[Radiobiological characteristics of cancer stem cells from esophageal cancer cell lines].

OBJECTIVE: To study the cancer stem cell populations in esophageal cancer cell lines KYSE150 and TE1 and identify whether resulting stem-like cell spheres display radiation resistance characteristics. METHODS: Serum-free medium (SFM) suspension was used to culture the esophageal cancer stem cell lines and enrich the esophageal stem-like cell spheres. RT-PCR assay was used to detect the stem cell gene expression in the sphere cells. Radiosensitivity of the sphere cells and parental cells were evaluated by clone formation assay. Different cells after irradiation at different doses were tested to evaluate the changes of sphere formation, and cell cycle and CD44(+)CD271(+) expression of the sphere cells were also analyzed by flow cytometry before and after irradiation. RESULTS: Cancer stem-like cell spheres were generated from KYSE150 and TE1 cells and enriched by culture in serum-free medium, and the number of spheres was increasing alone with the increase of cell passages. The numbers of spheres formed from the 1st, 2nd and 3rd generations of KYSE150 cells were 25 ± 2, 37 ± 2 and 47 ± 3, respectively. The numbers of spheres formed from the 1st, 2nd and 3rd generations of TE1 cells were 15 ± 3, 24 ± 3 and 36 ± 4, respectively. Certain doses of radiation increased the sphere formation rate. The average survival fraction (SF2) of the suspension-cultured KYSE150 stem-like cell spheres after 2 Gy irradiation were 0.81 ± 0.03 and 0.69 ± 0.04, while that of TE1 parental cells were 0.87 ± 0.01 and 0.80 ± 0.03 (P < 0.05 for all). In the esophageal parental KYSE150 and TE1 cells, arrest at G2 phase was induced after irradiation. After the same dose of irradiation, the inhibition of proliferation of the cancer stem cells was lower than that of the parent cells (P < 0.05). After 0, 4 and 8 Gy irradiation, the CD44(+)CD271(+) cell percentage of KYSE150 parental cells were (1.08 ± 0.03)%, (1.29 ± 0.07)% and (1.11 ± 0.09)%; the CD44(+)CD271(+) cell percentage of TE1 parental cells were (1.16 ± 0.11)%, (0.97 ± 0.08)% and (1.45 ± 0.35)% (P > 0.05 for all). After 0, 4 and 8 Gy irradiation, the percentage of CD44(+)CD271(+) cells of KYSE150 stem cell-like spheres were (35.83 ± 1.23)%, (44.90 ± 1.67)% and (57.77 ± 1.88)%, and that of TE1 stem cell-like spheres were (16.07 ± 0.91)%, (22.67 ± 1.12)% and (33.27 ± 1.07)%. Compared the 4 Gy and 8 Gy irradiated KYSE150 and TE1 stem-like cell spheres with the 0 Gy irradiated spheres, the differences were statistically significant (P < 0.05 for all). CONCLUSIONS: The cancer stem cells in KYSE150 and TE1 spheres are more radio-resistant than their parental cells. It may suggest that cancer stem cell populations in the esophageal cancer cells are related to the mechanism of occurrence of radioresistance.

18F-FLT and 125I-IdUrd uptake increase in human tumour cell lines induced by the thymidylate synthase inhibitor FdUrd.

AIM: 5-fluoro-2'-deoxyuridine (FdUrd) depletes the endogenous 5'-deoxythymidine triphosphate (dTTP) pool. We hypothesized whether uptake of exogenous dThd analogues could be favoured through a feedback enhanced salvage pathway and studied the FdUrd effect on cellular uptake of 3'-deoxy-3'-18F-fluorothymidine (18F-FLT) and 5-125I-iodo-2'-deoxyuridine (125I-IdUrd) in different cancer cell lines in parallel. METHODS: Cell uptake of 18F-FLT and 125I-IdUrd was studied in 2 human breast, 2 colon cancer and 2 glioblastoma lines. Cells were incubated with/without 1 µmol/l FdUrd for 1 h and, after washing, with 1.2 MBq 18F-FLT or 125I-IdUrd for 0.3 to 2 h. Cell bound 18F-FLT and 125I-IdUrd was counted and expressed in % incubated activity (%IA). Kinetics of 18F-FLT cell uptake and release were studied with/without FdUrd modulation. 2'-3H-methyl-fluorothymidine (2'-3H-FLT) uptake with/without FdUrd pretreatment was tested on U87 spheroids and monolayer cells. RESULTS: Basal uptake at 2 h of 18F-FLT and 125I-IdUrd was in the range of 0.8-1.0 and 0.4-0.6 Bq/cell, respectively. FdUrd pretreatment enhanced 18F-FLT and 125I-IdUrd uptake 1.2-2.1 and 1.7-4.4 fold, respectively, while co-incubation with excess thymidine abrogated all 18F-FLT uptake. FdUrd enhanced 18F-FLT cellular inflow in 2 breast cancer lines by factors of 1.8 and 1.6, respectively, while outflow persisted at a slightly lower rate. 2'-3H-FLT basal uptake was very low while uptake increase after FdUrd was similar in U87 monolayer cells and spheroids. CONCLUSIONS: Basal uptake of 18F-FLT was frequently higher than that of 125I-IdUrd but FdUrd induced uptake enhancement was stronger for 125I-IdUrd in five of six cell lines. 18F-FLT outflow from cells might be an explanation for the observed difference with 125I-IdUrd.

Role of sonography for implantation and sequential evaluation of a VX2 rabbit liver tumor model.

OBJECTIVE: We investigated the role of sonography in the implantation process of a VX2 rabbit liver tumor model and sequential evaluation. METHODS: Fifty rabbits were divided into 2 groups. Animals in group I underwent surgical implantation, whereas those in group II received percutaneous sonographically guided implantation. At 7, 14, 21, and 28 days after implantation, respectively, 5 rabbits in each group were examined with conventional, color Doppler (CD), contrast-enhanced (CE) pulse inversion harmonic (PIH), and CE CD sonography. Pathologic examination was performed with hematoxylin-eosin, nicotinamide adenine dinucleotide phosphate-diaphorase, and succinic dehydrogenase stains. RESULTS: Twenty-one rabbits with tumors survived in group I, and 22 with tumors survived in group II. The mean duration of implantation +/- SD in group II was 16.9 +/- 3.4 minutes, whereas that in group I was 21.5 +/- 4.1 minutes (P < .05). The tumor volume measured by conventional sonography increased from 0.28 +/- 0.14 cm(3) at 7 days to 16.49 +/- 5.50 cm(3) at 28 days in group I and from 0.31 +/- 0.19 to 19.79 +/- 4.70 cm(3) in group II, whereas no significant difference existed between the groups. On CD, CE PIH, and CE CD sonography, most tumors were hypervascular before 14 days and after 14 days had peripheral vessels and central hypovascular areas, which were shown as necrotic areas by pathologic examination. CONCLUSIONS: Sonographically guided implantation achieved a good success rate with convenient inoculation performance. Conventional gray scale, CD, CE PIH, and CE CD sonography were useful in sequential evaluation of tumor growth and characteristic vascularity.

Synchrotron-based in vivo tracking of implanted mammalian cells.

We have developed an X-ray imaging protocol that permits 3D visualisation of a small number of implanted cells within bulk tissue. The cells are marked using natural endocytosis of inert gold nano-particles. The resulting local increase in electron density allows high imaging contrast to be obtained from small clusters of these marked cells. Using this technique we have imaged C6 glioma cells within the brain of a model animal. The cells were marked by exposing them to colloidal gold incorporated in the growth media. Gold-loaded glioma cells were implanted into the brains of adult male Wistar rats. After tumours had been allowed to develop for up to 2 weeks, the animals were sacrificed and images of the intact cranium were acquired at the SYRMEP imaging station on the Elettra synchrotron in Italy. Computed tomography was performed using mixed absorption and phase contrast techniques at an X-ray energy of 24 keV. In the resulting volume datasets the tumour bulk is clearly visible and the infiltrating nature of the malignant growth is well demonstrated. Although the protocol was developed using this particular model of malignant brain tumour, it is believed that it will be possible to use it with other cell lines.

Lovastatin enhances phenylbutyrate-induced MR-visible glycerophosphocholine but not apoptosis in DU145 prostate cells.

In this study the effects of lovastatin on DU145 prostate cancer cells treated with phenylbutyrate (PB) was investigated in order to determine the NMR-detectable metabolic changes resulting from the cooperative activity of these two agents. DU145 cells were perfused with PB in the presence or absence of 10 microM of the HMG-CoA reductase inhibitor lovastatin, and the results monitored by 31P and diffusion-weighted 1H NMR spectroscopy. Lovastatin had additive effects on the PB-induced NMR-visible total choline in 1H spectra, and glycerophosphocholine in 31P spectra but no significant effect on NMR-visible lipid. Moreover, lovastatin had no effect on the ability of PB to either promote the formation of oil red O-detectable lipid droplets or arrest the cell cycle. The most remarkable observations from these studies were that lovastatin enhanced the increase in glycerophosphocholine while reversing late markers of apoptosis and the loss of NTP caused by PB. These results identify a branch point separating the neutral lipid production and the apoptotic cell death caused by the actions of differentiating agents.

In vitro and in vivo characterization of 64Cu-labeled Abegrin, a humanized monoclonal antibody against integrin alpha v beta 3.

Abegrin (MEDI-522 or Vitaxin), a humanized monoclonal antibody against human integrin alpha(v)beta(3), is in clinical trials for cancer therapy. In vivo imaging using Abegrin-based probes is needed for better treatment monitoring and dose optimization. Here, we conjugated Abegrin with macrocyclic chelating agent 1,4,7,10-tetra-azacylododecane N,N',N'',N'''-tetraacetic (DOTA) at five different DOTA/Abegrin ratios. The conjugates were labeled with (64)Cu (half-life = 12.7 hours) and tested in three human (U87MG, MDA-MB-435, and PC-3) and one mouse (GL-26) tumor models. The in vitro and in vivo effects of these (64)Cu-DOTA-Abegrin conjugates were evaluated. The number of DOTA per Abegrin varied from 1.65 +/- 0.32 to 38.53 +/- 5.71 and the radiolabeling yield varied from 5.20 +/- 3.16% to 88.12 +/- 6.98% (based on 2 mCi (64)Cu per 50 microg DOTA-Abegrin conjugate). No significant difference in radioimmunoreactivity was found among these conjugates (between 59.78 +/- 1.33 % and 71.13 +/- 2.58 %). Micro-positron emission tomography studies revealed that (64)Cu-DOTA-Abegrin (1,000:1) had the highest tumor activity accumulation (49.41 +/- 4.54% injected dose/g at 71-hour postinjection for U87MG tumor). The receptor specificity of (64)Cu-DOTA-Abegrin was confirmed by effective blocking of MDA-MB-435 tumor uptake with coadministration of nonradioactive Abegrin. (64)Cu-DOTA-IgG exhibited background level tumor uptake at all time points examined. Integrin alpha(v)beta(3)-specific tumor imaging using (64)Cu-DOTA-Abegrin may be translated into the clinic to characterize the pharmacokinetics, tumor targeting efficacy, dose optimization, and dose interval of Abegrin and/or Abegrin conjugates. Chemotherapeutics or radiotherapeutics using Abegrin as the delivering vehicle may also be effective in treating integrin alpha(v)beta(3)-positive tumors.

Membrane blebbing in cancer cells treated with various apoptotic inducers.

The dynamics of cell morphology, in particular membrane blebbing, was studied after induction of apoptosis by etoposide or camptothecin in four human stabilized cell lines (Hep2, Bowes, HT-29 and HL-60). Time lapse videomicroscopy and F-actin staining revealed various dynamic parameters of this process including its duration, maximal extent, stages and final endpoints in individual cells. Although generally occurring in predictable patterns, our results indicate a relatively significant variability both in appearance and temporal organization of blebbing not only between different cell lines but also within them.

Radiolabeling, biodistribution, and dosimetry of (123)I-mAb 14C5: a new mAb for radioimmunodetection of tumor growth and metastasis in vivo.

UNLABELLED: This study reports on the in vitro evaluation, biodistribution, and dosimetry of (123)I-labeled monoclonal antibody (mAb) 14C5, a new antibody-based agent proposed for radioimmunodetection of tumor growth and metastasis in vivo. METHODS: (123)I-mAb 14C5 was prepared by direct iodination and tested for stability in vitro. Binding assays were performed on human SK-BR-3 and HeLa carcinoma cells to investigate the antigen expression, antibody affinity, and kinetics of tracer binding. For the biodistribution and dosimetry study, 3- to 4-wk-old NMRI mice were injected intravenously with (123)I-mAb 14C5 (148.0 +/- 7.4 kBq per mouse) and killed at preset time intervals. Organs, blood, urine, and feces were counted for radioactivity uptake, and the data were expressed as the percentage injected dose per gram tissue (%ID/g tissue) or %ID. The MIRDOSE3.0 program was applied to extrapolate the estimated absorbed radiation doses for various organs to the human reference adult. RESULTS: (123)I-mAb 14C5 was obtained in radiochemical yields of 85.0% +/- 2.5% and radiochemical purities were >97%. The iodinated antibody demonstrated good in vitro stability with 93.6% +/- 0.1% of (123)I-mAb 14C5 remaining intact at 24 h after radiolabeling. (123)I-mAb 14C5 bound to SK-BR-3 cells (dissociation constant [K(d)] approximately 0.85 +/- 0.17 nmol/L) and HeLa cells (K(d) approximately 1.71 +/- 0.17 nmol/L) with nanomolar affinity and high specificity, whereas both cell types exhibited a high CA14C5 antigen expression (maximum number of binding sites [B(max)] = 40.6 +/- 5.2 and 57.1 +/- 9.6 pmol/L, respectively). In mice, (123)I-mAb 14C5 accumulated primarily in lungs (20.4 %ID/g), liver (15.1 %ID/g), and kidneys (11.1 %ID/g) within 5 min after injection. A delayed uptake was observed in stomach (12.8 %ID/g) and urinary bladder (8.7 %ID/g) at 3 and 6 h, respectively, after injection. Radioactivity clearance was predominantly urinary, with 44.9 +/- 4.5 %ID excreted during the initial 48 h after administration (cumulative amount). The highest absorbed radiation doses determined for the human reference adult were received by the urinary bladder wall (0.1200-0.1210 mGy/MBq), liver (0.0137-0.0274 mGy/MBq), uterus (0.0196-0.0207 mGy/MBq), and lower large intestine wall (0.0139-0.0258 mGy/MBq). The average effective dose resulting from a single (123)I-mAb 14C5 injection was estimated to be 0.017-0.022 mSv/MBq. CONCLUSION: (123)I-mAb 14C5 shows good in vitro biologic activity and favorable biodistribution properties for imaging carcinomas of different origin and provides an acceptable radiation dose to the patient.

Amplification targeting: a modified pretargeting approach with potential for signal amplification-proof of a concept.

UNLABELLED: Conventional nuclear medicine imaging with large radiolabeled molecules such as antitumor antibodies suffers from slow localization and clearance. Pretargeting is under active investigation as an alternative using either (strept)avidin/biotin, bispecific antibodies, or oligomers. However, only the use of oligomers such as phosphorodiamidate morpholinos (MORFs) in pretargeting offers the potential of signal amplification at the target. Amplification targeting is a multistep procedure with the potential to greatly improve target localization of radioactivity (and eventually drugs) through the intermediate use of polymers conjugated with multiple copies of oligomers. OBJECTIVE: This study was conducted to prove the concept in vivo in tumored mice of amplfication targeting. METHODS: Nude mice bearing LS174T tumors received in order: the anti-CEA antibody MN14 conjugated with MORF, a polymer conjugated with multiple copies of complementary MORFs (cMORFs), and, finally, (99m)Tc-MORF. RESULTS: In tumored animals, dual radiolabels ((99m)Tc and (111)In) were used to demonstrate that, after 18 h, about 25% of antibody MORFs in tumor were targeted with polymeric cMORFs and, after 3 h, about 12% of the polymeric cMORFs in tumor were targeted with (99m)Tc-MORF. Therefore, hybridization in tumor in both cases (i.e., polymeric cMORF to antibody MORF and radiolabeled MORF to polymeric cMORF) was surprisingly efficient given the barriers to targeting in vivo and the competition between targeting and clearance. Moles of radiolabeled MORF accumulating in tumor were more than tripled for study animals receiving all 3 injections compared with control animals not receiving the antibody or the polymer. Furthermore, MORF expression (on antibody) and cMORF expression (on polymer) were rapidly lost in normal organs such as liver, spleen, and kidneys but not in tumor, thus improving the target-to-nontarget ratios. CONCLUSION: Although signal amplification has not yet been convincingly demonstrated and amplification targeting will require further studies for optimization, the concept has now been shown to be feasible.

Tetraphenylphosphonium as a novel molecular probe for imaging tumors.

UNLABELLED: Mitochondrial membrane potential (DeltaPsim)-dependent enhanced uptake of phosphonium salts, including (3)H-tetraphenylphosphonium ((3)H-TPP), in tumor cells, suggests the potential use of phosphonium salts as tracers for tumor imaging. In this study, we characterize the tumor accumulation of (3)H-TPP and compare it with (18)F-FDG in cell culture and in xenograft, metastatic, and inflammation models in living animals. METHODS: (3)H-TPP and (3)H-FDG accumulation was compared in cell culture with a variety of cell lines in different glucose concentrations. Normal biodistribution and tumor uptake were assessed using nude mice with or without subcutaneous xenograft tumors (C6). To compare the accumulation of (3)H-TPP and (18)F-FDG in a metastatic tumor, severe combined immunodeficiency mice were tail-vein injected with human melanoma cell lines (A375-FL). To characterize the accumulation of (3)H-TPP and (18)F-FDG in inflammation, an inflammatory reaction was induced by subcutaneous injection of Complete Freund's Adjuvant in the left hind paw of Sprague-Dawley rat. RESULTS: The DeltaPsim data from a separate study and the current (3)H-TPP uptake data showed good correlation (r(2) = 0.82, P < 0.05). (3)H-TPP accumulation was significantly greater than that of (3)H-FDG for glucose >/=100 mg/dL. The biodistribution study of (3)H-TPP showed low uptake in most tissues but high accumulation in the heart and kidneys. (3)H-TPP accumulation in xenograft or metastatic tumors was comparable with that of (18)F-FDG, whereas (3)H-TPP accumulation in inflammatory tissues was markedly lower than that of (18)F-FDG. CONCLUSION: The sensitive tumor accumulation of (3)H-TPP with less propensity for inflammatory regions warrants further investigation of radiolabeled phosphonium analogs for tumor imaging in living subjects.

Evidence that 99mTc-(V)-DMSA uptake is mediated by NaPi cotransporter type III in tumour cell lines.

In vivo studies have demonstrated that pentavalent technetium-99m dimercaptosuccinic acid [(99m)Tc-(V)-DMSA] may be a useful tumour imaging agent. Several studies have suggested that (99m)Tc-(V)-DMSA uptake may be related to the structural similarity between the (99m)Tc-(V)-DMSA core and the PO(4)(3-) anion. As phosphate ions enter cells via NaPi cotransporters, we investigated whether (99m)Tc-(V)-DMSA uptake is mediated by NaPi cotransporters. (99m)Tc-(V)-DMSA and phosphate uptake kinetics were compared in three cancer cell lines (MCF-7, G152 and MG-63) under several conditions (with and without sodium and NaPi cotransporter inhibitor and at different pH). Determination of molecular NaPi cotransporter mRNA expression was performed by reverse-transcriptase polymerase chain reaction (Rt-PCR) assay. Results obtained in the presence of NaPi inhibitor, in sodium-free medium and at alkaline pH showed that (99m)Tc-(V)-DMSA accumulation is linked to NaPi cotransporter functionality. MCF-7 and G152 exhibited the same tracer uptake, whereas MG-63 showed the highest phosphate accumulation and the lowest (99m)Tc-(V)-DMSA uptake. These results were in accordance with mRNA NaPi expression, i.e. all cell lines expressed NaPi type III but MG-63 also co-expressed NaPi type I. The total level of NaPi cotransporter was highly correlated with phosphate accumulation, while the level of type III was related to (99m)Tc-(V)-DMSA uptake. We have demonstrated that (99m)Tc-(V)-DMSA uptake is specifically mediated by NaPi type III in cancer cells.

Monitoring tumor cell proliferation by targeting DNA synthetic processes with thymidine and thymidine analogs.

UNLABELLED: The use of radiolabeled thymidine (TdR) and thymidine analogs as PET-based tracers of tumor growth rate is based on the assumption that measurement of uptake of these nucleosides, a function primarily of thymidine kinase-1 (TK(1)) activity, provides an accurate measure of active cell proliferation in tumors. The goal of this study was to test this hypothesis and determine how well these tracers track changes in proliferation of tumor cells. METHODS: TK(1) activity; S-phase fraction; and uptake of TdR, 3'-deoxy-3'-fluorothymidine (FLT), and 2'-fluoro-5-methyl-1-(beta-D-2-arabino-furanosyl) uracil (FMAU) were determined in plateau-phase and exponentially growing cultures of 3 human and 3 murine tumor cell lines. RESULTS: TK(1) activity and S-phase fraction increased in all cell lines as cells moved from plateau-phase conditions to exponential growth. Some cell lines had relatively large TK(1) activities and S-phase fractions under plateau-phase conditions, consistent with a loss of normal cell cycle checkpoint control in these cells. There were also 2 cell lines in which TK(1) activity changed little as cells moved from the plateau phase to exponential growth, suggesting that in these cell lines, de novo nucleotide synthesis pathways predominate over salvage pathways. Both TdR and FLT detected changes in TK(1) activity. The slope of the relationship between TdR uptake and TK(1) activity was nearly twice that for FLT and more than 40-fold that for FMAU. CONCLUSION: Although not all tumors show a strong TK(1) dependence of proliferation, in all cell lines for which proliferation is highly TK(1) dependent, phosphorylation of TdR or FLT accurately reflects changes in TK(1) enzyme activity.