In vitro Technical Aspects of Anti-Gene IGF-I Vaccines against Glioma

Research on glioblastoma has demonstrated a significant increase in IGF-I gene expression in this brain tumor. After suppression of IGF-I expression using anti-gene IGF-I technologies, glioma cells become immunogenic, expressing MHC-I. While injected in vivo, they induce an immune response mediated by T-CD8 lymphocytes. These cells, applied as autologous anti-cancer vaccines have increased the median survival of glioblastoma patients up to 18 months, but often up to two-three years. These differences in clinical results could be explained by variability in vaccine preparation standards. For this reason, the conditions of in vitro preparation of anti-cancer vaccines were explored using antisense, AS, and triple helix, TH technology. The established human glioblastoma primary cell line, and an established rat cell line, C6 glioma, were transfected in vitro with IGF-I AS, and with IGF-I TH vectors producing arrest of the synthesis of IGF-I on transduction and transcription levels, respectively. An arrest in IGF-I gene expression was demonstrated in 50-60% of cultured cells. The AS and TH cells expressed MHC-I only in 30-40% of cells. To increase the immunogenic population, the transfected cells were cloned to obtain 100% IGF-I negative population. These cloned cells expressed MHC-I and B7 antigens in 60-70% of cells. The presented in vitro results will be applied for preparation of cell vaccines for cancer immunotherapy, with the objective of increasing median survival in glioblastoma treated patients.

plant the classical oligodeoxynucleotide approaches is still in question at the moment, because we do not yet have the final clinical results [10].
Using AS and TH technologies we have obtained in both approaches, similar experimental and clinical results. The transfected AS and TH cells-autologous anti-cancer 'vaccines'expressed MHC-I and B7.1 and B7.2 antigens, and moreover became apoptotic. These cells induced in vivo T-cell mediated immune reaction against their tumorigenic progenitor's cells in syngeneic animals, with tumor regression at distal sites [12,13]. The objective of here presented article was to increase the usefulness of this strategy [6], by making more performant anti IGF-I vaccines. The ultime goal would be to increase of median survival of treated glioblastoma patients.
To distinguish between neoplastic glial cells and neuroblasts, we used the IGF-I marker. In this case, glial cells gave positive reaction and neuroblastic cells, negative [5]. This observation suggested that targeting IGF-I gene expression we could stop the glial neoplastic development using anti-gene strategy [6].
The past forty years have accelerated the researches related to the treatment of tumors targeting the proteins using either antibodies i.e. anti AFP, or injection of radiolabelled oncoproteins was proposed [7]. Currently, the anti-gene strategies stopping the protein synthesis either on translation level (antisense, AS) [8] or transcription level (triple-helix, TH) [9] offer new possibilities for cancer therapy.
The antisense sequences are capable of blocking the messenger RNA translation conducting to block of specific protein synthesis. In another anti-gene approach, TH technology, specific oligonucleotide sequences also called triple helix-forming oligonucleotides, TFOs, while introduced into cells are linked to the genomic DNA forming the triple helix structure with the target gene and inhibiting its transcription [2,4,[8][9][10]. A novel development in oligodeoxynucleotide technology is the relatively recent finding that 21-23 mer double-stranded RNA molecules, known as siRNA, can effectively silence gene expression [11]. Whether or not siRNA technology will sup-

RT-PCR
RNA from cells was isolated using High Pure RNA Isolation Kit (Roche Diagnostics GmbH nr 1828665), and RT PCR was used according Reverse Transcription System Promega Corporation (nr A3500).
The following primers were used for RT PCR study of rat IGF-I and rat MHC-I:

Results
The following optimal conditions were established for cell cultures as well of rat C6 glioma as of primary cells

Vectors
The IGF-I antisense and triple helix technologies were used to construct episome based plasmids either pMT-Anti-IGF-I expressing IGF-I RNA antisense, or pMT-AG inducing the IGF-I RNA-DNA triple helix (Figure 1). For control experiments, the vectors suppressing MHC-I and B7 molecules, expressing antisense MHC-I and antisense B7 (MHC-I AS and B7 AS), as well as antisense IGF-II RNA expressing vector, were constructed as previously described. To stimulate the MT promotor, the cells were cultivated in presence of 60 uM ZnSO 4 [13,14].

In vitro cells
The rat cell line C6 was used for the experiments [8]. Primary cell cultures of human glioma were derived from tumors of glioblastoma multiforme patients during surgical resection in the INC-National Institute of Cancerology, Bogota. The human cell line, HG, was established according to the technique described previously [13]. As a control Hela cell line was used. In general, rat and human glioma cells were cultured in DMEM (GIBCO-BRL) supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin, at 37 C and 5% CO 2 .
Cells, 60-80% confluent, were transfected in 6-well plates in a ratio of 1 ug of plasmid per 3-4 × 10 5 cells. The FuGENE 6 Transfection Reagent (Boehringer Mannheim) was used according to the supplier's instructions (3 ul of Reagent per 1 ug DNA). Hygromycin B (Boehringer Mannheim) at a concentration of 0.05 mg/ml (C6) and 0.005 mg/ml (primary human glioma, HG) was added 48 hours after transfection to select for transfected cells. After one week, concentration of hygromycin B was changed to 0.15 mg/ml (C6) and 0.015 mg/ ml (HG) and maintained with each change of fresh medium over the next 2-3 months.

Immunocytochemistry
Parental, non-transfected cells and transfected cells were verified for IGF-I presence by immunoperoxidase technique using anti IGF-I antibodies. Immunocytochemical localization  (Table 2).

Discussion
In the current paper we have demonstrated the optimum conditions for cultivation as well rat glioma cells as primary originated from glioblastoma biopsy ( Figure 2): DMEM medium supplemented with Glutamax, 10% CO 2 , 15% NBCS (neonatal bovine calf serum), 200 U/ml of penicillin, 200 ug/ ml of streptomycin, 1.2 ug of amphotericin B, 10.000 ng/ml of insulin, 5 ug/ml of transferrin, at 37 C.
As to the transfection of glioma cells, the optimum 'timing' was as follows: One week after beginning of C6 cells culture, and three weeks after establishment of primary HG cell line. The confluence of cells as 85-90% was also the optimum factor. The optimum time to form a complex of transfection was 20 minutes, using transfection reagent: Vector as 1: 1 in the volume 1.5 ul each. The timing of cell transfection was minimum 4 hours, and maximum 6 hours. (The reagent of transfection should be eliminated before 20 hours because of toxicity).
The transfected cells of both antisense and triple helix types showed an inhibition of IGF-I in 50%-60% of cell population (Figure 3 and Table 1). They have expressed MHC-I in 30%-40% of cells (Figure 4 and Table 2). After cloning, the    The transfected cells, verified in permanently maintained cultures up to six months, were negative for IGF-I presence.
The absence of IGF-I synthesis in "antisense" and "triplehelix" transfected cells, could lead to a compensative increase in IGF-I receptor (tyrosine kinase); IGF-I and IGF-II present in fetal calf serum of culture medium, as well as intracellular IGF-II can interact with the type I receptor. Indeed, the increase of IGF-I receptor level could explain the expression of B7. There is a known relation between the signal transduction pathway of tyrosine kinase and the induction of B7 molecules [13,17,18].
The phenotypic modification of transfected cells concerns also the phenomenon of apoptosis [19] (the results not shown). Apoptotic cells, in the context of MHC-I, are recognized by dendritic cell activating lymphocytes T-CD8 [20][21][22]. B7 molecules may also be included in this mechanism, because both MHC-I and B7 molecules are necessary for T cell activation [15,23].
Different immunotherapies and immunogene therapies including antisense strategy to treat glioblastoma has been recently investigated especially in relation to growth factors human glioma cell line. Moreover, we have established the criteria for anti-cancer vaccines: the population of IGF-I AS and IGF-TH transfected cells became more immunogenic thanks to the cloning of cells negative for IGF-I expression. The culture of these transfected cells, totally negative for IGF-I, has permitted to increase two times the number of MHC-I positive cells: From 30% to 60-70% cells. The immunogenic character of the transfected cells expressing both MHC-I and B7 antigens, explain their vaccine effect in in vivo tumorigenicity [14-16] ( Table 2).
The expression of IGF-I in primary cultures established from glioblastoma biopsies vary between 80-100%. For these reasons, the cultures without total expression were cloned to obtain 100% IGF-I positive cell line. That was important for the transfection of these cultures using anti-gene IGF-I vectors. After transfection, as mentioned in the paper, only 50-60% of cells have showed an inhibition of IGF-I. Once more the cloning of cells was performed to obtain 100% cell population without expression of IGF-I (these transfected cells have also showed an increase in MHC-I expression). The necessity of cloning of cells as well of parental as of transfected cultures was verified in in vivo experimental animal model: Only the 'vaccines' prepared from cloned cells have stopped the development of tumors in 90-100% cases. No cloned 'vaccines' have stopped the development of tumors only in half of treated animals. For this reason, as far as prognostic importance in human glioblastoma is considered, we have kept the cloning procedure for preparation of vaccines in clinical trial.
As well the AS and TH vectors preparation, as the in The similar results were obtained using human glioma HG cell line. Concerning MHC-II and B7 expressions (using MHC-II primer and B7 primer, respectively), the results were identical to those shown for MHC-I.