Cytotoxic effector cells and regulatory T cells in GBM xenografts growing in immunocompetent rats.

<p>(A, B) Representative images show Foxp3 immunopositive perivascular lymphocytes in a rejected (A) and a tolerated (B) xenograft. Foxp3+ cells were scarce, most often found in the vicinity of tumor blood vessels. Scale bars: 50 μm (C, D). Granzyme B-positive cytotoxic cells in a GBM xenograft with rejection. Asterisks mark tissue lyzed by effector cells. Magnified view (E) shows granzyme-containing lysosomes (arrowheads) and target cells with apoptotic nuclei (arrows). (D) Generally, tolerated tumors were devoid of cytotoxic cells, but some were found perivascularly. Scale bars: 20 μm. (F) Quantification of Granzyme B expressing cells. (G) Quantification of Foxp3 expressing cells. Significant differences are denoted by asterisks (*** marks <i>P</i><0.001); n.s.: not significant. Circles represent outlier data points.</p

Serum levels of selected cytokines in immunocompetent rats implanted with GBM xenografts.

<p>Shown are the serum concentrations of cytokines that were consistently detected throughout the time course of the experiment. X axis indicates days from implantation (d = 0). The lines show average serum concentrations (± SEM) in the rejection group (black squares) vs. the engraftment group (open circles) of rats implanted with high generation P3 spheroids. Significant differences are denoted by asterisks (*** marks <i>P</i><0.001, * marks <i>P</i><0.05).</p

Leukocytes, therein T cells and monocytes are increased in the tumor bed in rats undergoing xenograft rejection.

<p>Box plots show the distribution of CD45+, CD4+, CD8a+ and ED1+ cells per high power field (HPF, x400) taken from immunostained sections. Three categories of tumors were considered, low generation xenografts undergoing rejection, high generation xenografts undergoing rejection and high generation xenografts with tolerance. The groups were compared using Mann-Whitney <i>U</i> Test. Significant differences are denoted by asterisks (*** marks <i>P</i><0.001); n.s.: not significant. Circles represent outlier data points.</p

Engraftment of Human Glioblastoma Cells in Immunocompetent Rats through Acquired Immunosuppression

<div><p>Transplantation of glioblastoma patient biopsy spheroids to the brain of T cell-compromised Rowett (nude) rats has been established as a representative animal model for human GBMs, with a tumor take rate close to 100%. In immunocompetent littermates however, primary human GBM tissue is invariably rejected. Here we show that after repeated passaging cycles in nude rats, human GBM spheroids are enabled to grow in the brain of immunocompetent rats. In case of engraftment, xenografts in immunocompetent rats grow progressively and host leukocytes fail to enter the tumor bed, similar to what is seen in nude animals. In contrast, rejection is associated with massive infiltration of the tumor bed by leukocytes, predominantly ED1+ microglia/macrophages, CD4+ T helper cells and CD8+ effector cells, and correlates with elevated serum levels of pro-inflammatory cytokines IL-1β, IL-18 and TNF-α. We observed that in nude rat brains, an adaptation to the host occurs after several <i>in vivo</i> passaging cycles, characterized by striking attenuation of microglial infiltration. Furthermore, tumor-derived chemokines that promote leukocyte migration and their entry into the CNS such as CXCL-10 and CXCL-12 are down-regulated, and the levels of TGF-β2 increase. We propose that through serial <i>in vivo</i> passaging in nude rats, human GBM cells learn to avoid and or/ suppress host immunity. Such adapted GBM cells are in turn able to engraft in immunocompetent rats without signs of an inflammatory response.</p></div

Outline of transfer experiments performed in nude and immunocompetent rats.

<p>Biopsy tissue from GBM patients or passaged xenograft tumors were minced into cubes and allowed to form spheroids in agar-overlay cultures before transplanting to the brain of animals. Comparisons in engraftment were made between 1) immunocompromised nude versus immunocompetent animals implanted with spheroids from the same culture; both primary, low generation and high generation material, 2) primary/low generation versus high generation spheroids in immunocompetent animals, 3) xenografts generated from tumors that engrafted in nude versus immunocompetent animals concerning subsequent engraftment rate in immunocompetent animals.</p

Monocytes and T cells infiltrate the tumor bed and the brain of immunocompetent rats that reject their xenografts.

<p>Panels show typical distribution of leukocyte subsets in the tumor and brain tissue of immunocompetent rats implanted with GBM xenografts. Brain slices were stained for the indicated markers and x400 fields were captured from representative areas. Shown are typical cases from immunocompetent rats with different engraftment outcomes. (A) An infiltrative GBM xenograft generated directly from patient tissue that elicited a strong immune response. (B) A diffusely growing, high generation GBM xenograft with significant immune response. (C) High-generation GBM xenograft, tolerance. Scale bar: 100 μm. ED1 antigen: CD68.</p

Engraftment outcome of GBM biopsy spheroids in immunocompetent rats.

<p>Engraftment outcome of GBM biopsy spheroids in immunocompetent rats.</p

Repeated <i>in vivo</i> passaging cycles of human GBM xenografts in nude rats lead to loss of immunogenicity.

<p>A. Left: In low generation xenografts, Allograft Inflammatory Factor-1 and Cathepsin S staining identified tumor-infiltrating microglia with predominantly intermediate and amoeboid morphology. Right: In high generation xenografts, immunopositive cells were less abundant. Box plots compare the numbers of AIF-1+ cells, or the area fractions occupied by Cat S+ cells per high power field (HPF, x400). Significant differences are denoted by asterisks (*** marks <i>P</i><0.001, ** marks <i>P</i><0.005). Circles represent outlier data points. B. Fold change comparison of transcript levels of selected human cytokines, chemokines and growth factors with relevance to immune responses in low- versus high generation xenografts. Of the panel of analysed transcripts in the array, only those with at least a three-fold change in levels between low and high generation in both patient xenografts are presented. C. Fold change of tumor-derived TGF-β2 transcripts in corresponding low and high generation xenografts. Mean ± SEM; three independent runs. Scale bars in A: 100 μm.</p

Tumors treated with lentiviral vectors and ganciclovir show complete remission on MRI.

<p>Representative three-dimensional MRI (T2 RARE). (A,F,K,P) Lentiviral LCMV-GP vectors with ganciclovir treatment. (B,G,L,Q) Lentiviral VSV-G vectors with ganciclovir treatment. (C,H,M,R) Lentiviral LCMV-GP vectors without ganciclovir treatment. (D,I,N,S) Lentiviral VSV-G vectors without ganciclovir treatment. (E,J,O,T) ganciclovir treatment only. Time points after tumor implantation: (A-E) 1 day before vector injection. (F-J) 1. week ganciclovir treatment. (K-O) 2. week ganciclovir treatment. (P-T) 4. week ganciclovir treatment.</p

Therapeutic efficiency of LCMV-GP and VSV-G pseudotyped lentiviral vectors <i>in vivo</i>.

<p>Intracranial gliomas were injected with LCMV-GP or VSV-G pseudotyped lentiviral vectors expressing HSV-1-<i>tk</i> fused to eGFP 3 weeks after tumor implantation. 7 days after vector infection, animals in both treated groups and in one control group were treated with ganciclovir for 30 days. (A) Kaplan-Meier survival curve. The survival benefit for both treatment groups compared to control groups was statistically significant (<i>P</i><0.001; log-rank test). There was no significant difference in survival between the two treatment groups. (B-D) Representative MRI (T2 RARE) of recurrent tumors in the LCMV- (B,C) and VSV-treated (D,E) group. (B) Invasive contralateral recurrence. (C) Invasive local and contralateral recurrence. (D) More circumscribed local recurrence. (E) Macroscopic picture of a rat brain with a recurrence in the cerebellum (red circle), treated with VSV-G pseudotyped vectors and GC. (F-M) Sections of recurrent tumors were stained with antibodies against human-specific nestin and analyzed by fluorescence (F,J) or confocal microscopy (G-I,K-M). Pictures show overlay of nestin (red) and eGFP transgene (green). (F-I) Recurrent tumors of animals treated with VSV-G pseudotyped lentiviral vectors. (F) Recurrent tumor with GFP-positive cells in the invasive area. (G) Higher magnification of (F). (H) GFP-positive tumor cells in the corpus callosum region. (I) GFP-positive normal brain cells at the tumor border. (J-M) Recurrent tumors of animals treated with LCMV-GP pseudotyped lentiviral vectors. (J) GFP-positive tumor cells in residual small lesion from the primary tumor. The recurrent tumor is growing from the contralateral hemisphere over the corpus callosum to the ipsilateral hemisphere (arrows). (K) Higher magnification of (J). (L) GFP-positive tumor cells in the solid ipsilateral recurrent lesion. (M) Few GFP-positive cells in a contralateral recurrent tumor. F,J: Magnification 40×. G,K,M: Magnification 200×. H,I,L: Magnification 100×.</p