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Microencapsulated iNOS-expressing cells cause tumor suppression in mice ¹

The Wolfson Institute for Biomedical Research, UCL, London, WC1E 6BT, UK

²Correspondence: The Wolfson Institute for Biomedical Research, The Cruciform Building, Gower Street, UCL, London, WC1E 6BT, UK. E-mail: rmgzigc{at}ucl.ac.uk

SPECIFIC AIMS

Macrophages are capable of killing tumor cells by releasing nitric oxide (NO) and reactive nitrogen species after expression of the inducible nitric oxide synthase (iNOS) gene. We intend to develop a novel cell-based therapy for tumor inhibition using microencapsulated cells capable of expressing iNOS under control of a regulated promoter.

1. Characterization of NO release by inducible microencapsulated cells
We cloned a human iNOS cDNA under the control of an ecdysone-inducible promoter and transfected the construct into the human fetal kidney cell line EcR293. The resulting transfected cell lines are able to express iNOS in a dose- and time-dependent manner after treatment with analogs of the insect hormone ecdysone (muristerone A or ponasterone A). Treatment of one of the subclones (EcR293 clone-11 sub-1 cells) with 1 µM and 10 µM ponasterone A results in 50 (±10) and 300 (±40) pmol/min/mg NOS activity, respectively, activity comparable to the levels of NOS measured in murine macrophages activated by treatment with -interferon/LPS.

For microencapsulation, the inducible NO-generating cells were entrapped within a semipermeable alginate-poly-L-lysine membrane. This technique allows small molecules such as nutrients and oxygen to freely diffuse into the cell and allows NO and reactive nitrogen species to diffuse out while preventing attack from the host immune system. Microencapsulated EcR293 clone-11 sub-1 cells can survive in vitro for at least 3 wk. Moreover, the encapsulated cells can express iNOS in a controlled fashion upon stimulation with ponasterone A or muristerone A at concentrations from 1 to 10 µM.

2. Inhibition of tumor cell growth in vivo by microencapsualtion of iNOS-expressing cells
A nude mouse/xenograft model was used to assess the ability of microencapsulated cells to inhibit tumor cell growth in vivo. The present studies used two human carcinoma cell lines that produce rapidly growing subcutaneous (s.c.) tumors in nude mice. DLD-1 is a human colon adenocarcinoma cell line and SKOV-3 is an ovarian carcinoma cell line. To produce s.c. tumors in female CD 1 nude mice, DLD-1 cells were injected s.c. along with microencapsulated EcR293 clone-11 sub-1 cells. The results are shown in Fig. 1 A and demonstrate that DLD-1 tumors in the presence of iNOS-expressing microencapsulated cells are significantly smaller (257±150 mm³) 28 days after s.c. injection than control tumors not generating NO (480±143 mm³).

View larger version (26K): [in this window] [in a new window]   Figure 1. A) iNOS-expressing cell delivery using microencapsulated EcR293 clone-11 sub-1 cells. Results of s.c. injection of human colon cancer DLD-1 cells (1x106/per mouse) concomitantly with microencapsulated EcR293 clone-11 sub-1 cells (5x106/per mouse) with either ponasterone A/sesame seed oil () or sesame seed oil alone (). Each point represents the mean (±SD) for 10 mice; *P < 0.05, 2-tailed Student’s t test. B) Results of s.c. injection of human ovarian cancer SKOV3 cells (5x105/per mouse) with microencapsulated EcR293 clone-11 sub-1 cells (3x106/per mouse) with either with ponasterone A/sesame seed oil () or sesame seed oil alone (). Each point represents the mean (±SD) for 10 mice. C) Western blot analysis of control and iNOS-expressing SKOV3/EcR293 clone-11 sub-1 tumors on day 10 postimplantation using human iNOS-specific antibody. Fas and FasL have also been shown to be concomitantly increased with increased human iNOS protein.

Mouse tumor experiments were repeated using SKOV3 human ovarian cancer cells. The results are shown in Fig. 1B and demonstrate that the growth of SKOV-3 tumors is almost completely inhibited by microencapsulated cells expressing iNOS.

Human iNOS protein expression was analyzed in a panel of five mice from the day 10 SKOV3/EcR293 clone-11 sub-1 group. For all five samples, human iNOS-specific protein could be detected in tumors receiving ponasterone A. An example is shown in Fig. 1C .

3. Tumor killing in iNOS-expressing cells is associated with concomitant up-regulation of the FAS/FASL proteins
Fas-mediated apoptosis represents an important mechanism by which tumor cells can be eliminated. To determine whether Fas-mediated events occur in our tumor-killing model, Western blotting experiments were carried out with anti-Fas and anti-FasL antibodies. In all five mice from the day 10 SKOV3/EcR293 clone-11 sub-1 group induced with ponasterone A, a considerably higher signal for Fas/FasL proteins was detectable in iNOS-expressing tumors than with controls. Figure 1C shows the results of a Western blotting experiment detecting Fas and FasL protein in tumor samples.

To support these findings, we examined Fas and FasL mRNA levels in our muristerone A-inducible cells in vitro. Treatment of EcR293 clone-11 sub-1 cells with muristerone A results in the generation of NO, a concomitant 3.3 (±0.5) -fold increase in FasL mRNA, and a 4.5 (±1.5) -fold increase in Fas mRNA compared with untreated cells. This result was further supported by transient transfection experiments using human Fas and FasL promoter-luciferase constructs.

4. Administration of tetracycline/doxycycline to suppress tumor growth
Although the insect hormone ecdysone and its synthetic analogs muristerone A and ponasterone A have no reported effects on mammalian cells, they have not yet been evaluated in human safety trials. To make our iNOS-expressing cells more acceptable for human use, an alternative induction system based on tetracycline was selected. Tetracycline has been used in humans for decades and represents a good orally bioavailable inducer. We cloned a 4 kb human iNOS cDNA sequence into a tetracycline-regulated mammalian expression vector pcDNA/TO. A panel of 25 cell lines was isolated showing tetracycline-dependent (10 ng/ml) expression of iNOS. Tex clone-22 can generate up to 100 µM nitrite/10⁶ cells in 24 h.

To determine whether established tumor growth can be reversed by treatment with iNOS-expressing cells, we tested the cells in a mouse xenograft model. Tex clone-22 cells were microencapsulated as describe above and then injected adjacent to the site of preestablished DLD-1 tumors. Results after 30 days s.c. implantation are shown in Fig. 2 A and are representative of two experiments. Treatment ofestablished tumors with Tex clone-22 iNOS-expressing cells and administration of doxycycline in the drinking water considerably slows the growth of DLD-1 tumors (100±74 mm³ compared with control DLD-1 cells (678±441 mm³. This represents an 86% reduction in tumor volume (Student’s t test, P<0.01). At the end of 30 days, five mice still showing measurable tumor mass (between 50–200 mm³) received a second dose of microencapsulated Tex clone-22 cells (10⁷ cells/mouse). Doxycycline was maintained in the drinking water, resulting in the maintenance of tumor growth inhibition for another month, with an average tumor volume of 100 (±86 mm³) at 56 days (Fig. 2B ). The control group of mice (carrying the same number of the DLD-1 cells without administration of doxycycline) was killed on day 45. The doxycycline-treated DLD-1 tumor animals were killed after another 30 days and showed no remaining tumor histology. Histological examination of host tissue flanking the site of microencapsulated cell implantation revealed no overt damage, suggesting that the reactive nitrogen species and NO-mediated effect on cell viability is tumor specific. Using Tex clone-22 iNOS-expressing cells to treat SKOV-3 tumors produces similar results (Fig. 2C ).

View larger version (29K): [in this window] [in a new window]   Figure 2. A) The use of microencapsulated Tex clone-22 cells to treat established tumors. 2 x 106 DLD-1 cells were implanted in the flank of a nude mouse and grown for 7 days. Microencapsulated cells were implanted adjacent to the established tumor; iNOS expression was induced by administration of 0.5 mg/ml doxycycline in the drinking water (in 1% ethanol ). In the control group, mice received vehicle only (1% ). All mice were treated for 30 days with drug (changed 2–3 times/wk) or vehicle alone. Each point represents means (±SD) for 10 mice. B) Second injection of microencapsulated Tex clone-22 cells into the remaining 5 DLD-1 tumor sites. Each point represents means (±SD) for 5 mice. C) The use of microencapsulated Tex clone-22 cells to treat established SKOV-3 tumors. 5 x 105 SKOV-3 cells were implanted in the flank of a nude mouse and grown for 10 days. The microencapsulated cells were implanted adjacent to the established tumor and iNOS expression was induced by administration of 0.5 mg/ml doxycycline in the drinking water (in 1% ethanol ). In the control group, mice received vehicle only (1% ). All mice were treated for 30 days with drug (changed 2–3 times/wk) or vehicle alone. Each point represents means (±SD) for 6 mice.

CONCLUSION AND SIGNIFICANCE

We have described a novel method to produce human cells capable of expressing high levels of iNOS under the control of two inducible promoters responsive to the insect hormone ecdysone and its analogs and to tetracycline and its analogs. Our results demonstrate that microencapsulated iNOS-expressing cells can be used to inhibit tumor growth. The NOS activity in our two inducible promoter systems is comparable to NOS measured in murine macrophages activated by treatment with -interferon/LPS, which have been demonstrated to possess anti-tumor activity. In a previous study, we reported the surprising finding that DLD-1 human carcinoma cells transfected with a murine iNOS cDNA cassette (iNOS-19 cells) actually resulted in increased tumor cell growth. The iNOS expression constructs reported here result in considerably higher NOS activity than that reported for the iNOS-19 clone. This level of expression is also one order of magnitude greater than retrovirally transfected human carcinoma cells, such as cau-6 and HT29. It appears that high levels of iNOS expression may be cytostatic/cytotoxic for tumor cells; lower activity can have the opposite effect and promote tumor growth and neovascularization, and provide radiation resistance and multidrug resistance through up-regulation of DNA-PKCs.

We have also demonstrated for the first time that the iNOS-directed tumor killing is associated with regulation of the FAS/FASL cell death pathway. It is possible that the high levels of NO and related reactive nitrogen species generated by our microencapsulated iNOS-expressing cells may trigger the up-regulation of Fas, FasL, and related proteins in target tumors, resulting in the activation of apoptotic pathways. The approach described here could have the potential to inhibit or kill many different types of tumors of various histological origins that are sensitive to the FAS/FASL death pathway.

View larger version (55K): [in this window] [in a new window]   Figure 3. Schematic

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0590fje; to cite this article, use FASEB J. (December 28, 2001) 10.1096/fj.01-0590fje

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