This study was designed to investigate differentiation of human pancreatic duct carcinoma cells (Capan-1) in vitro. Observations on live cells, and electron microscopic examination, together with enzymological and immunocytochemical methods, have demonstrated that these cells differentiate spontaneously at an early stage. The cells are seen to be joined by apical junctions. High ATPase activity can be detected in the basolateral membranes, and the cells secrete a gastric type mucin (MI) bearing acidic groups. During differentiation in culture, they form domes which are thought to be the morphological expression of trans-epithelial transport of water and electrolytes. This particular structure is transitory, since after 6 days in culture all the cells lose their adhesivity, and form into floating cords. Co-culture of Capan-1 cells and human, nude mice or chick embryo fibroblasts leads to a higher degree of differentiation of epithelial cells, reflected by the earlier appearance of numerous domes. In addition, the anchorage of Capan-1 cells to fibroblasts prevents retraction of the monolayer, and enables the domes to be maintained in the cultures for more than one month. These findings suggest that Capan-1 cells are able to carry out trans-epithelial movement of water and electrolytes. It is suggested that excretion of ions (bicarbonate and/or chloride) is preserved after transformation of pancreatic duct cells. Mucins (MI) and the recently described VIP receptor sites are also thought to play a part in these exchange processes.

It has been shown that adult pancreatic ductal cells can dedifferentiate and act as pancreatic progenitors. Dedifferentiation of epithelial cells is often associated with the epithelial-mesenchymal transition (EMT). In this study, we investigated the occurrence of EMT in adult human exocrine pancreatic cells both in vitro and in vivo. Cells of exocrine fraction isolated from the pancreas of brain-dead donors were first cultured in suspension for eight days. This led to the formation of spheroids, composed of a principal population of cells with duct-like phenotype. When cultivated in tissue culture-treated flasks, spheroid cells exhibited a proliferative capacity and coexpressed epithelial (cytokeratin7 and cytokeratin19) and mesenchymal (vimentin and alpha-smooth muscle actin) markers as well as marker of progenitor pancreatic cells (pancreatic duodenal homeobox factor-1) and surface markers of mesenchymal stem cells. The switch from E-cadherin to N-cadherin associated with Snail1 expression suggested that these cells underwent EMT. In addition, we showed coexpression of epithelial and mesenchymal markers in ductal cells of one normal adult pancreas and three type 2 diabetic pancreases. Some of the vimentin-positive cells were found to coexpress glucagon or amylase. These results point to the occurrence of EMT, which may take place on dedifferentiation of ductal cells during the regeneration or renewal of human pancreatic tissues.

Human pancreatic cells of the Capan-1 line form domes in culture during the stationary growth stage. The domes are thought to be a result of the transport of water and electrolytes by the Capan-1 cells. In older Capan-1 cultures, the epithelial sheets formed thickenings from several layers of cells of which the outermost ones were joined by tight type junctions. In the intracellular space, deposits of insoluble calcium salts were observed. Culture of Capan-1 cells in the presence of fibroblasts prolonged survival of the cultures with intact domes for more than 80 days. The Capan-1 cells proliferated forming multilayers and closed cavities which we called super-domes. X-ray spectrometry and electron diffraction analysis showed that the abundant deposits inside these cavities consisted of calcium phosphate in an apatite structure. The number of these deposits increased with time in culture, and they appeared to be formed at the sites of contact with an extracellular matrix consisting of cell debris. Deposits were not observed within the culture medium. Cells from domes were stained cytochemically for ATPases and alkaline phosphatases and examined by light and electron microscopy. The Capan-1 cells surrounding the domes were differentiated, polarized cells containing placental type alkaline phosphatases on their apical membranes and Ca2(+)-ATPases on their basolateral membranes. These enzymes were thought to play a role in the accumulation of phosphate and Ca2+ ions in the dome cavities, which then formed crystals in the presence of organic compounds produced by lysis of cells of the deepest layers of the super-domes. The crystals of hydroxyapatite observed in standard Capan-1 cell cultures and those cocultured with fibroblasts were assumed to be a result of transepithelial transport of Ca2+ and phosphate ions by these cells.

Alveolar type II pneumocytes are thought to be progenitor cells capable of self-renewal and differentiation into type I pneumocytes. Nevertheless, the existence of an alveolar stem cell has been postulated. In lungs from patients with cystic fibrosis, the alveolar epithelium is damaged with ulceration and subsequent regeneration. We characterized alveolar modifications histologically and immunohistochemically in the pulmonary tissue of a patient homozygous for the DF508 mutation. Alveoli were of variable size and surrounded by an inflammatory infiltrate. They were lined by a continuous layer of cuboidal cells with very weak proliferative activity. These cells resembled type II pneumocytes. They expressed thyroid transcription factor-1, cystic fibrosis transmembrane conductance regulator, cytokeratin 7 and contained lamellar bodies. Weak expression of cytokeratin 5 considered to be a marker of progenitor cells of the bronchial and bronchiolar epithelium was detected. Explantation of this alveolar epithelium produced primary cultures and subcultures of epithelial cells that had acquired proliferative properties showing signs of dedifferentiation with a loss of lamellar bodies and a lack of expression of thyroid transcription factor-1. Persistence of the expression of cytokeratin 7 and a strong expression of cytokeratin 5 were observed. The culture conditions were thought to have circumvented the inhibition of proliferation observed in vivo due to the inflammatory peri-alveolar environment. They thus favored the multiplication of a population of cells co-expressing cytokeratin 5 and certain characteristics of type II pneumocytes. The presence of these cells of intermediate phenotype is indicative of the existence of immature precursors for type II pneumocytes.

Low-density lipoproteins (LDL) labeled with indium via a lipid-chelating agent, the bis(stearylamide) of diethylenetri-aminepentaacetic acid (L), were evaluated as a potential radiopharmaceutical (111 In-L-LDL) for tumor localization by studying their internalization in human pancreatic cancer cells (Capan-1). Using DiI-LDL (1,18-dioctadecyl-3,3,38,38-tetramethylindodicarbocyanine perchlorate-LDL), this cell line was shown to bind human LDL with a high-affinity saturable component and a low-affinity non-saturable (40%) component. The single saturable high-affinity binding site had a K D of 27.5 6 2.1 mg/ml and a maximal binding of 610 6 7.5 ng/ml protein. Electron-microscopic examination of the In-L-LDL particles revealed the peripheral distribution of the electron-dense indium atoms at the outer surface of LDL. The modified LDL were then shown to be internalized by the cells. After conjugation of In-L-LDL to colloidal gold to follow the different stages of internalization, electron-microscopic examination showed that the In-L-LDL gold conjugates were stuck to the external sheet of the plasma apical and microvilli membrane, into earlier and later endosomes and into multi-vesicular bodies, suggesting the penetration of the In-L-LDL particles into lysosomal vacuoles. The observation of In-L-LDL-gold conjugates in deep-seated cytoplasm suggests that LDL could be employed as a drug-transport vehicle for targeting cytotoxics or radionuclides close to the cell nucleus. Int. J. Cancer, 70:315-322, 1997. r 1997 Wiley-Liss, Inc. Low-density lipoproteins (LDL) have attracted increasing attention as endogenous drug-transport vehicles to cancer cells since reports of higher receptor-mediated LDL uptake in tumor cells than in normal cells (Ho et al., 1978; Hynds et al., 1984; Lombardi et al., 1989; Samadi-Baboli et al., 1993). Various methods have been proposed for labeling LDL with a metal ion in order to use LDL as target imaging agents in diagnostic applications. For the radiomet-als detectable by gamma scintigraphy, the radioelement was affixed directly or indirectly to protein residues of Apoprotein B-100 (ApoB), the binding ligand to the LDL cell receptor (R-LDL) (Rosen et al., 1990; Virgolini et al., 1991; Ponty et al., 1993). In these procedures, the radiometal/LDL ratio was kept low to avoid disrupting the 3-dimensional structure of the recognition protein. To localize the radionuclide preferentially on the lipid layer and to minimize the structural modification of ApoB, we have developed a new labeling method using a lipid-chelating anchor (Jasanada et al., 1996). A lipophilic derivative of diethylenetriamine-pentaacetic acid (DTPA), the N,N9-bis(stearylamide) of DTPA (L), was first incorporated into LDL giving L-LDL conjugates before adding 111-indium to obtain the final tracer 111 In-L-LDL. In vitro experiments demonstrated the stability of the lipophilic anchorage of the chelator, the stability of the indium-111 complexation in plasma, while binding studies showed recognition of the 111 In-L-LDL particles by LDL...

The present study was designed to evaluate the potential of labeled low-density lipoprotein with 111 In using a lipid chelating agent (bis(stearylamide) of diethylenetriaminepentaacetic acid: L) to detect pancreatic tumors and melanoma in mice by gamma-scintigraphy. We compare the biodistribution of radioactivity and scintigraphic images in nude mice heterotransplanted with human cancerous pancreatic duct cells (Capan-1) and in mice transplanted with murine tumor cells (B16 melanoma). Biodistribution studies showed that radioactivity was twice as high in the Capan-1 xenograft after injection of the radiolabel than after injection of radiometal alone, and 34-fold higher in the B16 tumor. On gamma-scintigraphic imaging, the Capan-1 tumor was just visible, whereas the B16 melanoma was clearly imaged. The lack of contrast of the Capan-1 tumor compared with the B16 melanoma could be due to a poor vascularization.

Pancreatic cancer has an extremely poor prognosis, due, in part, to lack of methods for early diagnosis. The present study was designed to evaluate the potential of labeling low-density lipoprotein (LDL) with a radionuclide using a lipid chelating agent, bis(stearylamide) of diethylenetriaminepentaacetic acid (L), to detect pancreatic tumors by gamma-scintigraphy. Previous studies indicated that the difficulty of visualization of pancreatic tumors was due to their poor vascularization. This study compares the ability of two radiotracers, 111 In-L-LDL and 153 Gd-L-LDL to target highly vascularized rat pancreatic tumors (AR4-2J) implanted in nude mice. Biodistribution studies showed that the tumor uptake of 111 In-L-LDL and 153 Gd-L-LDL tracers was twofold and fivefold higher respectively than with the controls ( 111 In citrate and 153 Gd citrate respectively). These tracers would thus be suitable for scintigraphic imaging.We show here that LDL could be employed as a delivery system for tracers such as 111 In or 153 Gd when these two radionuclides are complexed by a lipid-chelating anchor, and that 111 In-L-LDL and 153 Gd-L-LDL enabled better visualization of the pancreatic tumor tissues, with a better result with 153 Gd-L-LDL.

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