Pulmonary fibrosis resulting from diverse etiologies is characterized by proliferation of fibroblasts and excessive accumulation of interstitial collagen. Whether fibrosis is associated with selective expansion of fibroblast subpopulations differing in amounts or types of collagens synthesized is unknown. We have previously isolated lines and clones of normal murine lung fibroblasts based on the presence of the Thy 1 surface antigen. These subpopulations differ in morphology, growth characteristics, and display of class II major histocompatibility complex antigens (R.P. Phipps, D.P. Penney, P. Keng, H. Quill, A. Paxhia, S. Derdak, and M. E. Felch. Am. J. Respir. Cell Mol. Biol. 1: 65-74, 1989). We evaluated the amounts and types of collagen and fibronectin synthesized by Thy 1+ (Fib2-T-3+) and Thy 1- (Fib2-T-4-) lung fibroblast lines and clones. Thy 1+ fibroblast line synthesized two- to threefold more collagen and noncollagen protein than the Thy 1- line. In contrast, both the Thy 1+ and Thy 1- lines synthesized similar amounts of fibronectin. Thy 1+ and Thy 1- lines and clones expressed mRNA for alpha 1(I)-and alpha 1(III)-procollagen and synthesized both types (predominantly type I and lesser amounts of type III) of collagen, protein, and mRNA. The fibroblast clones varied significantly in total collagen and fibronectin production, with one Thy 1- clone (D3) synthesizing the largest amount of collagen but relatively little fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Summary Hypoxic cells in KHT sarcomas were characterized using fluorescence activated cell sorting based on the diffusion properties of the fluorochrome Hoechst 33342. Tumour-bearing female C3H/HeJ mice were injected i.v. with lOjgg-g Hoechst 33342 and the cells derived from the tumours sorted on the basis of their staining intensities. For each sorted fraction the DNA histogram was evaluated using FCM analysis. The results indicated that the bright and dim cells were not equally distributed about the cell cycle. For example, a greater proportion of S phase cells were in the bright subpopulations whereas the dim subpopulations contained an increased proportion of cells in GV. When the tumours were irradiated with a single dose of radiation prior to cell sorting, the dim cells survived preferentially. Dose response curves for the 20% most dim and 20% most bright cells, sorted on the basis of fluorescence intensity, then were determined. The survival curves of the dim and bright cells were found to have slopes similar to those of KHT cells irradiated in situ in dead animals or in vitro under fully oxic conditions, respectively. In addition, when KHT sarcomabearing mice were given a 2.5mmolkg-1 dose of misonidazole (MISO) prior to irradiation and cell sorting, the dim subpopulation was sensitized whereas the bright subpopulation was not. These findings suggest that (i) compared to well-oxygenated areas, hypoxic regions of KHT tumours contain a smaller percentage of cells actively proliferating and (ii) Hoechst 33342 sorting may allow the detailed in situ evaluation of agents acting directly against hypoxic cells in solid tumours.

Summary Hoechst 33342 is a fluorescent dye used for cell selection from tumours based upon intratumour location. When the dye is administered i.v. to tumour-bearing animals, cellular fluorescence is directly related to the proximity of cells to blood vessels. The present study compared inherent Hoechst fluorescence between in vitro-stained EMT6/Ro (mouse mammary sarcoma) cells and host cells, to determine if these populations have different staining characteristics that may influence cell selection procedures. Tumour cell fluorescence exceeded host cell staining by 8-fold when pure cell populations (EMT6/Ro monolayer cells, mouse spleen and peritoneal cells) were compared, and 3-fold for tumour cell-enriched and host cell-enriched populations from solid tumours. Inherent uptake of HO 33342 appeared to be correlated with cell volume. These differences in inherent dye uptake between host and tumour cells were found to be minor in comparison to the fluorescence gradient between the 10% brightest and 10% dimmest (78-fold) cell populations from in vivostained tumours.The bisbenzamide dye Hoechst 33342 (HO 33342) has been used for the selection of cell populations from different locations within multicellular tumour spheroids (Durand, 1982; and solid tumours . Administration of HO 33342 to tumour-bearing animals (via i.v. injection), or incubation of multicellular tumour spheroids in the dye (via dilution in tissue culture medium), results in diffusion-limited delivery of the dye, based upon the proximity of cells to either blood vessels supplying the tumour, or surface of the spheroid, respectively. HO 33342 binds to cellular DNA, and at concentrations greater than 6pgml-1, staining of cells is based upon their DNA content; lower concentrations result in fluorescence intensity being determined by relative cellular metabolic activity (Loken, 1980), activation status (for antigen-or mitogen-stimulated lymphocytes) , and possibly other factors as well. Cell populations obtained from different intratumour locations by in vivo Hoechst staining, followed by cell sorting based upon fluorescence intensity, have been examined for parameters such as in vitro survival, adriamycin cytotoxicity, and resistance to radiotherapy , and for investigation of the cell cycle distribution of chronically hypoxic cells within solid tumours (Pallavicini et al., 1979).Many types of murine and human tumours are significantly infiltrated by host cells, primarily macrophages, neutrophils, and lymphocytes (Witz & Hanna, 1980). However, studies of HO33342 for cell selection in tumours have until now focused primarily upon staining of the tumour cell population, and direct comparisons of Hoechst staining of host and tumour cells have not been made. In order for HO 33342 to be useful for selection of cells based upon location with tumours, it is necessary that significant differences in fluorescence intensity between cells result only from differences in location and not from inherent variations in staining between host and tumour cells. The objecti...