College of Dentistry, Department of Oral and Maxillofacial Surgery and Pathology and
1 College of Pharmacy, Division of Pharmaceutics, Ohio State University, Columbus, OH 43210, USA
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Abstract |
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Abbreviations: AIDS-KS, AIDS-related Kaposi's sarcoma; bFGF, basic fibroblast growth factor; HAART, highly active anti-retroviral therapy; H&E, hematoxylin and eosin; IL-6, interleukin-6; PLGA, polylactideco-glycolide; VEGF, vascular endothelial growth factor.
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Introduction |
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Accurate preclinical assessment of pharmacological parameters and therapeutic efficacies of novel AIDS-KS therapies is highly dependent upon in vivo parameters. Currently, there are no animal models for AIDS-KS. In addition, mucocutaneous AIDS-KS cell isolates have proved nontumorigenic in animal hosts. While previous studies by Lunardi-Iskander et al. have reported establishment of a tumorigenic KS cell line (KS Y-1); this line was obtained from pleural effusion isolates from which the most aggressive phenotypes were stringently selected (6). Therefore, an animal model which permits in vivo transplantation of early passage `native' AIDS-KS strains, which are not selected for the most malignant phenotypes, would improve on previous work by permitting therapeutic evaluations on cells that more closely depict those comprising the patient KS lesions.
The angiogenic nature of AIDS-KS is manifest by its clinical and microscopic appearances. AIDS-KS lesions clinically present as red-purple patches, plaques or nodules, reflective of their prominent vascularity (7,8). A histological hallmark of AIDS-KS lesions is the proliferation of numerous, highly permeable, slit-shaped vascular channels (7,8). Incipient AIDS-KS lesions resemble well-vascularized, exuberant granulation tissue and contain few lesional KS cells. However, as with many malignancies, AIDS-KS cell growth and lesional promotion is dramatically facilitated by tumor-associated angiogenesis. Neovascularization supports KS cell proliferation both by perfusion (more efficient than diffusion for nutrient/catabolite exchange) and paracrine (endothelial cell production of growth factors) effects (9,10). Further, both cancer initiating and promoting events, e.g. increased mutations and forced expression of altered genes, are frequent sequelae of such sustained mitogenesis.
This report describes the development of a murine model that enables in vivo transplantation of `native' low population doubling level AIDS-KS cells from mucocutaneous sites. The angiogenic phenotype of in situ AIDS-KS lesions is reconstituted via the controlled release of a complete angiogenic peptide, recombinant human basic fibroblast growth factor (bFGF), from locally injectable, biodegradable poly- lactideco-glycolide (PLGA) implants. Consequential to the sustained local release of bioactive bFGF, a murine vascular network is established, which facilitates the in vivo transplantation of AIDS-KS cells. The transplanted human AIDS-KS cells and the extensive murine vascular network create lesions that retain a striking resemblance, at both the gross and microscopic levels, to in situ AIDS-KS tumors. Particularly desirable aspects of this model include: (i) the use of a low cost murine species; (ii) transplantation of non-selected patients' cells; (iii) use of animal species (BALB/c and NIH-bg-xidBR mice) that are T cell-deficient.
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Materials and methods |
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The AIDS-KS cells were isolated from the tissue specimen as previously described (11,12). These AIDS-KS strains have been shown to possess a normal XY karyotype. The KS spindle cell isolates from our laboratory show aspects of a transformed phenotype including: high production of `KS'-related cytokines such as interleukin-6 (IL-6), tumor necrosis factor- and bFGF, capacity to undergo multiple population doublings (>30) in reduced serum (0.5%) medium and loss of anchorage dependence. Furthermore, relative to matched, non-lesional cells from the AIDS-KS donors, the KS spindle cells possess unique biochemical features, including reduced cytoprotective enzyme function, increased responsiveness to proinflammatory cytokines and reduced tolerance to chemotherapeutic agents that function by redox cycling. The AIDS-KS cells were cultured at 37°C, 5% CO2 in `complete' medium, which consisted of M-199 (Gibco BRL, Grand Island, N.Y.) supplemented with 15 mM HEPES, 0.23 mg/ml L-glutamine, 11 µg/ml sodium pyruvate, 90 µg/ml sodium heparin (Sigma, St Louis, MO), endothelial cell growth supplement (prepared in-house, from bovine brain, 150 µg lyophilized powder/ml), 15% heat-inactivated fetal bovine serum (Hyclone, Logan, UT) and 5% heat-inactivated pooled male human serum (Sigma).
Preparation and characterization of the bFGF controlled release polymers
Recombinant human bFGF was encapsulated in PLGA millicylinders (i.e. injectable monolithic cylindrical implants of 0.8 mm diameter), which had been engineered to slowly release bioactive bFGF for >1 month (13). To prepare the bFGF-containing powder for encapsulation, bFGF was combined with heparin, sucrose and bovine serum additives at a weight ratio (additive:bFGF) of 1, 180 and 1000, respectively, in 0.5 mM EDTA and 10 mM sodium phosphate buffer (pH 7.4). The solution was lyophilized for 2 days at room temperature using a Labconco Freeze Dry System (Kansas City, MO) to a fine powder with 4% moisture (determined with a Karl Fisher Titrator, model DL 18; Mettler-Toledo Inc., Hightstown, NJ) and sieved. A uniform suspension of the sieved bFGF powder (<90 µm) and Mg(OH)2 in a 50% by weight PLGA 50/50 (inherent viscosity 0.63 dl/g) acetone solution was loaded into a syringe and extruded into silicone tubing (i.d. 0.8 mm) at ~0.1 ml/min (14). The final loading in the polymer was ~0.0025% bFGF and 3% Mg(OH)2 on a weight basis. The solvent extruded suspension was dried at room temperature (24 h) and then dried in a vacuum oven at 45°C (24 h). These preparations have been shown to retain >94% immunoreactivity and >60% bioactivity over 1 month of in vitro release under physiological conditions (13).
Animal protocols
In order to determine the conditions permissive for in vivo transplantation of AIDS-KS cells, a variety of experimental conditions, including use of two different strains of mice (BALB/c and NIH-bg-nu-xidBR), were examined (Table I). Because clinical and epidemiological data strongly suggest a contribution of male sex steroids in the development of KS, intact male mice were used for these studies. To enhance cell transplantation success rate, AIDS-KS cells were delivered in conjunction with the synthetic basement membrane Matrigel (1x106 AIDS-KS cells suspended in 200 µl Matrigel; Collaborative Research, Bedford, MA) (15,16). All animals received human recombinant growth factors that are accepted facilitators of AIDS-KS cell growth (20 ng/ml IL-6 and 2 ng/ml oncostatin M, suspended in the Matrigel) at the time of AIDS-KS cell implantation (17,18). To decrease natural killer cells, thereby diminishing host potential to eradicate the KS cell implants, all animals were given 50 µl containing 13 µg anti-asialo GM1 (Wako Bio- Products, Wako Chemicals, Richmond, VA) i.p. at the time of and 72 h following AIDS-KS cellular injections (19). All AIDS-KS cell and polymer injections were done with a 16 gauge trocar, at a subcutaneous (s.c.) flank site.
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Clinical photographs of all animals were taken at the time of initial cell placement and polymer delivery, at weekly intervals and at the time they were killed. Animals were evaluated daily for any signs of stress and examined every 34 days to observe neovascularization and tumor growth.
Animals were killed at selected time points (ranging from 7 to 45 days after KS transplantation) to permit assessment of time-dependent parameters such as neovascularization. After death, the tissues and associated millicylinders were placed in 10% buffered neutral formalin and processed for light microscopic, immunohistochemical and in situ hybridization analyses.
Confirmation that the proliferating spindle cells in the murine model were of human AIDS-KS origin
Standard hematoxylin and eosin (H&E) sections were prepared of every tissue section for light microscopic analyses. These sections were read in a blind fashion to assess the extent of spindle cell proliferation, tissue response at the site and degree (none > moderate > extensive) of angiogenesis. Sequential serial slide sections were then prepared for the in situ hybridization and immunohistochemical studies. The use of serial slide sections permitted cross-referencing between the H&E (structures most readily identifiable), the in situ hybridization (confirmation that spindle cells were of human origin) and the immunohistochemically (demonstration of ongoing proliferation) stained sections. Due to the high concentrations of growth factors delivered, we anticipated that our experimental conditions would stimulate the proliferation of a variety of mesenchymal origin cells. Because many of the stimulated cell populations have a spindle cell morphology (e.g. AIDS-KS cells and myofibroblasts and fibroblasts of murine origin), we conducted in situ hybridization (ISH Kit, specific for human DNA Alu elements; InnoGenex, San Ramon, CA) to confirm the human origin of the spindle cell proliferation at the cell implant site. Although the AIDS-KS spindle cells may remain viable in vivo, proliferative potential may be diminished. Therefore, additional evaluations were conducted to confirm ongoing KS cellular proliferation. Mitotic figures of cells confirmed to be human in origin (via the in situ studies) were determined on the matched (serial section) H&E sections. Further, cycle progression was determined by immunohistochemical staining (ImmunoCruz Staining System; Santa Cruz Biotechnology, Santa Cruz, CA) using antibodies specific for two cyclin markers, E and A. Cyclin E (C-19) is specific for human cells in a very narrow window of the cell cycle at late G1 phase. Although cyclin A detects a broader cell cycle range (late G1 through S phases), this antibody stains a range of mammalian cells including human and mouse. Therefore, for the cyclin A (H-432) immunostained sections, the spindle cells were first confirmed to be of human origin by in situ hybridization, then the sequential histological section was evaluated for positive expression of cyclin E. Immunohistochemical studies (anti-human rhVEGF165 antibody; R&D Systems, Minneapolis, MN) were also conducted to determine whether or not the in vivo transplanted AIDS-KS cells retained the capacity for vascular endothelial growth factor (VEGF) production.
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Results |
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In vivo transplantation of AIDS-KS cells is facilitated by bFGF and its associated neovascularization
All of the animals retained a visible s.c. swelling for ~5 days after the initial cell implantation procedure, probably due to the inclusion of Matrigel. However, the successful in vivo transplantation of AIDS-KS cells was contingent upon substantial bFGF doses. None of the mice from Groups 1 (no exogenous bFGF), 2 or 3 (lower dose PLGA-delivered bFGF) retained viable AIDS-KS cells at the transplant site. In contrast, those animals (Group 5) that received considerable bFGF doses [either a bFGF bolus (22 µg, left side) or controlled bFGF release (release rates of 5.4 and 7.7 µg bFGF over 7 and 14, days, respectively, right side)] retained AIDS-KS cells at both the 7 and 14 day harvests. At the 7 day point in the Group 5 mice both bFGF sites (5/5 bFGF bolus, 5/5 PLGA implant bFGF delivery) showed the presence of mitotically active KS spindle cells in all animals, with the highest KS cell mitotic activity noted at the bFGF bolus-delivered sites (left sides). These proliferating, cyclin A-positive spindle cells were confirmed to be of human origin via in situ hybridization and cyclin E staining at both the left and right implantation sites (Figure 2). Notably, these positive results were not restricted to in vivo transplantation of a single KS strain from one donor. Three characterized AIDS-KS cell strains, all obtained from donors with nodular tumors (that microscopically demonstrated a true sarcomatous appearance, i.e. numerous mitotic figures, invasive growth characteristics and cellular pleomorphism) have been successfully in vivo transplanted. The higher bFGF dose (22 µg bolus, 5.4 µg controlled release over 7 days) permitted in vivo transplantation of several different strains of low population doubling AIDS-KS cells that were all obtained from donors with mucocutaneous, microscopically confirmed AIDS-KS.
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The results of the 30 day experiments on the Group 5 mice reinforced the dependency of the AIDS-KS cell implants on sustained bFGF levels. Scant residual AIDS-KS cells, which were restricted to residual Matrigel foci, remained in 2 out of 3 mice on the left side of the animals (blank millicylinder with bolus bFGF). In contrast, the functional bFGF implant sites (right side, 13 µg bFGF released over 30 days) showed numerous aggregates of mitotically active AIDS-KS cells (3/3 mice) contained in a well-vascularized murine stroma. While some KS cells remained in association with residual Matrigel, infiltration by AIDS-KS cells into the surrounding murine tissues, including dispersion through skeletal muscle, was noted. (Figure 2)
While the initial angiogenic induction was most likely attributable to the bFGF implants, VEGF immunohistochemical studies of the tumors obtained at the 14 and 30 day harvests showed that the in vivo transplanted AIDS-KS cells express high levels of VEGF (data not shown).
AIDS-KS tumorigenicity is contingent upon sustained angio- genesis
A final series of experiments was undertaken (Group 6) to determine the aggressive potential of in vivo transplanted AIDS-KS cells, i.e. tumorigenicity, capacity for intravascular or perineural invasion during controlled release of bFGF. Results from these studies showed no significant differences between the murine strains and successful in vivo transplantation of AIDS-KS cells, with 5/6 mice retaining AIDS-KS tumor foci. Although the NIH-bg-nu-xidBR strain is genetically more immunodeficient (devoid of NK cells), comparable in vivo transplantation success rates [75% (3/4 mice) and 100% (2/2)] were noted for the NIH-bg-nu-xidBR and BALB/c mice, respectively. Further, comparable tumor burdens were noted in both strains and ranged between 560 and 1000 mm3 at death. Consistent with our previous results, locally invasive growth of the AIDS-KS cells into the surrounding murine connective tissues was noted at both the gross and microscopic levels. Clinically, the cell implantation sites were not freely movable, suggesting locally infiltrative growth and lack of a connective tissue capsule. However, none of the mice showed any indications of tumor-associated cachexia as all of the mice continued to gain weight over the experimental time course. Also, despite the abundant vascularity and number of cutaneous nerves at the transplantation sites, there was no evidence of perineural or intravascular invasion by the AIDS-KS cells.
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Discussion |
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Studies from our laboratory, as well as others, have shown that cultured AIDS-KS cells possess a `weakly malignant' phenotype (22,23). While cultured AIDS-KS strains demonstrate sustained in vitro growth under reduced serum conditions, these cells were not tumorigenic in nude mice (22). A variety of alternative approaches have been employed to circumvent KS cell transplantation difficulties and still allow investigation of the contribution of the in vivo environment in AIDS-KS pathogenesis (24-27). These studies have included induction of `KS-like' lesions in nude mice by employing an array of inflammatory cytokines (24,25), immunophenotyping `KS-like' spindle cells from transgenic mice (26) and implantation of endothelial cells chronically exposed to inflammatory cytokines to induce `KS-like' murine lesions (27).
Although Lunardi-Iskander et al. reported isolation of a tumorigenic AIDS-KS cell line (KS Y-1) (6), the histogenesis of this cell line has recently been questioned. Data obtained during DNA analyses for quality control procedures at American Type Cell Culture (ATCC), which distributes this patented cell line, showed that the short tandem repeats of the KS Y-1 line (ATCC designation CRL-11448) are identical to those of T-24, a human bladder cancer cell line. As a consequence of these findings, the current classification of `identity in question' and the disclaimer `reportedly from a Kaposi's sarcoma' have been applied to the KS Y-1/CRL-11448 line by ATCC. To our knowledge, prior to this report, in vivo transplantation of mucocutaneous AIDS-KS cells has never been accomplished.
Despite the inclusion of the synthetic basement membrane Matrigel (an established route to facilitate in vivo transplantation) (15,16), the results of our Group 1 studies (no exogenous bFGF delivered) confirmed the lack of AIDS-KS cellular tumorgenecity in nude mice. Therefore, we sought alternative approaches to facilitate AIDS-KS cell in vivo transplantation. For example, there is compelling evidence to support an integral role for bFGF in the development of AIDS-KS (24,25,28,29). First, bFGF is expressed at high levels by KS spindle cells both in vitro and in situ (25,28). Furthermore, both anti-bFGF antibodies and antisense oligonucleotides to bFGF mRNA markedly diminish the angiogenic and proliferative potential of AIDS-KS cells (29). In addition, inoculation of nude mice with bFGF induces lesions of murine origin that resemble early AIDS-KS lesions (24). Based on this evidence, we hypothesized that exogenously delivered bFGF would facilitate AIDS-KS in vivo transplantation by both initiating and sustaining the host vascular network and serving as a mitogen for the AIDS-KS cells.
Recently, our laboratories gained the technology to slowly deliver biologically active bFGF from biodegradable polymers (13). In this previous study we found that by neutralizing the highly acidic intrapolymer pH with an antacid (MgOH2), the encapsulated bFGF retained its stability (>60% bioactivity), resulting in controlled release of bioactive growth factor for >4 weeks (13). Therefore, our results, which show that PLGA controlled release of bFGF enhances in vivo angiogenic capacity relative to a comparable bFGF bolus dose, were not unexpected. The serum half-life of i.v. delivered bFGF has been reported to be of the order of 1.53 min (30,31), with an elimination half-life of ~50 min (32). However, concurrent administration of heparin with bFGF results in growth factor stabilization against deactivation and proteolytic cleavage and prolongs its serum half-life (31). Notably, the PLGA millicylinders used in this study deliver heparin-stabilized bFGF. In addition, studies by Lazarous et al. have reported that bFGF regional distribution is concentration-dependent, with most pronounced growth factor uptake at the point of delivery (33). Because the PLGA formulations provide controlled release of heparin-stabilized, bioactive growth factor over several weeks, the bFGF uptake at the AIDS-KS cell implant site would remain consistently high. Finally, although the initial millicylinder formulation (lowest bFGF dose) delivered sufficient bFGF to induce neovascularization, this bFGF level was inadequate to sustain the proliferation of in vivo transplanted AIDS-KS cells. These data suggest that the higher bFGF dose functioned in a dual capacity, i.e. both to initiate and sustain the murine vascular network and to provide mitogenic stimulation for the transplanted AIDS-KS cells. Finally, our results, which show residual foci of AIDS-KS cells at the 30 day harvest in the bolus-treated sites, suggest that more frequent and repeated injections of bFGF may be adequate to sustain the murine vascular network and support AIDS-KS in vivo transplantation. However, such repeated bFGF injections are more labor intensive and would still result in growth factor peak and valley concentrations, which are avoided with the use of the PLGA millicylinders.
Further, the retention of VEGF production by the AIDS-KS spindle cells comprising the tumors obtained at the 14 and 30 day harvests suggests that autologous production of angiogenic and mitogenic growth factors accompanies successful in vivo transplantation.
In contrast to the findings of van Weerden et al. (34), who showed that human tumor transplantation was strain-dependent, our final series of experiments revealed comparable in vivo transplantation success rates (5/6 total mice) of 75 and 100% regardless of murine strain. There are several explanations for these differences. We used anti-asialo GM1 to decrease natural killer cells, thereby making the BALB/c mice more immunodeficient. In addition, van Weerden et al. were conducting in vivo transplantation of tumor explants, not cell strains. Finally, our use of bFGF-containing PLGA millicylinders created a highly receptive in vivo environment.
While essential information can be derived from in vitro cell culture studies (35), experimental therapeutic development depends on complex in vivo factors. Determination of therapeutic efficacies of newly developed treatments is contingent on factors, such as drug clearance, only manifest in vivo. Until now, the development of novel AIDS-KS therapies has been hindered by the inability to conduct in vivo AIDS-KS cell transplantation. However, by reconstruction of the highly angiogenic phenotype of AIDS-KS lesions via controlled release of bFGF, our laboratory has developed a murine model that enables in vivo transplantation of `native' AIDS-KS cells. Because of its extensive murine vascular network (which simulates the abundant vascularity found in KS lesions in situ), this murine model should provide an excellent vehicle for assessment of a variety of important therapeutic parameters, such as drug clearance from AIDS-KS lesional sites. We intend to use this murine model to investigate two potentially synergistic routes for cancer therapy: local controlled release of chemotherapeutic agents in combination with either local or systemic controlled release of angiostatic agents. Such a combination therapy would concurrently treat both components of the cancer, i.e. lesional tumor cells and stromal endothelial cells, and is a promising approach for long-term remission or cure.
Finally, although this report presents a method to facilitate in vivo transplantation of AIDS-KS cells, this technology is not necessarily restricted to AIDS-related malignancies. Establishment of an extensive host vascular network should augment success rates for in vivo transplantation of numerous other human cell strains or lines. Finally, provision of other `tumor cell critical' factors, e.g. androgens for prostatic carcinoma cells, from PLGA implants to the transplant site could enable in vivo transplantation of fastidious human cell lines.
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Notes |
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Acknowledgments |
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References |
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