Surfactant protein C expression in urethane-induced murine pulmonary tumors

Abstract

Mice injected with urethane develop tumors with distinct histological patterns, which are classified as solid, papillary, or a mixture of these two patterns within the same tumor. Most investigators agree that solid tumors arise from alveolar type II cells, but the cellular origin of papillary tumors is less certain, being attributed to either type II cells or nonciliated bronchiolar epithelial (Clara) cells. To characterize the state of differentiation of these tumors more precisely and to provide additional information on gene expression, we used immunocytochemistry and/or in situ hybridization to determine the cellular localization of surfactant-associated proteins A (SP-A), SP-B, SP-C, and SP-D; Clara cell-associated protein CC-10; and thyroid transcription factor-1. In normal mouse lung, the messenger RNAs (mRNAs) for SP-A, SP-B, and SP-D were expressed in both type II cells and Clara cells. SP-C mRNA, however, was expressed only in type II cells, and CC-10 expression of mRNA was restricted to Clara cells. All tumors examined, both solid and papillary, expressed SP-A, SP-B, SP-C, SP-D, and thyroid transcription factor-1, but not CC-10. However, SP-C expression was slightly diminished in larger (older) papillary tumors. These results demonstrate that urethane-induced murine lung tumors express the type II cell phenotype.

Figure 1.

Immunocytochemistry for SP-A, -C, and -D and CC-10 in urethane-induced adenoma. A: Histology of representative solid tumors. B: Histology of representative papillary tumors. Tumor cells were stained with H&E. C, E, G, and I: Immunostaining for SP-A, -C, and -D and CC-10, respectively, in solid adenoma. D, F, H, and J: Immunostaining for SP-A, -C, and -D and CC-10, respectively, in representative papillary adenomas. The methods for immunostaining required different fixatives for different antigens as stated in Materials and Methods. Thus the micrographs are not all from the same tumors. All panels are at the same original magnification.

Figure 2.

Pro-SP-C immunocytochemistry and SP-C and CC-10 in situ hybridization in a large papillary tumor. This tumor was harvested from a mouse 50 weeks after intraperitoneal injection of urethane. A: Histology with staining with H&E; B: Immunostaining for pro-SP-C. C and D: In situ hybridization for CC-10. E and F: In situ hybridization for SP-C. All panels are at the same original magnification.

Figure 3.

In situ hybridization for SP-C, CC-10, and TTF-1 in the same tumors. Sections of a solid and papillary tumor were hybridized with riboprobes for SP-C (A–D), CC-10 (E–H), and TTF-1 (I–L). The pairs of micrographs are visualized by bright-field or dark-field illumination. These are consecutive sections from the same tumor fixed in 4% paraformaldehyde and processed as stated in the methods section. All panels are at the same original magnification.