Increase in tumor size following intratumoral injection of immunostimulatory CpG-containing oligonucleotides in a rat glioma model

Abstract

The immunosuppressive environment of malignant gliomas is likely to suppress the anti-tumor activity of infiltrating microglial cells and lymphocytes. Macrophages and microglial cells may be activated by oligonucleotides containing unmethylated CpG-motifs, although their value in cancer immunotherapy has remained controversial. Following injection of CpG-containing oligonucleotides (ODN) into normal rat brain, we observed a local inflammatory response with CD8+ T cell infiltration, upregulation of MHC 2, and ED1 expression proving the immunogenic capacity of the CpG-ODN used. This was not observed with a control ODN mutated in the immunostimulatory sequence (m-CpG). To study their effect in a syngeneic tumor model, we implanted rat 9L gliosarcoma cells into the striatum of Fisher 344 rats. After 3 days, immunostimulatory CpG-ODN, control m-CpG-ODN, or saline was injected stereotactically into the tumors (day 3 group). In another group of animals (day 0 group), CpG-ODN were mixed with 9L cells prior to implantation without further treatment on day 3. After 3 weeks, the animals were killed and the brains and spleens were removed. Rather unexpectedly, the tumors in several of the animals treated with CpG-ODN (both day 0 and day 3 group) were larger than in saline or m-CpG-ODN treated control animals. The tumor size in CpG-ODN-treated animals was more variable than in both control groups. This was associated with inflammatory responses and necrosis which was observed in most tumors following CpG treatment. This, however, did not prevent excessive growth of solid tumor masses in the CpG-treated animals similar to the control-treated animals. Dense infiltration with microglial cells resembling ramified microglia was observed within the solid tumor masses of control- and CpG-treated animals. In necrotic areas (phagocytic), activation of microglial cells was suggested by ED1 expression and a more macrophage-like morphology. Dense lymphocytic infiltrates consisting predominantly of CD8+ T cells and fewer NK cells were detected in all tumors including the control-treated animals. Expression of perforin serving as a marker for T cell or NK cell activation was detected only on isolated cells in all treatment groups. Tumors of all treatment groups revealed CD25 expression indicating T cells presumed to maintain peripheral tolerance to self-antigens. Cytotoxic T cell assays with in vitro restimulated lymphocytes ((51)chromium release assay) as well as interferon-gamma production by fresh splenocytes (Elispot assay) revealed specific responses to 9L cells but not another syngeneic cell line (MADB 106 adenocarcinoma). Surprisingly, the lysis rates with lymphocytes from CpG-ODN-treated animals were lower compared to control-treated animals. The tumor size of individual animals did not correlate with the response in both immune assays. Taken together, our data support the immunostimulatory capacity of CpG-ODN in normal brain. However, intratumoral application proved ineffective in a rat glioma model. CpG-ODN treatment may not yield beneficial effects in glioma patients.

Fig. 1

Effect of immunostimulatory CpG-ODN or CpG-ODN mutated in the immunostimulatory sequence (m-CpG) in normal rat brain. a After 2 days, focal staining for ED1 was observed at the injection site and in the leptomeninges (magnified in the inserted image) of the CpG-ODN-treated hemisphere. b After 5 days, ED1 staining at the inoculation site is more pronounced (the ED1 positive area is magnified in the inserted image). Similarly, CpG-ODN (d, f) but not mCpG-ODN (c) treatment resulted in MHC 2 induction 5 days after ODN administration. Magnification revealed MHC 2 positive microglial cells or macrophages (f) and perivascular inflammation with CD8 positive cells representing microglia, macrophages, or lymphocytes (e). Similarly, in the leptomeninges of the CpG-ODN-treated hemisphere CD8 positive (g) and MHC 2 positive (h) cells were detected

Fig. 2

Effect of CpG-ODN on 9L cells grown in culture. Although exponential cell proliferation was observed in vehicle-treated cultures, the addition of CpG-ODN (10⁻⁶ M) and CpG-ODN mutated in the immunostimulatory sequence (m-CpG; 10⁻⁶ M) resulted in growth inhibition. A decrease in cell numbers indicating cytotoxicity was observed with CpG-ODN when added at a concentration of 10⁻⁵ M

Fig. 3

Effect of intratumoral CpG-ODN treatment on tumor volumes 3 weeks after intracerebral implantation of rat 9L cells. On day 3 after tumor cell implantation, intratumoral injections were performed with saline (control), a control oligonucleotide mutated in the immunostimulatory sequence (m-CpG), or a CpG-containing immunostimulatory ODN (CpG). In treatment group ‘CpG (day 0)’, the 9L cells were mixed with immunostimulatory CpG-ODN prior to tumor cell inoculation to simulate an optimal distribution of CpG-ODN within the tumor. These animals received no additional CpG treatment on day 3. In this group, one animal had died prior to killing because of excessive tumor growth (not included). No statistically significant differences were detected between the treatment groups (p > 0.05; ANOVA). The letters on top of the bars indicate the plot in Fig. 5 depicting the CTL response of the same animal, e.g., the CTL response of the saline-treated animal labeled with an a is shown in panel a of Fig. 5

Fig. 4

Immunohistochemical staining of tumor sections following intratumoral application of CpG-ODN (CpG) or saline (control). a–e the same region revealing the tumor margin and the adjacent normal brain was stained. In particular, in perivascular areas, dense infiltrates with TCR+ cells (a) were observed which consisted predominantly of CD8+ lymphocytes (c). The tumors contained numerous CD25 positive cells indicating CD4+ (or CD8+) regulatory T cells implicated in peripheral tolerance to self-antigen (d). The same region also contained NK cells which were less abundant (e). Perforin serving as a marker for cytotoxic effector activity of NK and CD8+ cells was only detected on few cells within CpG- and saline-treated (f) tumors. Staining for CD4 revealed dense infiltrates (b) consisting of lymphocytes and microglia. More specific staining (Iba1 and ED1) distinguished two types of microglial cells (g–q). In particular, around necrotic areas which were more prevalent in CpG-treated tumors, patchy areas were found revealing cells which resembled macrophages and which showed strong expression of ED1, a marker for phagocytic activity (l, m, p, q). To a lesser extent, focal expression of ED1 was also observed in saline-treated control animals (j, k). An abundance of more evenly distributed microglial cells with a ramified morphology was found within the tumors and peritumoral brain parenchyma (g, h, n, o). Such ramified microglia was predominant in the non-necrotic areas of both control-treated or CpG-treated animals

Fig. 5

Cytotoxic T cell responses assessed by ⁵¹chromium release (a–l) and IFN-γ synthesis quantified by the ELISPOT assay (m). a–l ⁵¹Cr release lymphocytes were restimulated with 9L cells for 1 week and added to ⁵¹Cr-labeled 9L target cells or another syngeneic adenocarcinoma cell line (MADB 106) in different effector to target ratios (E:T ratio). In all animals (a–l), specific lysis was observed with 9L cells but not with syngeneic MADB 106 cells. In both control groups (saline, a–d, and m-CpG, e–g), high lysis rates (>80–90%) were detected. In CpG-treated animals similar levels were measured following CpG-ODN treatment on day 0 concomitant with tumor cell grafting (n, o). Rather surprisingly, intratumoral application of immunostimulatory CpG-ODN 3 days after tumor cell grafting resulted in markedly lower lysis rates in 4 of 5 animals (h–m). IFN-γ synthesis in freshly isolated splenocytes following exposure to 9L cells or syngeneic MADB 106 cells was determined by Elispot assays. The letters on the x-axis indicate the panel showing the ⁵¹chromium release response of the same animal. For example, “c” corresponds to panel c depicting the CTL response of the same animal. This represents a saline-treated animal, and the tumor volume measured in the same animal can be determined from Fig. 3. IFN-γ synthesis was observed in all treatment groups following exposure to 9L cells. Reduced levels of IFN-γ synthesis were observed upon stimulation with the syngeneic rat adenocarcinoma cell line MADB 106 suggesting a 9L specific response. In most animals (c, f, g, k, l, n, o) the amount of IFN-γ synthesis detected was similar to the CTL response obtained after restimulation of lymphocytes in culture