Cytoreductive effects of anti-transferrin receptor immunotoxin in a multicellular tumor spheroid model.

We have evaluated the sensitivity to immunotoxins (IT) of monolayer and of 200-250 microns multicellular tumor spheroid (MTS) cultures obtained with human breast (MCF7) and glioblastoma (U118) tumor cells and with rat glioblastoma (9L) cells. Monolayer MCF7 and U118 cells were highly sensitive to antitransferrin receptor (anti-TfnR) ricin A chain (RTA)-IT (Tfn-RTA and MAb OKT9-RTA) treatment in the presence of the intracellular RTA-IT enhancing agent human serum albumin-monensin (HSA-Mo) conjugate. A 790- to 2000-fold higher concentration of anti-TfnR IT was instead required to reduce by 50% the volume of individually treated MCF7 spheroids, as evaluated by applying the Gompertz growth model. Monolayer 9L cells showed 230- to 5700-fold lower sensitivity to Tfn-RTA IT than MCF7 and U118 monolayers, yet 9L spheroid cells were almost as sensitive to anti-TfnR IT as monolayer 9L cultures. Binding studies performed with [125I]-Tfn and FITC-labelled anti-TfnR MAb revealed that 9L monolayers and MTS expressed 4.1-fold and 8.8-fold lower amounts of TfnR than MCF7 monolayers and MTS, respectively. However, Tfn bound to TfnR sites of 9L and of MCF7 cells with comparable affinity. Experiments carried out with the diphtheria toxin mutant CRM107 linked to Tfn confirmed the pattern observed with RTA-IT. Monolayers and spheroids showed no considerable differences in sensitivity to ricin toxin. Collectively, these results indicated that the efficacy of IT against 3-D tumors is heavily influenced by the number of target Ag expressed by the tumor cells, as well as by the affinity of IT/toxin-cell interaction.

Phenotypic analysis of human peripheral blood lymphocytes by automatic sampling flow cytometry after stimulation with mitogens or allogeneic cells.

Human peripheral blood lymphocyte (PBL) phenotypes have been analyzed before and after stimulation with phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM) for 3 days and in mixed lymphocyte culture (MLC) for 7 days. PBL labeled with each of 10 fluorescent monoclonal antibodies were automatically sampled for flow cytometry from 96-well microtiter plates using a microsample delivery system. The reference phenotypic ranges were determined in fresh cells and control cultures. PHA was mostly mitogenic for T PBL bearing the CD3, CD5, CD7, CD8 and CD25 differentiation clusters, and a low density of CD1 and CD4 had a small effect on human natural killer cells (HNK) and also did not stimulate B (CD19) and HLA-DR+ PBL. There was an incomplete phenotypic overlapping between PHA- and ConA-stimulated cultures, ConA being more mitogenic for CD4 and less mitogenic for CD8 PBL. The mitogenic effect of PWM was evident on CD3, CD5, CD7, CD4, CD25 and CD8, but not on HNK, HLA-DR and CD19 B PBL, which presumably had already differentiated into antibody-secreting cells. After MLC stimulation all T, B and HNK PBL subsets tested were increased, but the cells bearing CD1, CD4, CD5, CD7, CD25, HNK, CD19 and HLA-DR had the greatest proliferation with respect to the unmixed control PBL. The present approach to the phenotyping of PBL subsets could offer more complete and accurate data for monitoring and follow-up of patients in transplantation and immunopathology hospital wards.

DNA analysis of stimulated lymphocytes by automatic sampling for flow cytometry.

A microsample delivery system (MSDS) was tested for automatic flow cytometry (FCM) analysis of DNA synthesis in stimulated human peripheral blood lymphocytes (PBL) cultivated in wells of microtiter plates. After incubation, either for 1-3 days with phytohemagglutinin, concanavalin A, and pokeweed mitogen, or for 7 days with allogenic PBL, the cells, while in the wells, were washed in hypotonic Tris buffer and stained with ethidium bromide-RNAse solution. The results obtained from quintuplicate replicated wells, each of the five containing the same control or stimulated cultures, were reproducible in terms of the number of nuclei counted in each histogram of control, mitogen-stimulated PBL, and mixed lymphocyte cultures (MLC). Using a computer program that superimposes histograms and calculates their differences on the scale of fluorescence intensity, it was possible to quantify the intensity of the response to the mitogenic stimuli. This approach to the study of lymphocyte proliferation offers not only a simpler and faster analysis of DNA synthesis than the method of 3H-thymidine incorporation, but it also allows for the analysis of other FCM parameters, such as forward and 90 degrees light scatter and double fluorescence labelling of PBL nuclei.

Establishment and characterization of a new human melanoma cell line (HU 214) with a high growth potential and stable properties.

A human melanoma cell line (HU 214) with high growth potential was established from a lymph node metastasis of a patient with advanced cutaneous melanoma. The cells of this line were able to grow in monolayer (according to the Rosenblum technique) and in agar (according to the Courtenay-Mills method), and formed tumors when injected in nude mice. The line has been maintained in culture for more than 47 passages. The cell cultures were periodically characterized (every 6-8 passages) by immunohistochemistry using a panel of monoclonal antibodies (MoAbs) including MoAbs against tumor-associated antigens (antimelanoma, antiglioma and anti-LLA), against vimentin, and against major histocompatibility antigens, and including also Ki 67, a MoAb which reacts with a nuclear antigen associated with cell proliferation. The results of this characterization indicate that we have established a human melanoma cell line with a stable antigenic phenotype during subculturing, poorly differentiated cells, and a high growth potential.

Human glioma cell lines: tumour associated antigens distribution and sensitivity to antibody-toxin or ligand-toxin conjugates. A preliminary report.

We have investigated the phenotype of seven human glioma cell lines established in vitro from primary tumour explants. Indirect immunofluorescence and flow cytofluorimetry revealed a heterogeneous distribution of surface GE 2 and CG 12 Tumour Associated Antigens (TAA). In one group of cell lines TAA were detected both at the cell surface and in the cytosol, whereas in a second group of glioma cell lines TAA were found only in the cytosol. We have also investigated the sensitivity of glioma-derived cell lines to antibody-toxin and ligand-toxin conjugates (Immunotoxins). Monoclonal antibodies anti GE 2 antigen linked to ricin toxin A subunit (RTA) showed poor cytotoxicity, which increased about 50 fold when the whole toxin was linked to anti GE 2 monoclonals. Treatment with human recombinant interferon gamma (IFN-gamma) greatly augmented the percentage of HLA-DR+ cells and the amount of HLA-DR antigens per cell. IFN-gamma treatment resulted in a net increase of sensitivity to anti HLA-DR Immunotoxins (IT). Human diferric transferrin linked to RTA exhibited a potent cytotoxic effect against human glioma-derived cells when used in the presence of the lysosomotropic carboxylic ionophore monensin.

In vitro analysis of BCNU-sensitivity in human malignant gliomas. II. Cross-resistance studies with cisplatinum and nitrosoureas.

With the use of the Human Brain Tumor Stem Cell Assay (HBTSCA) in a cross-resistance study, four early (3-4) culture passages of human malignant gliomas (glioblastoma multiforme) were tested for in vitro chemosensitivity with three of the most effective single agents for brain tumor chemotherapy: BCNU, CCNU and cisplatinum (DDP). The shapes of the dose-response curves indicated complete cross-resistance between BCNU and CCNU, i.e. two chloroethyl-nitrosoureas sharing a common alkylating-carbamoylating activity, with no evident cross-resistance between the two nitrosoureas and the DDP, a DNA binder with a putatively different antitumor action. Probably because of differences in drug delivery kinetics or in the cytotoxic mechanism, DDP might play a role in the treatment of nitrosourea-resistant gliomas.

In vitro analysis of BCNU-sensitivity in human malignant gliomas. I. A model study with alkylating, cross-linking and carbamoylating agents in anaplastic astrocytomas of pediatric age.

Like all chloroethyl-nitrosoureas of major clinical use, 1,3 bis-(2-chloroethyl)-1-nitrosourea (BCNU) - which is one of the most effective chemotherapeutic agents for CNS malignancies - biologically degrades into active alkylating and carbamoylating moieties. Using a human brain tumor stem cell assay, we analyzed a series of anaplastic astrocytomas of pediatric age, characterized by different degrees of BCNU-resistance. Early (2-4) passage cultures from these tumors were treated in vitro with model drugs for alkylation (BCNU, CHLZ (2-[3-(2-chloroethyl)-3-nitrosoureido]-2-deoxy-D-glucopyranose), ENU (N-ethyl-N-nitrosourea), cross-linking (BCNU, CHLZ) and carbamoylation BHCNU (1,3 bis (trans-4-hydrocyclohexyl)-1-nitrosourea): dose-schedules were compatible with clinically achievable levels. Results of chemosensitivity tests confirmed that - as previously reported in malignant gliomas of the adult - cellular resistance to BCNU was closely related to the cross-linking activity of alkylating species. However, in pediatric gliomas the levels of cell kill after treatment with the purely carbamoylating agent BHCNU, even at the highest doses tested, were lower than expected.