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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 3, 2003 as doi:10.1096/fj.03-0483fje. |
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* Dept of Biotechnology Engineering and The Institute for Applied Biosciences, Faculty of Engineering Sciences, and
Dept of Microbiology and Immunology and The Cancer Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel; and
University of Colorado, Health Science Center, Denver, CO 80262, USA
2Correspondence: Department of Biotechnology Engineering and The Institute for Applied Biosciences, Bldg. 39, Room 222, Ben-Gurion University of the Negev, Beer Sheva, Israel 84105. E-mail: scohen{at}bgumail.bgu.ac.il
SPECIFIC AIM
The involvement of interleukin-1ß (IL-1ß) in inflammation, tumor growth and metastasis makes it an attractive target for therapeutic intervention. In the present study, we tested the hypothesis that effective neutralization of the proinflammatory cytokine IL-1 by the delivery of a low, but steady-state level of the naturally occurring IL-1 receptor antagonist (IL-1Ra) may prevent in vivo, in experimental animals, phenomena that are dependent on IL-1, such as tumor growth, angiogenesis and inflammation. This sets up the stage for the use of such delivery systems in patients.
PRINCIPAL FINDINGS
1. Microencapsulated genetically-engineered cells can provide sustained, steady state delivery of IL-1Ra, in vitro and in vivo
To provide for a sustained, steady state delivery of IL-1Ra, we genetically engineered NIH/3T3 cells to continuously secrete IL-1Ra. The cells were encapsulated within alginate-poly (L-lysine)-alginate (APA) microspheres (initial cell density of 3x106 cells/ml microspheres), which provide an immunoisolation barrier for the entrapped cells. In addition, the APA microspheres are permeable to allow free diffusion of nutrients to the cells, which maintain their viability, and the exit of IL-1Ra once it is secreted from the cells. Viable encapsulated cells were detected for at least two weeks in culture, and the microspheres were releasing IL-1Ra at a rate of 19-25 ng/ml microspheres/day, according to enzyme linked immunosorbent assay (ELISA) using specific antibodies to IL-1Ra. In vivo, the viability of the microencapsulated cells, injected subcutanously (s.c.) into syngeneic NFS/N mice, was maintained close to the pre-implantation level for at least two weeks, and the microspheres delivered a continuous source of IL-1Ra during this time period, at levels of 24–28 ng/ml microspheres/day.
2. Continuous delivery of IL-1Ra inhibits tumor growth and prolongs survival
We examined the inhibitory effect of the continuously–delivered IL-1Ra on the development of a tumor from fibrosarcoma cells that were transfected with IL-1ß (Clone 3-ssIL-1ß). This tumor cell line was transfected with a fused gene containing the mature form of IL-1 ß linked to a signal peptide, to enable its efficient secretion, as IL-1ß does not bear a signal peptide and is secreted only to a limited manner by fibroblasts. Our previous studies have demonstrated that active secretion of IL-1ß by the malignant cells significantly increased their invasiveness and metastasis and also induced potent tumor-mediated angiogenesis. In culture, Clone 3-ssIL-1ß cells secreted IL-1ß at a rate of 1 ng/1x 106cells/day and after injecting 1 million cells into syngeneic NFS/N mice, the serum level of IL-1 ß on day 10 were at the range of 2ng/ml. NFS/N mice, injected with a minimal tumor dose of 7.5 x 104 of IL-1ß expressing cells, developed highly angiogenic tumors compared with tumors of mock-transfected or parental cells. Blood vessels in the IL-1ß expressing tumors were irregular and larger in size compared with those observed in tumors derived from the mock-transfected cells of a similar size.
Mice were injected with Clone 3-ssIL-1ß tumor cells and then treated with two s.c. injections of IL-1Ra secreting microspheres at tumor vicinity, on days 6 and 11 post tumor cell injection. Each injected dose of microspheres secreted 25ng of IL-1Ra per day near the tumor cell site. Controls consisted of tumor-bearing mice treated with microencapsulated wild-type NIH/3T3 cells or without treatment. The continuous delivery of IL-1Ra from the microencapsulated cells resulted in complete tumor eradication in 82% of the tumor-bearing mice (Fig. 1
). In the 18% of remaining mice, tumor growth rate was significantly inhibited as compared with the other groups (Fig. 1A)
. The survival rates (Fig. 1B)
reveal that 45 days after tumor cell injection, all tumor-bearing mice in the control groups had died. In contrast, at this time point, all mice treated with microencapsulated IL-1Ra secreting cells were alive. Microencapsulated wild-type NIH/3T3 or mock-transfected NIH/3T3 (NIH/3T3-LXSN) did not have any effect on tumorigenicity patterns.
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3. IL-1Ra secreting microspheres reduce in vivo inflammation-related angiogenesis
Microspheres encapsulating the wild-type NIH/3T3 cells were coated with a hypervascularized fibrotic sac after their retrieval from the s.c site, while much less vascularized tissue surrounded the microspheres that contained the IL-1Ra secreting cells (Fig. 2
A). Histology of thin sections from the fibrotic capsule of the 3T3/NIH-loaded microspheres revealed an inflammatory response characterized by active neo-angiogenesis, as manifested by a large number of capillaries of different sizes and the infiltration of inflammatory cells (Fig. 2B)
. A different picture was seen with the implanted IL-1Ra-secreting microspheres; the fibrotic sac was noticeably thinner and the capillaries were smaller in number and size. Quantification of the capillary density in the fibrotic tissue surrounding the IL-1Ra secreting microspheres, revealed two–fold less capillaries compared with that counted in the tissue surrounding 3T3/NIH microspheres (Fig. 2C
, significance P=0.01).
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CONCLUSIONS AND SIGNIFICANCE
In the present work, we demonstrate that the continuous delivery of IL-1Ra from microencapsulated engineered cells inhibited tumor development in mice injected with fibrosarcoma cells that constitutively express and secrete IL-1ß (Clone 3-ssIL-1ß) and as a result are highly malignant. In contrast, control mice injected with tumor cells overexpressing IL-1ß, but were not treated or treated with microspheres encapsulating the wild-type 3T3 cells, developed hypervascularized violent tumors.
IL-1Ra is the naturally occurring inhibitor of pre-formed IL-1. In mice deficient in IL-1Ra, IL-1 activities are unopposed and severe inflammatory diseases spontaneously develop, such as rheumatoid arthritis-like multiple joint disease. In our tumor model, the continuous local secretion of IL-1ß has led to the development of hypervascularized tumors with large and branched blood vessels, indicating that the cytokine incuses in vivo pronounced angiogenesis. Our conclusion is supported by a recent work of our group, showing the cardinal role of host-derived IL-1 in the process of tumor angiogenesis. The pro-angiogenic effects of IL-1ß may include direct effects of IL-1 on endothelial cell proliferation and secretory function or may be mediated via a cascade of IL-1-induced angiogenic factors, such as bFGF and VEGF that are secreted from the malignant or stromal cells through autocrine and paracrine pathways. Indeed, we have recently described in IL-1ß–knockout mice impaired tumor invasiveness and angiogenesis.
An additional support to the effects of IL-1 in angiogenesis is our findings that the continuous delivery of the IL-1Ra significantly reduced the number of blood capillaries in the fibrotic sac that surrounded the microencapsulated cell systems. In this case, microenvironmental IL-1 of host origin possibly induces this angiogenic response. The decline in the capillary density was associated with a significant reduction in the inflammatory response, thus leading to a much thinner fibrotic sac.
Of considerable importance was the observation that the near total prevention of this aggressive angiogenic response, induced by the violent tumors of IL-1ß secreting fibrosarcoma cells, was accomplished by microspheres locally secreting low, but steady-state levels of IL-1Ra (25 ng/day). One can conclude that the pro-angiogenic and pro-tumorigenic properties of IL-1ß are taking place at relatively low levels of the host- or tumor cell-derived cytokine, so that 25 ng per day of the IL-1Ra are sufficient to oppose its effects. Apte et al showed that Clone 3-ssIL-1ß cells secrete IL-1ß at a rate of 1 ng/1 x 106 cells/day, in culture, and after injecting 1 million cells into NFS/N mice, the serum level of IL-1ß on day 10 is 2ng/ml (unpublished results). Yet, these malignant cells developed into highly-angiogenic tumors when s.c injected into mice, much more than the mock-transfected cells or the wild-type parental cells.
Another important conclusion drawn from our results is that local steady state secretion of IL-1Ra is more effective than the peak-and-valley values obtained after bolus injections of the cytokine. Two injections of IL-1Ra secreting microspheres, administered on days 6 and 11 after tumor cell inoculation, were able to prevent tumor growth in 80% of the mice and in the remaining 12%, a significant delay in tumor growth was achieved. The two microsphere injections provided an effective therapeutic level of the IL-1 inhibitor that was able to neutralize the activity of IL-1ß and the cascade of the downstream angiogenic factors induced by it. Thus, neutralizing of a signal upstream pro-inflammatory cytokine, i.e., IL-1ß, may be sufficient to prevent or attenuate the cascade of angiogenic factors. We hypothesize that this is advantageous as compared with neutralizing of single factors, as angiogenesis is mediated by multiple pro-angiogenic factors with overlapping and redundant functions that are all induced by a single stimuli, such as IL-1.
These results point to the possibility of using the IL-1Ra in anti-cancer therapies, in addition to its effective use in chronic inflammatory diseases, such as rheumatoid arthritis, as many tumors have been shown to produce and secrete IL-1 or to induce its production by stromal cells in the vicinity of the tumor. Thus, the use of the IL-1Ra in cancer therapy may not only reduce tumor growth, but also alleviate side effects that are induced by tumor-associated inflammatory responses, such as cachexia and tissue damage. Our strategy directed at the blockade of IL-1 activity alone or as an adjunct to the existing therapies may therefore be beneficial in the treatment of tumors using DFA approved IL-1Ra and PLGA microspheres. We believe that the use of such a delivery system would help adapting this treatment to clinical situations and enhance patient compliance.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0483fje 
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) microencapsulated IL-1Ra secreting cells (each microsphere injection consisted of 0.5 ml microspheres releasing 25 ng/day of IL-1Ra), or (
) microencapsulated wild-type NIH/3T3 cells. (
) are control mice, which received tumor cell injection but no further treatment.