Aerogenous metastasis (AM) is a form of lung cancer that spreads in a unique fashion, but its mechanisms are still unclear. Annexin A2 (ANX A2), a membrane-binding protein, promotes cancer invasion and is involved in cell adhesion and polarity. The relationship between ANX A2 and cancers with poor stromal invasion capacity has not been studied. We immunohistochemically analyzed ANX A2 expression in AM observed in a patient with pulmonary invasive mucinous adenocarcinoma. In the primary site, ANX A2 immunopositivity on the cell-cell borders weakened as tumor cells projected and separated into alveolar spaces. In AM, tumor cell aggregates with ANX A2 immunopositivity near the surface and within the cytoplasm attached to alveolar epithelial cells, then engulfed them and formed a protrusion. As tumor cell aggregates adhered to the alveolar wall and formed a single layer, cytoplasmic ANX A2-positive products accumulated in the lateral sides of the tumor cells and exhibited distinct membranous positivity. These results indicated that ANX A2 near the tumor cell surface was related to alveolar wall attachment. Furthermore, the translocation of cytoplasmic ANX A2 to cell-cell borders changed cell morphology, adhesion, and polarity restoration.
Aerogenous metastasis (AM) is a characteristic form of lung cancer progression [
Annexin A2 (ANX A2) belongs to a family of Ca2+/membrane-binding proteins [
Invasive mucinous adenocarcinoma (IMAC) of the lung, formerly referred to as mucinous bronchioloalveolar carcinoma, is a rare type of lung AC [
The subject of this case study provided written informed consent for participation. A 75-year-old male with a 50 pack-year smoking history presented for evaluation of abnormal findings on chest computed tomography (CT). Chest CT demonstrated a poorly demarcated, 4 cm-diameter mass with air bronchogram in the left lateral basal lung. The solid mass was accompanied by infiltrative and reticular shadows. Enlargement of hilar or mediastinal lymph nodes and organ metastasis were not seen. A follow-up chest CT two months later showed expansion of the mass, and a shadow suspected to be a skip lesion was also found near the mass (Figure
Axial computed tomography (CT) images. (a) A follow-up chest CT image obtained before left lobectomy shows a solid mass accompanied by infiltrative and reticular shadows, and the shadow of a suspected skip lesion (arrow) is also found near the mass. (b) Chest CT image obtained 1 month before partial resection of the right lung shows a widespread infiltrative shadow.
Both resected tumors were nearly macro- and microscopically identical. Macroscopically, both tumors were ill-defined and gelatinous with a diffuse pneumonia-like consolidation (Figure
Left lung cancer. (a) Gross view shows an ill-defined gelatinous tumor with a diffuse pneumonia-like consolidation. Bar, 1 cm. (b) Columnar tumor cells with intracytoplasmic vacuoles reveal a papillary growth pattern. H&E stain, ×200. (c) Multiple mucin-producing tumor cells. Alcian blue stain, ×200. (d) Several tumor cells are positive for MUC 5AC. MUC 5AC immunostain, ×200.
Immunohistochemical analyses were performed on serial sections prepared from 20% buffered formalin-fixed, paraffin-embedded tissues and carried out with Leica Bond-Max (Leica Biosystems, Australia).
Mouse anti-human ANX A2 antibody (clone 5/Annexin II, BD Transduction Laboratories, USA) [
Endogenous peroxidase was blocked with 3% hydrogen peroxide for 5 min. The primary antibodies were applied to the sections at dilution ratios of 1/2000, 1/100, and 1/100. Reaction products were visualized with 3,3
Immunohistochemical results for ANX A2 were nearly identical in both tumors, with the details noted in the following section. Multiple tumor cells were positive for MUC 5AC (Figure
Detachment of tumor cells in the primary site of left lung cancer. (a) Villous-shaped tumor cells project and separate into the alveolar space. H&E stain, ×100. (b) Immunopositivity on the cell-cell borders is weakened as tumor cells project or separate. Annexin A2 immunostain, ×400. (c) Tumor cells are negative, whereas the remaining alveolar epithelial cells are positive. TTF-1 immunostain, ×400.
Aerogenous metastasis of left lung cancer I. (a) Many isolated lesions are seen. H&E stain, ×40. (b) Small tumor cell aggregates are attached to alveolar epithelial cells, forming a protrusion. Some of the alveolar epithelial cells have an obscure nucleus and are detached within the alveolar spaces. H&E stain, ×400. (c) Tumor cell aggregates represent the immunopositivity near the cell surface with granular cytoplasmic positivity. Alveolar epithelial cells also show a weak positivity near the cell surface. Annexin A2 immunostain, ×400. (d) Alveolar epithelial cells are positive. The arrangement of these cells is well kept; however, some show weakened immunopositivity. TTF-1 immunostain, ×400.
Aerogenous metastasis of left lung cancer II. (a) Tumor cell aggregates adhere to the alveolar wall, and pseudoluminal gaps are seen inside the aggregates. H&E stain, ×400. (b) Cytoplasmic-positive products accumulate in the lateral sides, especially on the underside, forming membranous positivity. Annexin A2 immunostain, ×400. (c) Many alveolar epithelial cells disappear or are indistinct compared with those observed in Figure
Cells from both tumors were positive for CK7 (clone OV-TL 12/30, DAKO, USA) and MUC 1 (clone Ma695, Leica Microsystems, Germany) and negative for CK20 (clone Ks20.8, DAKO), Napsin A (clone IP64, Leica Microsystems), CDX2 (clone DAK-CDX2, DAKO), MUC 2 (clone Ccp58, Leica Microsystems), and ALK (clone 5A4, Abcam, UK) (data not shown, respectively).
The results are shown in Figures
The results are shown in Figures
Aerogenous metastasis of left lung cancer III. (a) Tumor cells arrange in one layer along the alveolar wall, and intracytoplasmic mucins are localized on the luminal sides. H&E stain, ×400. (b) Distinct membranous positivity on the cell-cell borders is seen. Annexin A2 immunostain, ×400.
In AM, tumor cell aggregates must spread within the alveolar spaces to adhere to AECs, to eliminate them and to stick to the alveolar wall [
The tumor cells also expressed MUC 5AC. It was reported previously that MUC 5AC promotes cell-ECM adhesion in various cancers [
In this case, we observed morphological changes of tumor cell aggregates.
Pseudoluminal gaps might derive from aggregate fusion or separation. In this process, irregularly arranged mucins were translocated to the luminal sides, suggesting restoration of cell polarity [
Recent reports have suggested that ANX A2 contributes to cancer invasion. It is derived from ECM degradation and from the promotion of the epithelial-mesenchymal transition (EMT) [
Indistinct ANX A2-immunoreactivity, in contact with the alveolar wall, may represent poor stromal invasion capacity [
It has been previously reported that AM in goblet cell-type IMAC originates during tumor cell detachment from the basement membrane of the alveolar wall [
It is debatable whether the right lung tumor originated due to AM from the left lung tumor or as a second primary lesion [
The present report had some limitations. We analyzed one patient only and lacked a functional assay. However, to our knowledge, ANX A2 expression in the cancer progression to free space, unlike stromal invasion, has not been reported yet. Further investigations are needed to confirm our suppositions.
Ethical approval for the study was provided by the ethics committee of Shizuoka General Hospital.
Written informed consent was obtained from an object patient.
The authors declare that there is no conflict of interest regarding the publication of this article.
The authors would like to thank Enago (