simultaneously in GC tissues with nanoprobes QDs-525, QDs-585 and QDs-655, respectively. The unmixed composite multiple spectrum image shows more information. Magnification: 200× (A1–F2). Scale bar: 50 μm for (A1–F2).

Abbreviations: QD, quantum dot; LOX, lysyl oxidase; GC, gastric cancer.

Table 1 Patient demographics and clinicopathologic characteristics

Items	Value (%)	LOX expression		P-valuea
		Negative (n=92), n (%)	Positive (n=92), n (%)
Age (mean ± SD, years)	57.9±12.9
Gender				0.009
Male	132 (71.7)	74 (80.4)	58 (63.0)
Female	52 (28.3)	18 (19.6)	34 (37.0)
Tumor location				0.768
Distal stomach	98 (53.3)	50 (54.3)	48 (52.2)
Non-distal stomach	86 (46.7)	42 (45.7)	44 (47.8)
Histological grade				0.496
1/2	46 (25.0)	21 (22.8)	25 (27.2)
3/4	138 (75.0)	71 (77.2)	67 (72.8)
Tumor size (tumor area, cm^2)b				0.135
<16	76 (46.3)	46 (51.7)	30 (40.0)
≥16	88 (53.7)	43 (48.3)	45 (60)
Serosal invasion				0.002
No (T0, T1, T2)	27 (14.7)	21 (22.8)	6 (6.5)
Yes (T3, T4)	157 (85.3)	71 (77.2)	86 (93.5)
Lymph node metastasis				<0.0001
No (N0)	53 (28.8)	40 (43.5)	13 (14.1)
Yes (N1, N2, N3)	131 (71.2)	52 (56.5)	79 (85.9)
Distant metastasis				0.054
M0	177 (96.2)	91 (98.9)	86 (93.5)
M1	7 (3.8)	1 (1.1)	6 (6.5)
TNM stages				<0.001
Early (stages I, II)	58 (31.5)	42 (45.7)	16 (17.4)
Advanced (stages III, IV)	126 (68.5)	50 (54.3)	76 (82.6)
Surgery				0.519
Subtotal resection	159 (86.4)	81 (88)	78 (84.8)
Non-subtotal resection	25 (13.6)	11 (12)	14 (15.2)
Chemotherapy				0.039
No	35 (19.0)	23 (25)	12 (13)
Yes	149 (81.0)	69 (75)	80 (87)
Recurrence				<0.001
No	124 (67.4)	82 (89.1)	42 (45.7)
Yes	60 (32.6)	10 (10.9)	50 (54.3)
Recurrence locationc				0.024
Local–regional	37 (61.7)	3 (30.0)	34 (68.0)
Distant	23 (38.3)	7 (70.0)	16 (32.0)

Notes:

a Bold indicates values with a significant difference (P<0.05).

b Analysis was performed based on 164 patients with complete data of tumor size.

c In all, 60 recurrent cases were included.

Abbreviation: LOX, lysyl oxidase.

Table 2 Relationship between LOX expression and stromal parameters

Stromal parameters	n (%)	LOX expression		P-valuea
		Negative (n=92), n (%)	Positive (n=92), n (%)
Infiltrating macrophage density
Low	125 (67.9)	69 (75)	56 (60.9)	0.04
High	59 (32.1)	23 (25)	36 (39.1)
MVD
Low	42 (22.8)	27 (29.3)	15 (16.3)	0.035
High	142 (77.2)	65 (70.7)	77 (83.7)
Neovessel maturity (%)		61.6±18.5	73.3±22.3
Low	87 (47.3)	50 (42.2)	55 (54.3)	0.038
High	97 (52.7)	37 (59.8)	42 (45.7)
MMP-9 expression
Negative	92 (50.0)	59 (64.1)	33 (35.9)	<0.001
Positive	92 (50.0)	33 (35.9)	59 (64.1)
Type IV collagen
Negative	92 (50.0)	39 (42.4)	53 (57.6)	0.039
Positive	92 (50.0)	53 (57.6)	39 (42.4)

Note:

a Bold indicates values with a significant difference (P<0.05).

Abbreviations: LOX, lysyl oxidase; MVD, microvessel density; MMP-9, matrix metalloproteinase 9.

Figure 5 QD probe-based molecular imaging showed the relationship between LOX and other stromal markers.

Notes: (A) The expression of LOX was low (yellow arrows), and the MVD (red arrows) was low. (B) The expression of LOX was high (yellow arrows), and the MVD (red arrows) was high. (C) The expression of LOX was high (yellow arrows), and the MVD (red arrows) and infiltrating macrophages (green arrows) were high. (D) The expression of LOX was low (yellow arrows), and type IV collagen (green arrows) was high. (E) The expression of LOX was high (yellow arrows), and type IV collagen (green arrows) was low. (F) The expression of LOX was high (yellow arrows) in the invasion front, and type IV collagen (green arrows) was low. Magnification: 200× (A–F). Scale bar: 50 μm for (A–F).

Abbreviations: QD, quantum dot; LOX, lysyl oxidase; MVD, microvessel density.

Table 3 Analyses of factors regarding OS

Variables	n (%)	Number of death (%)	Estimate median OS (range)	3-year survival rate (%)	5-year survival rate (%)	Log-rank test χ^2 value	P-valuea
Age (years)							0.058
<60	99 (53.8)	52 (52.5)	53.1 (25.5–80.6)	55.58	45.10	3.584
≥60	85 (46.2)	56 (65.9)	31.0 (21.9–40.1)	44.56	28.58
Gender							0.146
Male	132 (71.7)	74 (56.1)	40.5 (29.7–51.4)	53.57	40.0	2.114
Female	52 (28.3)	34 (65.4)	27.4 (19.9–35.0)	42.27	30.25
Tumor size (cm^2)b							0.003
<16	76 (41.3)	35 (46.1)	70.1 (42.8–97.4)	65.06	53.77	8.773
≥16	88 (47.8)	57 (64.8)	33.8 (17.9–49.7)	45.77	29.20
Histological grade							0.001
1/2	46 (25.0)	32 (69.6)	42.3 (30.6–53.9)	34.70	19.28	10.301
3/4	138 (75.0)	76 (55.0)	19.9 (8.1–31.8)	55.73	43.60
Lymph nodes metastasis							0.015
Yes	131 (71.2)	83 (63.4)	31.03 (19.9–42.2)	46.56	30.53	5.895
No	53 (28.8)	25 (47.2)	76.3 (47.6–104.9)	60.15	52.93
Serosal invasion (T stage)							<0.001
T1–2	27 (14.7)	7 (25.9)	38.7 (30.3–47.0)	79.72	74.89	14.742
T3–4	157 (85.3)	101 (64.3)	30.1 (20.2–40.1)	45.43	30.78
TNM							0.002
Early (stage I, II)	58 (31.5)	26 (44.8)	76.3 (50.1–102.5)	63.0	54.00	9.823
Advanced (stage III, IV)	126 (68.5)	82 (65.1)	30.1 (15.7–44.5)	44.72	29.20
Recurrence							<0.001
No	124 (67.4)	52 (41.9)	94.8 (43.9–145.7)	64.86	55.44	46.442
Yes	60 (32.6)	56 (93.3)	19.3 (16.6–22.0)	23.0	5.90
Infiltrating macrophages density							0.349
Low	125 (67.9)	70 (56.0)	39.87 (25.2–54.5)	52.31	39.35	0.878
High	59 (32.1)	38 (64.4)	34.1 (20.3–47.9)	46.58	29.74
MVD							0.048
Low	42 (22.8)	21 (50.0)	70.0 (53.0–87.0)	67.94	49.66	3.922
High	142 (77.2)	87 (61.3)	31.0 (21.4–40.6)	45.25	33.84
Neovessels’ maturity							<0.001
Low	87 (47.3)	66 (75.9)	19.7 (12.5–26.9)	26.72	16.09	33.442
High	97 (52.7)	42 (22.8)	70.1 (43.2–97.0)	70.44	54.94
Type IV collagen							<0.001
Negative	92 (50.0)	64 (69.6)	25.8 (19.0–32.7)	39.17	22.95	15.152
Positive	92 (50.0)	44 (47.8)	70.1 (44.7–95.5)	61.53	51.02
LOX							<0.0001
Negative	92 (50.0)	36 (39.1)	94.8 (35.9–153.7)	71.42	57.56	33.312
Positive	92 (50.0)	72 (78.3)	20.2 (14.2–26.2)	30.64	17.38
MMP-9							<0.0001
Negative	92 (50.0)	34 (37.0)	38.7 (30.4–47.0)	73.39	57.00	52.341
Positive	92 (50.0)	74 (80.4)	16.8 (12.3–21.3)	27.19	17.18

Notes:

a Bold indicates values with a significant difference (P<0.05).

b Analysis was performed based on 164 patients with complete data of tumor size.

Abbreviations: OS, overall survival; MVD, microvessel density; LOX, lysyl oxidase; MMP-9, matrix metalloproteinase 9.

Table 4 Univariate and multivariate analyses of factors associated with OS and DFS

Factors	OS			DFS
	UP	Multivariate		UP	Multivariate
		HR (95% CI)	P-value		HR (95% CI)	P-value
Age: <60 vs ≥60 years	0.058	NA		0.170	NA
Gender: male vs female	0.146	NA		0.659	NA
Tumor size (cm^2): <16 vs ≥16	0.003	0.324		0.003	2.006 (1.077–3.736)	0.028
Adenocarcinoma: differentiation vs undifferentiation	<0.001	0.227		0.212	NA
T stage: T1, T2 vs T3, T4	0.001	3.285 (1.496–7.215)	0.003	0.097	NA
LN metastasis: no vs yes	0.015	0.831		0.255	NA
Distant metastasis: no vs yes	0.005	4.250 (1.791–10.086)	0.001	NA	–
TNM stage: I, II vs III, IV	0.002	0.889		0.342	NA
Recurrence: no vs yes	<0.001	2.296 (1.473–3.576)	<0.001	NA	–
Positive rate of resected LN: ≤0.33 vs <0.33	<0.001	2.615 (1.689–4.048)	<0.001	0.002		0.426
Macrophages density: low vs high	0.349	NA		0.005		0.659
MVD: low vs high	0.048	0.695		0.108	NA
Neovessels maturity: low vs high	<0.001	2.736 (1.756–4.262)	<0.001	<0.001	1.870 (1.027–3.405)	0.028
Type IV collagen: negative vs positive	<0.001	0.485 (0.311–0.756)	0.001	0.349
LOX: negative vs positive	<0.001	0.606		<0.001	6.455 (2.980–13.984)	<0.001
MMP-9: negative vs positive	<0.001	3.340 (2.132–5.233)	<0.001	<0.001	2.625 (1.405–4.867)	0.002

Note: “–” indicates not applicable.

Abbreviations: OS, overall survival; DFS, disease-free survival; UP, univariate P-value; HR, hazard ratio; NA, not adopted; LN, lymph node; MVD, microvessel density; LOX, lysyl oxidase; MMP-9, matrix metalloproteinase 9.

Figure 6 Cumulative OS and DFS curves of GC patients.

Notes: (A and B) LOX expression was related to the increased risk of death and recurrence for GC. (C and D) Stromal activation was related to the increased risk of death and recurrence for GC.

Abbreviations: OS, overall survival; DFS, disease-free survival; GC, gastric cancer; LOX, lysyl oxidase.

Table 5 Relationship between LOX expression and stromal parameters

Stromal activation	n (%)	LOX expression		P-valuea
		Negative (n=92), n (%)	Positive (n=92), n (%)
Weak	26 (14.1)	16 (17.4)	10 (10.9)	0.001
Moderate	106 (57.6)	61 (66.3)	45 (48.9)
Intense	52 (28.3)	15 (16.3)	37 (40.2)

Note:

a Bold indicates value with a significant difference, P<0.05.

Abbreviation: LOX, lysyl oxidase.