Separation and characterization of neoplastic cell subpopulations of a transplantable rat pancreatic acinar carcinoma.

The transplantable pancreatic acinar carcinoma established in F344 rats demonstrates heterogeneity of cytodifferentiation with cell types ranging from those containing no zymogen granules to cells with mature zymogen differentiation. Two relatively homogeneous subpopulations of cells have been isolated by isopyknic Percoli gradient centrifugation from the heterogeneous cell population of this tumor. The subpopulation obtained at a density of 1.0987 g/ml was designated the granule-enriched fraction (GEF) and contained morphologically differentiated cells with abundant mature zymogen granules. The other subpopulation, obtained at a density of 1.0789 g/ ml, consisted of poorly differentiated cells lacking zymogen maturation and was thus termed the granule-deficient fraction (GDF). In both fractions, greater than 97% of the cells were viable and maintained linear rates of [3H]leucine and [3H]thymidine incorporation into protein and DNA for up to 3 hr following isolation. High-resolution autoradiographic analysis of [3H]thymidine incorporation revealed that 20% of the cells in GDF subpopulation and 12% of the cells in GEF subpopulation synthesize DNA. Morphological and morphometric analyses of the isolated GEF and GDF subpopulations of the acinar carci noma revealed distinct differences in nuclearcytoplasmic ratio, secretory granule content, and degree of polarity. The GEF subpopulation contained significant amounts of stored zymo gen, as evidenced by higher levels of amylase and lipase activities. In contrast, the GDF subpopulation displayed very low levels of these enzymes. Both GDF and GEF subpopulations produced tumors when injected s.c. into F344 rats. Characterization of concanavalin A (Con A)-binding sites on the plasmalemma of the GDF and GEF subpopulations by the Con A:peroxidase method indicated the presence of Con A receptors on pancreatic carcinoma cells regardless of the extent of cytodifferentiation in contrast to normal pancreatic embryogenesis, in which Con A receptors are discerned only in acinar cells containing mature secretory granules. The pres ence of Con A receptors on all cells of the GDF and GEF acinar carcinoma cell subpopulations suggests either that neoplastic transformation results in altered genetic expression whereby progenitor "stem" cells, which lack Con A receptors during normal pancreatic embryogenesis, acquire such receptors or that the pancreatic carcinoma arises from dedifferentiation of mature acinar cell which normally possesses Con A receptors. INTRODUCTION In recent years, several studies have dealt with the isolation and identification of purified populations of neoplastic cells from several experimental and human solid tumors using den sity gradient centrifugation. For the most part, these investi gations were aimed at isolating various nontumor cells from the tumor cells (1, 12, 30-35, 45, 52) but not at separating the generally heterogeneous tumor cell population into subpopu lations that depict differentiative and metastasizing potential (3, 13, 19). Highly purified subpopulations of neoplastic cells that are morphologically distinct and exhibit identifiable markers of gene expression would greatly facilitate investiga tions of cellular and biochemical events controlling not only differentiation in neoplastic cell population but also mainte nance of the malignant phenotype. Furthermore, comparative studies of tumor cell subpopulations with normal cellular com ponents of the tissue of origin of a given tumor may provide important clues about initiation and progression of neoplasia. The distinct morphological and enzymatic parameters of differentiation in cells of the transplantable pancreatic acinar carcinomas of rats (22,36, 39) provide a suitable model system to study tumor heterogeneity and cytodifferentiation (21, 48). Morphological analysis of the transplantable pancreatic acinar carcinoma established in the F344 strain of rats by Reddy and Rao (39) has revealed 4 cell subtypes, designated types 1 to 4, that display varying degree of cytodifferentiation (41). Type 4 neoplastic acinar cells possess all the characteristics of mature pancreatic acinar cells including abundant, well-formed secretory (zymogen) granules. Type 1 cells are undifferentiated and lack secretory granules and the Golgi apparatus. Tumor cell types 2 and 3 are considered as intermediate stages in this spectra of heterogeneous pancreatic acinar tumor cell populations. Biochemical and immunocytochemical studies have demonstrated the presence of at least 19 exocrine pan creatic enzymes in the secretory granules of these neoplastic cells (17, 42).3 Whether these cell types represent well-defined stages of differentiation or dedifferentiation sequence remains unclear (41 ). We now report the separation of 2 major subpopulations of cells from this pancreatic acinar carcinoma by isopyknic gradient centrifugation: one subpopulation, termed GEF*, consists predominantly of type 3 and 4 (secretory gran ule-containing) cells described previously (41). The second subpopulation, termed GDF, consists of type 1 and 2 cells that lack granules. In this report, we describe the separation tech nique with Percoli as a gradient medium (29, 31) and present ' This work was supported by Grant CA-23055, awarded by the National Cancer Institute, Department of Health and Human Services. This manuscript constitutes a part of doctoral thesis to be submitted by Michael J. Becich to the faculty of Northwestern University in September 1982. 2 To whom requests for reprints should be addressed, at the Department of Pathology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, III. 60611. Received March 9, 1982; accepted June 11,1982. 3 Presented at the Joint Meeting of the American Pancreatic Association/ National Pancreatic Cancer Project. Chicago, III., November 1981 (4). 4 The abbreviations used are: GEF, granule-enriched fraction; GDF, granuledeficient fraction; STI, soybean trypsin inhibitor; Con A, concanavalin A; BSA, bovine serum albumin; KRB, Krebs-Ringer bicarbonate; HSS, Hank's salt solu