Human colon tissue in organ culture: preservation of normal and neoplastic characteristics

Abstract

Normal and neoplastic human colon tissue obtained at surgery was used to establish conditions for organ culture. Optimal conditions included an atmosphere of 5% CO2 and 95% O2; tissue partially submerged with mucosa at the gas interface; and serum-free medium with 1.5 mM Ca2+ and a number of growth supplements. Histological, histochemical, and immunohistochemical features that distinguish normal and neoplastic tissue were preserved over a 2-d period. With normal tissue, this included the presence of elongated crypts with small, densely packed cells at the crypt base and mucin-containing goblet cells in the upper portion. Ki67 staining, for proliferating cells, was confined to the lower third of the crypt, while expression of extracellular calcium-sensing receptor was seen in the upper third and surface epithelium. E-cadherin and β-catenin were expressed throughout the epithelium and confined to the cell surface. In tumor tissue, the same disorganized, abnormal glandular structures seen at time zero were present after 2 d. The majority of cells in these structures were mucin-poor, but occasional goblet cells were seen and mucin staining was present. Ki67 staining was seen throughout the abnormal epithelium and calcium-sensing receptor expression was weak and variable. E-cadherin was seen at the cell surface (similar to normal tissue), but in some places, there was diffuse cytoplasmic staining. Finally, intense cytoplasmic and nuclear β-catenin staining was observed in cultured neoplastic tissue.

Figure 1

Histological features of normal colon tissue in organ culture. Formalin-fixed tissue is hematoxylin- and eosin-stained. Normal colon crypt structure is maintained after 2 d in culture. Original magnification left panel, ×125; right panel, ×340.

Figure 2

Histological features of neoplastic colon tissue in organ culture. Formalin-fixed tissue is hematoxylin- and eosin-stained. Tumor component of the tissue (sessile adenoma) is preserved after 2 d in culture. Original magnification left panel, ×125; right panel, ×340.

Figure 3

Alcian Blue staining of normal and neoplastic colon tissue in organ culture. Normal (a) and a sessile adenoma (b) continue to express mucin after 2 d in culture. Original magnification, ×300.

Figure 4

Immunoperoxidase staining of normal tissue with antibodies to growth and differentiation markers after 2 d in organ culture. a, Ki67: intense staining is observed in rapidly growing cells at the base of the crypt. As the cells move upward in the crypt, cells start to differentiate and little or no staining for Ki67 is seen in these regions. b, E-cadherin: a marker of differentiation and present at cell to cell contacts. Well-defined membrane staining of E-cadherin is observed. Intensity of E-cadherin staining progressively increases towards upper regions of the tissue where more differentiated cells are present. c, β-catenin: well-defined membrane staining of β-catenin is observed in normal tissue. Little or no nuclear staining of β-catenin is seen. Nuclear β-catenin staining would be an indicator of uncontrolled proliferation of epithelial cells. Original magnification left panel, ×170; right panel, ×540.

Figure 5

Immunoperoxidase staining of malignant colon tissue after 2 d in organ culture (adenocarcinoma). a, Ki-67: Intense staining, indicating proliferation, is seen throughout the section. b, E-Cadherin: Junctional and intracellular staining is observed. Cells in some areas of the section have little or no membrane staining, with diffuse intracellular staining—a marker of tumor malignancy. c, β-catenin: strong cytoplasmic and nuclear staining is evident. The distinct membrane staining is lost in places. At higher magnification, nuclear staining can be seen (bottom panel, arrows). Original magnification left panel, ×170; right panel, ×540.

Figure 6

Calcium-sensing receptor staining of normal and malignant colon tissue in organ culture. After 2 d in organ culture, normal tissue a expresses CaSR at in the upper part of the crypt and the malignant tissue b (adenocarcinoma) exhibits diffuse staining with reduced intensity. Original magnification, ×320.

Figure 7

Matrix metalloproteinase-1 elaboration (MMP-1; interstitial collagenase). 24 h culture supernatant fluids were collected on day 2 and analyzed for MMP-1 by Western blot. A higher level of MMP-1 was present in culture fluid from tumor compared with the normal tissue. The figure represents averages±ranges of two independent experiments.