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. 2017 Dec 27:13:161-174.
doi: 10.2147/IJN.S143871. eCollection 2018.

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

Chunwei Peng  1 , Jiuyang Liu  1 , Guifang Yang  2 , Yan Li  3
Affiliations

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

Chunwei Peng et al. Int J Nanomedicine. .

Abstract

Purpose: Semiconductor quantum dots (QDs) are a promising alternative to organic fluorescent dyes for multiplexed molecular imaging of cancer stroma, which have great advantages in holistically analyzing the complex interactions among cancer stromal components in situ.

Patients and methods: A QD probe-based multiplexed spectral molecular imaging method was established for simultaneous imaging. Three tissue microarrays (TMAs) including 184 gastric cancer (GC) tissues were constructed for the study. Multispectral analyses were performed for quantifying stromal biomarkers, such as lysyl oxidase (LOX). The stromal status including infiltrating of immune cells (high density of macrophages), angiogenesis (high density of microvessel density [MVD], low neovessel maturation) and extracellular matrix (ECM) remodeling (low density of type IV collagen, intense expression of matrix metalloproteinase 9 [MMP-9]) was evaluated.

Results: This study compared the imaging features of the QD probe-based single molecular imaging method, immunohistochemistry, and organic dye-based immunofluorescent methods, and showed the advantages of the QD probe-based multiple molecular imaging method for simultaneously visualizing complex components of cancer stroma. The risk of macrophages in high density, high MVD, low neomicrovessel maturation, MMP-9 expression and low type IV collagen was significantly increased for the expression of LOX. With the advantages of the established QD probe-based multiplexed molecular imaging method, the spatial relationship between LOX and stromal essential events could be simultaneously evaluated histologically. Stromal activation was defined and then evaluated. Survival analysis showed that the stromal activation was correlated with overall survival and disease-free survival (P<0.001 for all). The expression of LOX was significantly increased in the intense activation subgroup (P<0.001).

Conclusion: Quantifying assessment of the stroma indicates that the LOX may be a stromal marker for GC and stromal activation, which is not only responsible for the ECM remodeling morphologically, but also for the formation of invasive properties and recurrence. These results support the possibility to integrate morphological and molecular biomarker information for cancer research by the biomedical application of QDs.

Keywords: cancer stromal; gastric cancer; multiplexed molecular imaging; nanomedicine.

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Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
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
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
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
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.
Figure 5
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.
Figure 6
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.
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