Anti-Podocalyxin Monoclonal Antibody 47-mG 2a Detects Lung Cancers by Immunohistochemistry

Introduction

Lung cancer is one of the leading causes of cancer-related deaths in the world.⁽¹⁾ The number of new cases and deaths respectively reaches about 1.8 and 1.6 million per year. There are two main subtypes: small-cell lung cancer (SCLC; 15%) and non-SCLC (NSCLC). NSCLC includes adenocarcinoma (AC), squamous cell carcinoma (SCC), and large cell carcinoma.⁽²⁾ Regardless of the advances in lung cancer treatment, the prognosis is still poor with 5-year survival rates at approximately 15%.⁽³⁾ In the early stages, lung cancer is treated via surgical removal, which can be complemented by radiotherapy and/or chemotherapy. In the advanced stages, many types of anticancer agents, including cisplatin and docetaxel, are used for chemotherapy. In recent years, some techniques for the detection and identification of cell types and genetic mutations have been established that enable accurate investigations. Some molecular targeted drugs have displayed efficacy for lung cancer under specific conditions and have been approved for treatment. These drugs include bevacizumab,⁽⁴⁾ a mouse-human IgG₁ chimeric antibody against vascular endothelial growth factor; nivolumab,⁽⁵⁾ a fully human IgG₄ monoclonal antibody (mAb) against programmed cell death 1 (PD-1); pembrolizumab,^(6,7) a mouse-human IgG₄ chimeric antibody against PD-1; and atezolizumab,⁽⁸⁾ a mouse-human IgG₁ chimeric antibody against programmed cell death ligand-1.

Podocalyxin (PODXL) is a highly N-glycan- and O-glycan-glycosylated type I transmembrane protein with a molecular weight of 150,000–200,000.^(9–12) It was originally discovered in renal podocytes and is expressed in many kinds of tissues, including the heart, pancreas, and breast. PODXL is also found in cancer tissues.⁽¹³⁾ It has been reported to be a diagnostic marker and a prognostic indicator in some cancers, such as renal,⁽¹⁴⁾ brain,⁽¹²⁾ breast,⁽¹⁵⁾ oral,⁽¹⁶⁾ and lung cancers.^(17,18) This protein is involved in tumor growth, invasion, and metastasis,⁽¹⁹⁾ and its overexpression can cause undesirable outcomes. Furthermore, Kusumoto et al. reported that PODXL overexpression induces epithelial–mesenchymal transition in lung AC and contributes to tumor progression.⁽²⁰⁾ Therefore, more precise tools for detecting PODXL in lung cancer are necessary for optimal analysis and treatment.

Previously, we established specific and sensitive anti-PODXL mAbs, PcMab-47 (mouse IgG₁, kappa), and mouse IgG_2a-type (47-mG_2a).⁽²¹⁾ Herein, we investigated the utility of these two antibodies for immunohistochemical analysis of lung cancers.

Materials and Methods

Results and Discussion

To produce PcMab-47 (mouse IgG₁, kappa), we previously immunized mice with recombinant PODXL purified from the culture supernatant of the extracellular domain of PODXL-expressing LN229 cells.⁽²¹⁾ We further engineered PcMab-47 into a mouse IgG_2a-type mAb (47-mG_2a). This antibody exhibited a higher binding affinity than PcMab-47 for PODXL-expressing oral SCC (OSCC) cells. Immunohistochemical analysis of oral cancer tissues using these two antibodies revealed that 47-mG_2a stained OSCC cells in a cytoplasmic pattern at a much lower concentration. PcMab-47 and 47-mG_2a detected PODXL in 163/201 (81.1%) and 197/201 (98.0%) OSCC samples, respectively.

In this study, we performed immunohistochemical analyses using PcMab-47 and 47-mG_2a against several types of lung cancers. Typical staining results of PcMab-47 and 47-mG_2a against SCC, adenosquamous cell carcinoma (ASCC), small-cell carcinoma (SC), AC, bronchioloalveolar carcinoma (BC), and papillary carcinoma (PC) are shown in Figure 1 and Supplementary Figures S1–S5. Both PcMab-47 and 47-mG_2a stained cancer cells of all types of lung cancer in a cytoplasmic staining pattern. Furthermore, both PcMab-47 and 47-mG_2a stained endothelial cells in lung cancers (Fig. 2).

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FIG. 1.

Immunohistochemical analysis by anti-PODXL antibodies against lung SCCs. Sections (Case-7) were incubated with 5 μg/mL of PcMab-47 (A, D) and 0.5 μg/mL of 47-mG_2a (B, E) for 1 hour at room temperature, followed by treatment with the Envision+ kit for 30 minutes. Color development was performed using 3,3-diaminobenzidine tetrahydrochloride for 2 minutes. Sections were then counterstained with hematoxylin. (C, F) Hematoxylin & eosin staining; scale bar = 100 μm.

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FIG. 2.

Anti-PODXL antibodies detect endothelial cells in lung SCCs. Sections (Case-25) were incubated with 5 μg/mL of PcMab-47 (A, D) and 0.5 μg/mL of 47-mG_2a (B, E) for 1 hour at room temperature, followed by treatment with the Envision+ kit for 30 minutes. Color development was performed using 3,3-diaminobenzidine tetrahydrochloride for 2 minutes. Sections were then counterstained with hematoxylin. (C, F) Hematoxylin & eosin staining; scale bar = 100 μm; Arrows, endothelial cells.

As shown in Table 1 and Supplementary Table S1, PcMab-47 stained 51/70 cases (72.9%) of lung cancers. Among them, 35/41 cases (85.4%) of SCC, 6/10 cases (60%) of ASCC, 3/7 cases (42.9%) of SC, 4/5 cases (80%) of AC, 3/5 cases (60%) of BC, and 0/2 cases (0%) of PC were stained. In contrast, 47-mG_2a stained 59/70 cases (84.3%) of lung cancers. Among them, 38/41 cases (92.7%) of SCC, 8/10 cases (80%) of ASCC, 4/7 cases (51.7%) of SC, 4/5 cases (80%) of AC, 4/5 cases (80%) of BC, and 1/2 cases (50%) of PC were stained.

In conclusion, our immunohistochemical results demonstrate that 47-mG_2a is significantly more sensitive than the original PcMab-47 for the detection of PODXL in LSCCs and could be applied to other SCCs in the future.