Growth characteristics of human melanoma xenografts

Abstract. The growth of twelve human malignant melanomas in athymic nude mice was studied. Gompertz curves were fitted to volumetric growth data. DNA histograms were obtained with flow cytometry. Each of the twelve melanomas exhibited a characteristic growth pattern, indicating that inherent properties of the tumours are important for the growth control. The theoretical maximum volumes (Vmax) ranged from 208 to 12,900 mm³, the volume doubling times ( T d) from 2.8 to 15.3 days (^V= 50 mm³) and from 3.8 to 64.6 days ( V = 200 mm³), and the fraction of cells in S from 5 to 21%. Tumours with short T d were characterized by a higher growth fraction and probably by a lower cell loss factor than those with long T d. The growth was also influenced by the nude mouse host, as indicated by the values for V max which were similar to those reported for mouse tumours (geometric mean = 8100 mm³), but considerably lower than the volumes of many tumours in man. Also the T d-values for the xenografts were generally lower than those reported for tumours in man, presumably due to a lower cell loss factor. During serial transplantation the growth rate of one of the melanomas increased abruptly, probably because of both an increased growth fraction and a reduced cell loss factor. The latter result demonstrates the necessity of keeping basic biological parameters of xenografts under observation during serial transplantation.     

Fluctuations in pO2 in irradiated human melanoma xenografts

Several studies have demonstrated that untreated tumors may show significant fluctuations in tissue oxygen tension (pO(2)). Radiation treatment may induce changes in the tumor microenvironment that alter the pO(2) fluctuation pattern. The purpose of the present study was to investigate whether pO(2) fluctuations may also occur in irradiated tumors. A-07 human melanoma xenografts were irradiated with single doses of 0, 5 or 10 Gy. Fluctuations in pO(2) were recorded with OxyLite probes prior to irradiation and 24 and 72 h after the radiation exposure. Radiation-induced changes in the tumor microenvironment (i.e. blood perfusion and extracellular volume fraction) were assessed by dynamic contrast-enhanced magnetic resonance imaging. Seventy-two hours after 10 Gy, tumor blood perfusion had decreased to approximately 40% of that prior to irradiation, whereas the extracellular volume fraction had increased by approximately 25%. Fluctuations in pO(2) were seen in most tumors, irrespective of radiation dose and time after irradiation. The mean pO(2), the number of fluctuations around the mean pO(2), the number of fluctuations around threshold pO(2) values of 1, 2, 3, 5, 7 and 10 mmHg, and the amplitude of the fluctuations were determined for each pO(2) trace. No significant differences were detected between irradiated and unirradiated tumors. The results showed that pO(2) fluctuations may occur in irradiated tumors and that the pO(2) fluctuation pattern in A-07 tumors exposed to 5 or 10 Gy is similar to that in untreated tumors. Consequently, these doses did not induce changes in the tumor microenvironment that were sufficient to cause detectable alterations in the pO(2) fluctuation pattern.     

Selection of invasive and metastatic subpopulations from a heterogeneous human melanoma cell line

The formation and propagation of several subpopulations of human melanoma cells from a heterogeneous parental population was accomplished with the use of the Membrane Invasion Culture System (MICS) in vitro under sterile conditions. Five sequentially selected subpopulations of melanoma cells showed an increasing ability to do the following: a) invade reconstituted basement membranes in vitro; b) form experimental lung metastases in vivo; and c) express steady-state levels of human type IV collagenase, a marker for metastatic potential. In addition, the morphology and expression of 35S-methionine-labeled cell surface proteins changed with sequential selection. The adaptation of the MICS assay for studying tumor cell subpopulations allows the morphological, biochemical and molecular characterization of events associated with tumor progression in an in vitro model.     

Growth and vascular structure of human melanoma xenografts

The growth and the vascular structure of five human melanomas grown in athymic nude mice were studied. Four growth parameters (tumour volume doubling time, fraction of cells in S-phase, growth fraction, cell-loss factor) were analysed against each of four vascular parameters (length of vessels with diameters in the range 5-15 micron, total vessel length, total vessel surface, total vessel volume--all per unit of histologically intact tumour volume). Statistically significant linear correlations between the parameters were found for any of the combinations. However, there was a consistent trend in the data: the tumour volume doubling time and the cell-loss factor tended to decrease while the fraction of cells in S-phase and the growth fraction tended to increase with increasing vascular density, whichever vascular parameter was considered. This finding indicates that the vascular density is among the factors which are decisive for the growth rate of tumours. However, the present work does not exclude the possibility that intrinsic properties of the tumour cells may also be important.     

Radioimmunodetection of human melanoma tumor xenografts with human monoclonal antibodies

We established a human IgM monoclonal antibody that defines a tumor-associated membrane antigen expressed on human melanoma cells. The antigen has been identified as the ganglioside GD2. In this paper, we describe the potential usefulness of the human monoclonal antibody for radioimaging. Nude mice bearing tumors derived from a human melanoma cell line were used as a model. Antibody activity was degradated significantly after labeling with ¹³¹I by the use of a modified chloramine-T method. After testing various concentrations, labeled antibody of a specific activity of 2.8 μCi/ μg produced the best results. Balb/c nude mice bearing a GD2-positive M14 melanoma cell line were injected with 10–30 μg of labeled antibody, and its radiolocalization in different organs and in the whole body were evaluated. The best tumor image was obtained on Day 6. The labeled antibody uptake ratio between tumor and muscle was 9.2:1; the ratio between tumor and liver was 1.4:1. These studies represent the first report of experimental tumor imaging with human monoclonal antibody. Human monoclonals will probably prove to be superior reagents for tumor imaging in melanoma patients if the problem of antibody radiolysis is resolved.     

Micronucleus formation in human melanoma xenografts following exposure to hyperthermia

Summary The formation of micronuclei in two human melanoma xenografts (E. E. and V. N.) following hyperthermic treatment (42.5° C for 60 min) was studied and compared to that following single dose irradiation. The melanomas were grown in the hind leg of athymic mice and heated by immersing the tumour-bearing leg into a water-bath. Histological sections were prepared from tumours removed from the mice at predetermined times after treatment and the fraction of abnormal mitotic figures and the number of micronuclei per nucleus were scored. During the first 24 h after treatment, the fraction of abnormal mitotic figures increased abruptly to 90%–100% followed by a rapid decrease to 40%–50%. It then decreased slowly towards about twice the level in untreated tumours. The number of micronuclei started to increase at about the same time as the fraction of abnormal mitotic figures was highest, reached a maximum at about 2–3 days after treatment, and then decreased slowly. The number of micronuclei seen after the hyperthermic treatment was lower than that seen after radiation treatments causing similar tumour regrowth delays. The same hyperthermic treatment resulted in more micronuclei and larger regrowth delays for E. E. than for V. N. melanoma. The present results indicate that DNA damage is involved in heat-induced cell death in tumours treated in vivo.     

Assessment of fraction of hypoxic cells in human tumor xenografts with necrotic regions by dynamic contrast-enhanced MRI

The potential usefulness of gadopentetate dimeglumine (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) for assessing hypoxia in tumors with significant necrosis was investigated. Small (100-350 mm(3)) and large (500-1000 mm(3)) D-12 and U-25 tumors were subjected to DCE-MRI, measurement of the fraction of necrotic tissue, and measurement of the fraction of radiobiologically hypoxic cells. Images of E.F (E is the initial extraction fraction of Gd-DTPA and F is perfusion) and lambda (lambda is proportional to extracellular volume fraction) were produced by subjecting the DCE-MRI data to Kety analysis. Necrotic tissue could be identified in lambda images but not in E.F images of the tumors. Most voxels in viable tissue showed lambda values of 0.15-0.70, whereas the lambda values of most voxels in necrotic tissue were either <0.15 or >0.70. The E.F and lambda frequency distributions of the viable tissue, but not the E.F and lambda frequency distributions of the whole tissue, were consistent with the observation that the four groups of tumors showed similar fractions of radiobiologically hypoxic cells. E.F and lambda images may thus provide useful information on the extent of hypoxia in tumors provided that voxels in necrotic tumor regions are identified and excluded from the images.     

Phenotypic and functional changes of human melanoma xenografts induced by DNA hypomethylation: immunotherapeutic implications

Emerging in vitro evidence points to an immunomodulatory activity of DNA hypomethylating drugs in human malignancies. We investigated the potential of 5-aza-2'-deoxycytidine (5-AZA-CdR) to modulate the expression of cancer testis antigens (CTA) and of HLA class I antigens by melanoma xenografts, and the resulting modifications in immunogenicity of neoplastic cells. Three primary cultures of melanoma cells, selected for immune phenotype and growth rate, were grafted into BALB/c nu/nu mice that were injected intraperitoneally with different dose- and time-schedules of 5-AZA-CdR. Molecular analyses demonstrated a de novo long-lasting expression of the CTA MAGE-1, -2, -3, -4, -10, GAGE 1-6, NY-ESO-1, and the upregulation of MAGE-1, MAGE-3, and NY-ESO-1 levels in melanoma xenografts from 5-AZA-CdR-treated mice. Serological and biochemical analyses identified a de novo expression of NY-ESO-1 protein and a concomitant and persistent upregulation of HLA class I antigens and of HLA-A1 and -A2 alleles. Immunization of BALB/c mice with 5-AZA-CdR-treated melanoma cells generated high titer circulating anti-NY-ESO-1 antibodies. Altogether, the data obtained identify an immunomodulatory activity of 5-AZA-CdR in vivo and strongly suggest for its clinical use to design novel strategies of CTA-based chemo-immunotherapy for melanoma patients.     

Evaluation of thymidylate synthase protein expression by Western blotting and immunohistochemistry on human colon carcinoma xenografts in nude mice.

In this study we investigated the relationship between thymidylate synthase (TS) protein expression, evaluated by Western blotting analysis and by immunohistochemistry (IHC), and growth rate in human colon xenograft tumors in nude mice. Human colon cancer cell lines were used to induce xenograft tumors and the tumor mass growth rate was calculated by measuring tumor size variations over time. TS 106 monoclonal antibody was used for both Western blotting and IHC TS detection. Tumor cell growth fraction was measured by Ki67/MIB1 immunolabeling and tumor cell growth rate by evaluating the mean nucleolar size in silver-stained sections. TS Western blotting values were related to tumor mass growth rate (p<0.001) and cell growth rate (p=0.002) but not to cell growth fraction (p=0.676). The degree of the IHC staining showed only a trend to be associated with TS protein expression measured on Western blotting, and was not related either to tumor mass growth or cell proliferation rate. Tumor xenografts were also characterized for TS promoter tandem repeat and p53 status. No relationship was observed between these variables and TS expression evaluated by both Western blotting and IHC analysis. Our results demonstrate that TS expression evaluated by Western blotting analysis is directly related to the tumor mass growth rate and question the use of the IHC approach to obtain precise quantitative information on TS expression in tumor samples.     

Production of a human T lymphocyte chemotactic factor by T cell subpopulations

Concanavalin A-stimulated human peripheral blood mononuclear cells release a lymphocyte chemotactic factor. This lymphocyte chemotactic factor is produced optimally after 24 to 48 hr of culture and is not found before 3 hr of culture, which suggests that the factor is synthesized de novo and is not preformed and secreted after Con A stimulation. This is further supported by experiments showing that the protein synthesis inhibitors cycloheximide and puromycin totally prevent the production of the chemotactic factor. Experiments using cultured and uncultured T lymphocytes as responding cells show that cultured T cells respond more efficiently than uncultured T cells to this factor. Furthermore, the lymphocyte chemotactic factor preferentially stimulates T lymphocyte locomotion as compared to peripheral blood non-T lymphocyte migration. Fractionation of mononuclear cells into glass nonadherent lymphocytes, monocyte-enriched preparations, T lymphocytes, and non-T lymphocytes shows that lymphocyte chemotactic factor is produced by Con A-stimulated, glass nonadherent lymphocytes and T cells but not by monocytes or non-T lymphocytes. Further fractionation of T lymphocytes into Leu-2 and Leu-3 T cell subpopulations shows that the production of T lymphocyte chemotactic factor can be attributed to the Leu-2 suppressor/cytotoxic T cell subset. The generation of a T lymphocyte chemotactic factor by Leu-2 T cells may represent a means of recruiting other T cells to the site of its release.     

Detection of HBD1 peptide in peripheral blood mononuclear cell subpopulations by intracellular flow cytometry

Production of human beta-defensin1 (HBD1) in response to LPS in monocytes, myeloid dendritic cells and plasmacytoid dendritic cells (PDC) was examined. Since PDC make up only 0.1-0.5% of the peripheral blood mononuclear cell population, we developed a method to determine HBD1 peptide levels using four-color flow cytometry, which can examine several cell surface or intracellular markers at once. Coupled with intracellular flow cytometry, we determined that PDC and monocytes only made significant amounts of HBD1 when exposed to >50ng/ml LPS for 2h. This response was limited to monocytes when ultrapure LPS was used, and was inhibited in PDC by chloroquine treatment.     

Fourier-Transform InfraRed (FT-IR) spectroscopy to show alterations in molecular composition of EV subpopulations from melanoma cell lines in different malignancy

BackgroundMelanoma cells release extracellular vesicles (EVs) subpopulations which differ in size, phenotype and molecular content. Melanoma derived EVs play a role in the development and progression of cancer by delivering surface receptors and bioactive (proteins, lipids, nucleic acids) or signaling molecules to target cells.MethodsWe applied Fourier Transform Infrared Spectroscopy (FTIR) to compare infrared spectra of absorption for different subpopulations of EVs originating from normal human melanocytes, primary cutaneous melanoma (WM115) and metastatic cutaneous melanoma (WM266-4).ResultsFTIR results showed that exosome and ectosome populations differ in content of protein and lipid components. We obtained higher lipid to protein ratio for ectosomes in comparison with exosomes what confirms that exosomes are very densely packed with protein cargo. We identified the lowest value of saturated fatty acids/unsaturated fatty acids parameter in the metastatic WM266-4 cell line and ectosomes derived from WM266-4 cell line in comparison with normal melanocytes and the primary WM115 cell line. We identified the alterations in the content of secondary structures of proteins present in EV subpopulations originating from melanocytes and melanoma cells in different malignancy.ConclusionsObtained results revealed differences in the molecular composition of melanoma derived EVs subtypes, including protein secondary structure, and showed progressive structural changes during cancer development.     

Detection of human papillomavirus in cervical cell specimens by hybrid capture and PCR with different primers

The purpose of this study was to compare hybrid capture assay with PCRs using different primers for the L1, E6-E7 regions for the detection of human papillomavirus (HPV) genome. One hundred twenty-five cervical smears with normal (n=42) and abnormal (n=83) cytology were investigated. Those at high-risk for HPV were studied by hybridization antibody capture assay and PCR with the pU-1M/pU-2R primers. Target DNA from the HPV L1 region was amplified by SPF10 primer set and home-PCR with MY09/MY11 primers. The presence of HPV DNA in cervical smears was detected by SPF10 (in 72% of cases), MY09/MY11 (58%), hybrid capture (55%) and pU-1M/pU-2R (39%). Results obtained with the SPF10 and MY09/MY11 consensus primer sets as well as hybrid capture and pU-1M/pU-2R specific for high-risk types differed significantly (chi2, P<0.0005). The correlation between assays with the use of SPF10 and MY09/MY11 was 86% and between hybrid capture and the pU-1M/pU2R technique--78%. In 49% of samples HPV DNA was detected by the four methods, whereas in 12% only by the SPF10 primers. The most sensitive technique was found to be PCR with the use of SPF10 primers, while the most specific--the MY09/11 PCR method. It seems that home-PCR with MY09/MY11 primers could be applied in screening tests.     

Detection of factors of differentiation in human malignant melanoma

Factors of differentiation (FD) have been detected and isolated from human melanomas cloned in nude mice as well as from amphibian embryos from neurula--early tail bud stage. It has been shown that each source contains two factors of differentiation--mesodermalizing (MF) and melanogenic (MgF). Biological activity of MF has been determined by its capacity to initiate in early embryonic multipotent cells the appearance of various cell types, normally arising from mesoderm. Biological activity of MgF have been determined by its capacity to initiate melanogenesis both in epithelial cells and in dermal melanophores of clawed toad. It has been shown that both factors, isolated from both sources are heterogeneous by their molecular weights. Mr values determined for MgF, isolated from both sources, are nearly identical whereas Mr values of MF from sources coincide partially. Resemblance of FD detected in remote representatives of animal kingdom suggest the high evolutionary conservatism of factors, which switch on cell differentiation.     

Cytolytic activity of human natural killer cell subpopulations isolated by four-color immunofluorescence flow cytometric cell sorting

The natural killer activity and phenotypic properties of six different subpopulations of normal human peripheral blood lymphocytes obtained by four-color immunofluorescence cell sorting were examined. Phycoerythrin-conjugated CD16 and CD56 were used simultaneously to identify (CD16 + CD56+) NK cells. The cells most effective in mediating NK cytolysis against K562 target cells were CD3-(16 + 56+)57 -8+. Although most of the K562 killing was found in the CD3-(16 + 56 +) groups of cells, a substantial degree of NK activity was detected in the CD3+(16 + 56 +) subpopulations of some individuals. The level of expression of CD57 and CD8 was significantly higher on CD3+(16 + 56 +) than on CD3-(16 + 56 +) cells.