Firstly, we examined distribution of free 99mTc-HMPAO. 1 hour after 99mTc-HMPAO administration radioactivity was visible in the brain, heart, and kidneys (Fig. 6). 17 hours after administration radioactivity was visible in liver and spleen as well. High amount of radioactivity was bound to red blood cells (SUV was 3.7) as showed by ex vivo biodistribution data 19 hours postinjection (Table 4). SUV were high for brain, heart, liver, lungs and spleen as well (Table 4). Rats treated with 99m Tc-HMAPO-labeled stem cells showed an absence of radioactivity in the brain both at 1 and 17h following injection (Fig. 6), which indicates that there is no significant leakage of
99mTc-HMPAO in a form that can be transferred to other cells/tissues. Moreover, the absence of radioactivity in the brain suggests that C17.2 cell do not spontaneously migrate to the healthy brain, as anticipated. Accumulation of radioactivity is visible in lungs, liver, spleen and kidneys at 1 hour following injection of labeled cells. The whole body scans at 17h show less accumulation of radioactivity in the lungs compared to the scans at 1 hour. The biodistribution data (t = 19±1h) show that 99m Tc-HMPAO-labeled cells accumulated in liver, spleen and lungs (Table 4). Some cells migrate to the site of inflammation and tumor site immediately after administration. The tumor-to-muscle ratio and inflammation-to-tumor-to-muscle ratio for 99mTc-HMPAO-labeled cells are similar (Table 3), and higher than the ratios found in free 99mTc-HMPAO-treated rats, indicating the accumulation of C17.2 cells at the tumor site and the inflamed muscle. It should be noted that the activity in kidneys is likely not due to an accumulation of cells, but presumably to the elimination of free 99mTc-pertechnetate via the renal system [20].
Figure 6. Gamma camera images obtained at 1 hour and 17 hours after administration of free 99mTc-HMPAO (15 - 25 MBq per animal) and 99m Tc-HMPAO labeled stem cells (10 – 20 MBq labeled cells per animal) through a tail vein. Planar images were obtained using a 10 min acquisition time frame during the first scan and one of 20 min during the second scan. Dotted regions of interest indicate the position of the tumors, as well as the inflammation and some organs.
Tissue Free 99m
In this study, we investigated the possibility of labeling C17.2 NSCs with [18F]-FDG and 99mTc-HMPAO the subsequent application of this approaches to examine the in vivo distribution of the radiolabeled C17.2 cells by microPET and gamma camera imaging in a tumor inflammation model after intravascular administration.
The PET tracer [18F]-FDG is a glucose analogue that is actively transported into cells by glucose transporters, where it is phosphorylated by hexokinase. The phosphorylated form, 2-[18F] fluoro-2-deoxy-D-glucose-6-phosphate ([18F]-FDG-6P), is a poor substrate for glucose-6-phosphate dehydrogenase, an enzyme in the pentose phosphate pathway, and therefore causes its entrapment within the cells [21].
C17.2 NSCs at high, that is, ≈90%, confluency showed a lower uptake of [18F]-FDG than C17.2 cells at median, that is, ≈50%, confluency (see Figure 1A). This distinction
Table 4. Biodistribution of free 99mTc-HMPAO and neural stem cells labeled with
99mTc-HMPAO in rat tumor inflammation model at 19±1 hours after administration.
Results are represented as SUV.
can be explained by the fact that C17.2 NSCs display contact inhibition on reaching total confluency, resulting in a lower metabolic activity. Moreover, incubation of cells with [18F]-FDG in the absence of glucose (PBS++) resulted in a significantly higher lysates, whereas in the medium, only free [18F]-FDG was detectable. A decrease in the amount of phosphorylated [18F]-FDG in cell lysates occurred over time. Although glucose-6-phosphatase, an enzyme that dephosphorylates glucose 6-phosphate, is abundantly expressed in liver and kidney cells, its expression has been demonstrated in other cell types as well [22-25]. Since we observed a decrease in the amount of phosphorylated [18F]-FDG in the cell lysate over time, as revealed by TLC, dephosphorylation of [18F]-FDG-6-P apparently also occurs in C17.2 cells. To inhibit the dephosphorylation of [18F]-FDG-6-P, cells were incubated with chlorogenic acid.
Chlorogenic acid inhibits hepatic glucose-6-phosphatase by blocking transport of glucose 6-phosphate to the endoplasmic reticulum, where dephosphorylation occurs [16]. Unfortunately, it did not show inhibitory effect on [18F]-FDG-6-P dephosphorylation in our hands in both C17.2 NSCs and HepG2 cells. One reason for that could be that chlorogenic acid does not affect intact cells as its effect was shown only on microsomes. On the other hand, Leuzzi and colleagues showed that glucose-6-phosphate transport in fibroblast- and HeLa cell–derived microsomes is not influenced by chlorogenic acid either [26]. As glucose 6-phosphate accumulates in the endoplasmic reticulum of the latter cell types, the authors suggested the existence of a second endoplasmic reticulum glucose-6-phosphate transport system. Although we did not investigate this further, owing to high [18F]-FDG efflux and the inability of chlorogenic acid to block the efflux, we cannot exclude the existence of such glucose-6-phosphate transport system in C17.2 NSCs.
Unfortunately, [18F]-FDG leakage from the cells was evident both in vitro and in vivo.
Whereas in vitro [18F]-FDG leakage from the cells could be prevented by the use of phloretin (see Figure 2), phloretin could not prevent the loss of cellular radioactivity in vivo (see Table 2). High levels of radioactivity in plasma and metabolically active organs such as the brain and heart in animals pretreated with phloretin indicated that phloretin (at 75 mg/kg) is not effective in the prevention of [18F]-FDG leakage from C17.2 cells following tail vein injection. As the brain and heart are organs with high metabolic activity but exhibit no inflammatory signals, the radioactivity in the brain and heart does not reflect accumulation of labeled cells but should be ascribed to
leakage of [18F]-FDG from the cells. Leakage of [18F]-FDG from labeled cells is not uncommon, and efflux from other cell types has been reported as well [27-29]. The inability of phloretin to inhibit [18F]-FDG efflux in vivo might be due to the dosage of phloretin administered in this study, which may be insufficient for completely blocking all glucose transporters. Phloretin is only very slightly soluble in water or ethanol. The inhibitor can be dissolved in DMSO, but to keep the injection volume of this organic solvent small within reasonable limits, we had to limit the dosage of the inhibitor to 75 mg/kg.
The majority of [18F]-FDG-labeled C17.2 cells were trapped in the capillary network of lungs, liver, and spleen (see Figure 4). Interestingly, PET images from animals treated with phloretin showed faster clearance of stem cells from the lungs compared to the group of animals that had not been treated with phloretin (see Figure 4). In accordance, the biodistribution data showed that the activity in lungs, liver, and spleen was significantly lower in animals pretreated with phloretin compared to the activity in animals that received no pretreatment (see Table 2). The measured activities in these organs in the phloretin-treated animals resembled the values of animals injected with free [18F]-FDG, suggesting avoidance of cell retention in the capillary beds of these organs in the presence of phloretin. However, this decrease in cell retention cannot be explained by an effect of phloretin per se, as the same decrease of radioactivity in lungs, spleen, and liver was attained when the animals had been pretreated with DMSO, that is, the solvent for phloretin (see Table 2). Interestingly, it has been shown that administration of sodium nitroprusside, a strong vasodilator, reduces mesenchymal stem cell trapping in lungs after intravenous injection [30]. Given that DMSO-induced relaxation in porcine pulmonary and coronary arteries has been described [31], it appears plausible that a vasodilatation effect of DMSO could have alleviated cell retention in lung, liver, and spleen in our study.
In future work, it will therefore be of interest to further investigate the effect of more vasodilation compounds on the distribution of intravenously injected stem cells as they could markedly improve the migration of stem cells to the target site [32].
Unlike [18F]-FDG that is internalized by cells via a transporter-mediated route, 99m Tc-HMPAO is a lipophilic radiotracer, primarily intercalating in cellular membranes or when acquiring cytosolic access, being retained within the cell after glutathione conversion to a hydrophilic form [33]. The labeling efficiency of C17.2 cells with
99mTc-HMPAO showed a linear correlation with 99mTc-HMPAO concentration confirming its cellular association being accomplished via a passive non-receptor/transporter-mediated mechanism (Fig 4b). As predicted based on the literature [19], 99mTc-HMPAO stem cells labeling efficiency has greatly improved in the absence of serum. Furthermore, labeling of cells in suspension led to desirable combination of
speed and efficiency without signs of radiotoxicity. Due to showed retention of radiolabel within the cell 99mTc-HMPAO labeling is preferred over [18F]-FDG.
Intravenous administration of free 99mTc-HMPAO led to extensive labeling of blood cells (Table 4) and typical biodistribution with highest values that resemble extent of organ perfusion [20]. Since 99mTc-HMPAO is very well retained by the cells, the body distribution of 99mTc-HMPAO-labeled cells reflects the genuine location of the C17.2 cells. Kidney SUV levels equally high for both 99mTc-HMPAO and cell administered animals confirm these organs as route of elimination of radioactive metabolites.
Labeling of C17.2 NSCs with 111In-oxine for long term in vivo imaging was showed not to be feasible. Similar to 99mTc-HMPAO, 111In-oxine is a lipid soluble complex.
The mechanism of labeling is not clear, but most likely indium attaches to components of the cytoplasm while oxine is released by the cell. Labeling of the cells with 111 In-oxine caused a toxic response after incorporation of 100 times less radioactivity
We have shown that labeling of C17.2 NSCs with [18F]-FDG is feasible, but labeling efficiency remains relatively low, despite optimization of the procedure. Still, sufficient quantities of labeled cells for in vivo cell tracking can be produced. However, the major drawback of [18F]-FDG-labeled NSCs was the substantial loss of radioactivity observed both in vitro and in vivo, which is presumably due to the glucose-6-phosphatase activity of the stem cells. In vitro, release of radioactivity from the labeled stem cells could be inhibited with the glucose transport inhibitor phloretin, but only partial reduction of efflux of radioactivity was observed in vivo. As a consequence, [18F]- FDG-labeled NSCs are not suitable for tracking of cell migration, not even after administration of phloretin. An incidental but interesting finding in this study was that DMSO, the matrix in which phloretin was administered, strongly reduced the entrapment of labeled cells in the microvasculature of lung, spleen, and liver, which might be due to the vasodilatation activity of DMSO. This could be an important finding for new strategies on improving cell replacement therapy protocols with intravenously administered stem cells. In contrast, 99mTc-HMPAO appears to be a better label for tracking of C17.2 stem cell migration in rats for ~ 24 hours. 99m Tc-HMPAO labeling revealed that C17.2 NSCs are equally attracted to tumor and inflammation site. For long-term (days) studies appropriate radiotracers with a longer
half-life, should be selected. In this context 111In-oxine is not suitable, as it shows
[1] Burnstein RM, Foltynie T, He X, Menon DK, Svendsen CN, Caldwell MA (2004) Differentiation and migration of long term expanded human neural progenitors in a partial lesion model of Parkinson's disease. Int J Biochem Cell Biol 36:702-13.
[2] Carbajal KS, Schaumburg C, Strieter R, Kane J, Lane TE (2010) Migration of engrafted neural stem cells is mediated by CXCL12 signaling through CXCR4 in a viral model of multiple sclerosis.
Proc Natl Acad Sci U S A 107:11068-73.
[3] Kim DE, Schellingerhout D, Ishii K, Shah K, Weissleder R (2004) Imaging of stem cell recruitment to ischemic infarcts in a murine model. Stroke 35:952-7.
[4] Zhao D, Najbauer J, Garcia E, Metz MZ, Gutova M, Glackin CA, Kim SU, Aboody KS (2008) Neural stem cell tropism to glioma: critical role of tumor hypoxia. Mol Cancer Res 6:1819-29.
[5] Aboody KS, Brown A, Rainov NG, Bower KA, Liu S, Yang W, Small JE, Herrlinger U, Ourednik V, Black PM et al. (2000) Neural stem cells display extensive tropism for pathology in adult brain:
evidence from intracranial gliomas. Proc Natl Acad Sci U S A 97:12846-51.
[6] Tang Y, Shah K, Messerli SM, Snyder E, Breakefield X, Weissleder R (2003) In vivo tracking of neural progenitor cell migration to glioblastomas. Hum Gene Ther 14:1247-54.
[7] Jin K, Sun Y, Xie L, Mao XO, Childs J, Peel A, Logvinova A, Banwait S, Greenberg DA (2005) Comparison of ischemia-directed migration of neural precursor cells after intrastriatal, intraventricular, or intravenous transplantation in the rat. Neurobiol Dis 18:366-74.
[8] Benedetti S, Pirola B, Pollo B, Magrassi L, Bruzzone MG, Rigamonti D, Galli R, Selleri S, Di Meco F, De Fraja C et al. (2000) Gene therapy of experimental brain tumors using neural progenitor cells. Nat Med 6:447-50.
[9] Heese O, Disko A, Zirkel D, Westphal M, Lamszus K (2005) Neural stem cell migration toward gliomas in vitro. Neuro Oncol 7:476-84.
[10] De Vries IJ, Krooshoop DJ, Scharenborg NM, Lesterhuis WJ, Diepstra JH, Van Muijen GN, Strijk SP, Ruers TJ, Boerman OC, Oyen WJ et al. (2003) Effective migration of antigen-pulsed dendritic cells to lymph nodes in melanoma patients is determined by their maturation state. Cancer Res 63:12-7.
[11] Gratz S, Rennen HJ, Boerman OC, Oyen WJ, Mast P, Behr TM, Corstens FH (2002) 99mTc-HMPAO-labeled autologous versus heterologous leukocytes for imaging infection. J Nucl Med 43:918-24.
[12] Tamura M, Unno K, Yonezawa S, Hattori K, Nakashima E, Tsukada H, Nakajima M, Oku N (2004) In vivo trafficking of endothelial progenitor cells and their possible involvement in the tumor neovascularization. Life Sci 75:575-84.
[13] Rini JN, Bhargava KK, Tronco GG, Singer C, Caprioli R, Marwin SE, Richardson HL, Nichols KJ, Pugliese PV, Palestro CJ (2006) PET with FDG-labeled leukocytes versus scintigraphy with 111In-oxine-labeled leukocytes for detection of infection. Radiology 238:978-87.
[14] van Waarde A, Cobben DC, Suurmeijer AJ, Maas B, Vaalburg W, de Vries EF, Jager PL, Hoekstra HJ, Elsinga PH (2004) Selectivity of 18F-FLT and 18F-FDG for differentiating tumor from inflammation in a rodent model. J Nucl Med 45:695-700.
[15] Lemaire C, Damhaut P, Lauricella B, Mosdzianowski C, Morelle J, Monclus M, Naemen Jv, Mulleneers E, Aerts J, Plenevaux A et al. (1997) Fast [18F]FDG synthesis by alkaline hydrolysis on a low polarity solid phase support. Journal of Labelled Compounds and Radiopharmaceuticals 45:435-447.
[16] Arion WJ, Canfield WK, Ramos FC, Schindler PW, Burger HJ, Hemmerle H, Schubert G, Below P, Herling AW (1997) Chlorogenic acid and hydroxynitrobenzaldehyde: new inhibitors of hepatic glucose 6-phosphatase. Arch Biochem Biophys 339:315-22.
[17] Yamada S, Kubota K, Kubota R, Ido T, Tamahashi N (1995) High accumulation of fluorine-18-fluorodeoxyglucose in turpentine-induced inflammatory tissue. J Nucl Med 36:1301-6.
[18] Szabo Z, Xia J, Mathews WB, Brown PR (2006) Future Direction of Renal Positron Emission Tomography. Seminars in Nuclear Medicine 36:36-50.
[19] Peters TJ (1997) All About Albumin: Biochemistry, Genetics, and Medical Applications. All About Albumin: Biochemistry, Genetics, and Medical Applications., San Diego, California.
[20] Neirinckx RD, Canning LR, Piper IM, Nowotnik DP, Pickett RD, Holmes RA, Volkert WA, Forster AM, Weisner PS, Marriott JA (1987) Technetium-99m d,l-HM-PAO: a new radiopharmaceutical for SPECT imaging of regional cerebral blood perfusion. J Nucl Med 28:191-202.
[21] Gallagher BM, Fowler JS, Gutterson NI, MacGregor RR, Wan CN, Wolf AP (1978) Metabolic trapping as a principle of radiopharmaceutical design: some factors resposible for the biodistribution of [18F] 2-deoxy-2-fluoro-D-glucose. J Nucl Med 19:1154-61.
[22] Chatelain F, Pegorier JP, Minassian C, Bruni N, Tarpin S, Girard J, Mithieux G (1998) Development and regulation of glucose-6-phosphatase gene expression in rat liver, intestine, and kidney: in vivo and in vitro studies in cultured fetal hepatocytes. Diabetes 47:882-9.
[23] Khan A, Hong-Lie C, Landau BR (1995) Glucose-6-phosphatase activity in islets from ob/ob and lean mice and the effect of dexamethasone. Endocrinology 136:1934-8.
[24] Surholt B, Newsholme EA (1981) Maximum activities and properties of glucose 6-phosphatase in muscles from vertebrates and invertebrates. Biochem J 198:621-9.
[25] Ghosh A, Cheung YY, Mansfield BC, Chou JY (2005) Brain contains a functional glucose-6-phosphatase complex capable of endogenous glucose production. J Biol Chem 280:11114-9.
[26] Leuzzi R, Fulceri R, Marcolongo P, Banhegyi G, Zammarchi E, Stafford K, Burchell A, Benedetti A (2001) Glucose 6-phosphate transport in fibroblast microsomes from glycogen storage disease type 1b patients: evidence for multiple glucose 6-phosphate transport systems. Biochem J 357:557-62.
[27] Ritchie D, Mileshkin L, Wall D, Bartholeyns J, Thompson M, Coverdale J, Lau E, Wong J, Eu P, Hicks RJ et al. (2007) In vivo tracking of macrophage activated killer cells to sites of metastatic ovarian carcinoma. Cancer Immunol Immunother 56:155-63.
[28] Adonai N, Nguyen KN, Walsh J, Iyer M, Toyokuni T, Phelps ME, McCarthy T, McCarthy DW, Gambhir SS (2002) Ex vivo cell labeling with
64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography.
Proc Natl Acad Sci U S A 99:3030-5.
[29] Botti C, Negri DR, Seregni E, Ramakrishna V, Arienti F, Maffioli L, Lombardo C, Bogni A, Pascali C, Crippa F et al. (1997) Comparison of three different methods for radiolabelling human activated T lymphocytes. Eur J Nucl Med 24:497-504.
[30] Gao J, Dennis JE, Muzic RF, Lundberg M, Caplan AI (2001) The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs 169:12-20.
[31] Lawrence RN, Dunn WR, Wilson VG (1998) Endothelium-dependent relaxation in response to ethanol in the porcine isolated pulmonary artery. J Pharm Pharmacol 50:885-90.
[32] Sigot V, Mediavilla MG, Furno G, Rodriguez JV, Guibert EE (2004) A simple and effective method to improve intrasplenic rat hepatocyte transplantation. Cell Transplant 13:775-81.
[33] Ballinger JR, Reid RH, Gulenchyn KY (1988) Technetium-99m HM-PAO stereoisomers:
differences in interaction with glutathione. J Nucl Med 29:1998-2000.
[34] ten Berge RJ, Natarajan AT, Hardeman MR, van Royen EA, Schellekens PT (1983) Labeling with indium-111 has detrimental effects on human lymphocytes: concise communication. J Nucl Med 24:615-620.
[35] Kuyama J, McCormack A, George AJ, Heelan BT, Osman S, Batchelor JR, Peters AM (1997) Indium-111 labelled lymphocytes: isotope distribution and cell division. Eur J Nucl Med 24:488-496.
[36] Brenner W, Aicher A, Eckey T, Massoudi S, Zuhayra M, Koehl U, Heeschen C, Kampen WU, Zeiher AM, Dimmeler S et al. (2004) 111In-labeled CD34+ hematopoietic progenitor cells in a rat myocardial infarction model. J Nucl Med 45:512-518.