Image-guided, tumor stroma-targeted 131I therapy of hepatocellular cancer after systemic mesenchymal stem cell-mediated NIS gene delivery

Abstract

Due to its dual role as reporter and therapy gene, the sodium iodide symporter (NIS) allows noninvasive imaging of functional NIS expression by (123)I-scintigraphy or (124)I-PET imaging before the application of a therapeutic dose of (131)I. NIS expression provides a novel mechanism for the evaluation of mesenchymal stem cells (MSCs) as gene delivery vehicles for tumor therapy. In the current study, we stably transfected bone marrow-derived CD34(-) MSCs with NIS cDNA (NIS-MSC), which revealed high levels of functional NIS protein expression. In mixed populations of NIS-MSCs and hepatocellular cancer (HCC) cells, clonogenic assays showed a 55% reduction of HCC cell survival after (131)I application. We then investigated body distribution of NIS-MSCs by (123)I-scintigraphy and (124)I-PET imaging following intravenous (i.v.) injection of NIS-MSCs in a HCC xenograft mouse model demonstrating active MSC recruitment into the tumor stroma which was confirmed by immunohistochemistry and ex vivo γ-counter analysis. Three cycles of systemic MSC-mediated NIS gene delivery followed by (131)I application resulted in a significant delay in tumor growth. Our results demonstrate tumor-specific accumulation and therapeutic efficacy of radioiodine after MSC-mediated NIS gene delivery in HCC tumors, opening the prospect of NIS-mediated radionuclide therapy of metastatic cancer using MSCs as gene delivery vehicles.

Figure 1

In vitro analysis of mesenchymal stem cells (MSCs) stably expressing the sodium iodide symporter (NIS). (a) ¹²⁵I uptake was measured in NIS-MSCs compared to wild-type (WT)-MSCs. NIS-MSCs showed a 12-fold increase in perchlorate-sensitive ¹²⁵I accumulation. In contrast, no perchlorate-sensitive iodide uptake above background level was observed in WT-MSCs (P < 0.001). (b) Analysis of NIS protein expression in NIS-MSCs as compared to WT-MSCs by western blot analysis. NIS protein was detected as a major band of a molecular mass of 80–90 kDa which was not detected in WT-MSCs. MW, molecular weight. (c) Time course of iodide uptake in NIS-MSCs and WT-MSCs. Iodide accumulation reached half-maximal levels in NIS-MSCs within 10–15 minutes and became saturated at 40–50 minutes, while WT-MSCs showed no iodide accumulation. (d) In an in vitro clonogenic assay mixed populations of WT-MSCs and hepatocellular cancer (HCC) cells as well as NIS-MSCs and HCC cells (ratio 1:1) were exposed to 29.6 MBq ¹³¹I. In HCC cells cocultured with NIS-MSCs a 55% reduction of cell survival was measured, whereas HCC cells cocultured with WT-MSCs survived the ¹³¹I incubation to almost 100% (P < 0.001). Results represent means of three plated cell densities ± SD (100, 500 and 1,000 cells per well).

Figure 2

In vivo radioiodine biodistribution studies. ¹²³I γ-camera imaging of mice harboring Huh7 tumors after mesenchymal stem cell (MSC)-mediated sodium iodide symporter (NIS) gene delivery 3 hours following ¹²³I administration. (a) After three intravenous (i.v.) applications of NIS-MSCs significant tumor-specific iodide accumulation was induced (7–9 % ID/g ¹²³I), which was completely abolished upon (b) pretreatment with NaClO₄. (c) In contrast, mice injected with WT-MSCs showed no tumoral iodide uptake. (a,c) Iodide was also accumulated physiologically in thyroid, stomach and bladder. (d) Time course of ¹²³I accumulation in Huh7 tumors after three i.v. NIS-MSC applications followed by injection of 18.5 MBq ¹²³I as determined by serial scanning. Maximum tumoral radioiodine uptake was 7–9% ID/g tumor with an average effective half-life of 3 hours for ¹³¹I. (e,f) ¹²⁴I PET imaging of mice harboring Huh7 tumors after MSC-mediated NIS gene delivery. After three i.v. applications of NIS-MSCs significant tumor-specific iodide accumulation was confirmed by PET imaging (left: sagittal slice orientation, right: coronal slice orientation).

Figure 3

Evaluation of iodide biodistribution ex vivo 5 hours following injection of 18.5 MBq ¹²³I. Tumors in NIS-MSC-injected mice showed high perchlorate-sensitive iodide uptake activity (~2.5–3% ID/organ), while no significant iodide accumulation was measured in tumors after injection of WT-MSCs or in nontarget organs. Results are reported as percent of injected dose per organ ± SD.

Figure 4

Analysis of sodium iodide symporter (NIS) mRNA expression in Huh7 tumors and nontarget organs by quantitative real-time PCR (qPCR). While only a low background level of NIS mRNA expression was detected in untreated tumors (which was set as one arbitrary unit) or tumors injected with wild-type mesenchymal stem cells (WT-MSC), significant levels of NIS mRNA expression were induced in Huh7 tumors after three applications of NIS-MSCs with or without NaClO₄ pretreatment (P < 0.05). In addition, no significant NIS mRNA expression was detected in nontarget organs after three applications of NIS-MSCs. Results are reported as NIS/GAPDH ratios.

Figure 5

Immunohistochemical staining of Huh7 tumors and spleen after application of NIS-MSCs or WT-MSCs. (a) After application of NIS-MSCs Huh7 tumors revealed NIS-specific immunoreactivity throughout the tumor stroma, which was most prominent in the vicinity of blood vessels, (b) with a similar distribution of SV40 large T Ag-positive cells. (c) After application of WT-MSCs no NIS-specific immunoreactivity was detected in Huh7 tumors, (d) while strong cytoplasmic SV40 large T Ag staining was detected, in particular in the vicinity of blood vessels. Other organs like lung, liver, and kidneys showed no detectable NIS protein expression and no SV40 large T Ag staining (data not shown). (e–h) In contrast, strong accumulation of SV40 large T Ag-expressing cells was detected in the spleen of mice that were injected with (f) NIS-MSCs or (h) WT-MSC, (e,g) while no NIS-specific immunoreactivity was detected.

Figure 6

Radioiodine therapy studies in vivo after MSC-mediated systemic NIS gene transfer. Two groups of mice were established receiving 55.5 MBq ¹³¹I 48 hours after the last of three NIS-MSC (n = 15) or wild-type (WT)-MSC (n = 15) applications in 2-day intervals, respectively. This cycle was repeated once 24 hours after the last ¹³¹I application. Twenty-four hours after these two treatment cycles, one additional MSC injection was administered followed by a third ¹³¹I (55.5 MBq) injection 48 hours later. (a) ¹³¹I therapy after NIS-MSC application resulted in a significant delay in tumor growth as compared with the control groups, that were injected with WT-MSC followed by ¹³¹I (P = 0.008103; n = 15), with NIS-MSC followed by saline (P = 0.001703; n = 15) or with saline only (n = 15). Immunofluorescence analysis using a Ki67-specific antibody (green) and an antibody against CD31 (red, labeling blood vessels) showed decreased proliferation (Ki67, 25 ± 4.1 %) and reduced blood vessel density (CD31, 1.85 ± 0.25%) in tumors of mice treated with (b) NIS-MSC followed by ¹³¹I treatment as compared to tumors of mice injected with (c) WT-MSC and ¹³¹I or mice treated with (d) NIS-MSC and saline (Ki67, 45 ± 8.7%; CD31, 5 ± 0.45%). Slides were counterstained with Hoechst Nuclear stain. Magnification ×200.