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. 2016 Dec;107(12):1909-1918.
doi: 10.1111/cas.13089. Epub 2016 Dec 12.

Highly sensitive detection of invasive lung cancer cells by novel antibody against amino-terminal domain of laminin γ2 chain

Kaoru Miyazaki  1   2 , Jun Oyanagi  3 , Atsuko Sugino  1 , Hiroki Sato  1 , Tomoyuki Yokose  3 , Haruhiko Nakayama  4 , Yohei Miyagi  2
Affiliations

Highly sensitive detection of invasive lung cancer cells by novel antibody against amino-terminal domain of laminin γ2 chain

Kaoru Miyazaki et al. Cancer Sci. 2016 Dec.

Abstract

The laminin γ2 chain, a subunit of laminin-332 (α3β3γ2), is a molecular marker for invasive cancer cells, but its pathological roles in tumor progression remain to be clarified. It was recently found that the most N-terminal, domain V (dV) of γ2 chain has activities to bind CD44 and stimulate tumor cell migration and vascular permeability. In the present study, we prepared a mAb recognizing γ2 dV. Immunoblotting with this antibody, for the first time, showed that proteolytic fragments containing dV in a range of 15-80 kDa were highly produced in various human cancer cell lines and lung cancer tissues. In immunohistochemistry of adenocarcinomas and squamous cell carcinomas of the lung, this antibody immunostained the cytoplasm of invasive tumor cells and adjacent stroma much more strongly than a widely used antibody recognizing the C-terminal core part of the processed γ2 chain. This suggests that the dV fragments are highly accumulated in tumor cells and stroma compared to the processed γ2 protein. The strong tumor cell staining with the dV antibody correlated with the tumor malignancy grade. We also found that the laminin β3 and α3 chains were frequently overexpressed in tumor cells and tumor stroma, respectively. The cytoplasmic dV detection was especially prominent in tumor cells infiltrating stroma, but low in the cells surrounded by basement membranes, suggesting that the active tumor-stroma interaction is critical for the aberrant γ2 expression. The present study suggests important roles of laminin γ2 N-terminal fragments in tumor progression.

Keywords: Biomarker; laminin γ2; lung cancer; monoclonal antibody; tumor invasion.

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Figures

Figure 1
Figure 1
Domain structure of laminin γ2 (Lm‐γ2) chain. Lm‐γ2 consists of domains I/II (or coiled‐coil domain), III (or LEb), IV (or L4), and V (or LEa) from the C‐terminus.2 Domain V (dV) consists of three N‐terminal EGF‐like repeats (NE1–3). Domain I/II is associated with the laminin β3 and α3 chains to form the Lm332 heterotrimer. P2H recognizes NE2 of dV, and D4B5 recognizes an unidentified EGF‐like repeat of domain III (dIII). Solid arrows, cleavage sites by endogenous proteinases; open arrows, epitopes for mAbs (D4B5 and P2H); γ2pf, a 45‐kDa fragment released by a major proteolytic cleavage.
Figure 2
Figure 2
Production of laminin γ2 (Lm‐γ2) fragments in cultures of cancer cell lines and lung cancer tissues. (a) Immunoblotting with the anti‐domain V (dV) antibody P2H and the anti‐dIII antibody D4B5 of γ2 fragments released into conditioned media by the two lung carcinoma lines VMRC‐LCP (VMRC) and Lu65. Arrowheads indicate γ2 or its fragments with their approximate molecular sizes in kDa. Note that P2H specifically detect γ2pf as a major band and small dV fragments of 15–30 kDa. The faint 150‐kDa band corresponds to the uncleaved γ2 chain, while the 105‐kDa major band detected by D4B5 corresponds to the cleaved, or processed, γ2 chain. (b) Immunoblotting of conditioned media from nine cancer cell lines with the P2H antibody. (c) Immunoblotting of soluble and insoluble fractions from an adenocarcinoma (ADC) and a squamous cell carcinoma (SCC) tissues with the anti‐dV (P2H) antibody. Note that both soluble fractions contain an 80‐kDa fragment as a major band, and γ2pf and small dV fragments can be faintly detected.
Figure 3
Figure 3
Immunohistochemical staining of acinar adenocarcinoma (#15) for two domains of γ2 (dV and dIII) and α3 and β3 chains of laminin‐332 (Lm332). Serial or close sections from the same tissue specimen were immunostained with antibodies against γ2 dV (P2H) (a,e), γ2 dIII (D4B5) (b, f), α3 (BG5) (c, g), and β3 (12C) (d, h). Microscopic fields showing lepidic pattern (a–d) and papillary pattern (e–h) were photographed. Insets show enlarged views of the areas shown by small dashed squares (a, e). Note that the cytoplasm of tumor cells is significantly stained only for γ2 dV in (a–d). N, non‐neoplastic basement membranes (BMs); T, tumor cells. Black arrows, positive cytoplasmic signals; green arrows, BM‐like structures surrounding tumor cells (tumor BMs). Scale bar = 100 μm.
Figure 4
Figure 4
Immunohistochemical staining of solid adenocarcinoma #5 (a–d) and lepidic adenocarcinoma #10 (e–h). In (a–d), tumor cells singly invading stroma show strong immunoreactivity for both domain V (dV) (a), dIII (b), and β3 (d), but not α3 (c). Stromal staining is seen for dV (a) and α3 (c). In (e–h), stroma adjacent to tumor cells was strongly stained for γ2 dV (a) and α3 (c), weakly for γ2 dIII (b), but not at all for β3 (d). Insets show enlarged views of the areas shown by small dashed squares (a,e). Black arrows, positive cytoplasmic or stromal signals; green arrows, tumor basement membranes. T, tumor cells. Scale bar = 100 μm.
Figure 5
Figure 5
Immunohistochemical staining of poorly differentiated squamous cell carcinoma #6 for γ2 domain V (dV) (a), γ2 dIII (b), α3 (c), and β3 (d). Tumor cells (black arrows) collectively invading stroma show immunoreactivity strongly for dV (a) and moderately for γ2 dIII (b) but poorly for α3 (c) and β3 (d). Basement membrane‐like structures (green arrows) are seen for α3 (c) and β3 (d). Insets show an enlarged view of the area shown by a small dashed square (a). Scale bar = 100 μm.
Figure 6
Figure 6
Immunohistochemical staining of four squamous cell carcinomas with different grades for γ2 domain V (dV). (a) Squamous cell carcinoma (SCC) #9, poorly differentiated; (b) SCC #5, poorly differentiated; (c) SCC #10, moderately differentiated; (d) SCC #4, well differentiated. Strong immunoreactivity (black arrows) is seen in tumor cells infiltrating stroma (a), surrounded by basement membrane‐like structures (b) and at the tumor–stroma interface (c), but scarcely in the cells surrounded by continuous basement membrane structures (green arrow) (d). In (d), marked deposition of dV‐containing fibrils (black arrow) is seen in the stroma. Insets show enlarged views of the areas shown by small broken squares (a, b, d). Scale bar = 100 μm.
Figure 7
Figure 7
Summary of immunohistochemistry with four antibodies against laminin‐332 subunits. Each column indicates the mean score ± SD (bar) for the relative immunoreactivity with the indicated antibody in 15 adenocarcinoma (ADC; upper panels) and 14 squamous cell carcinoma (SCC; lower panels) tissue samples. Antibodies used are P2H (γ2 dV), D4B5 (γ2 dIII), BG5 (α3), and 12C (β3).
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