The authors have declared that no competing interests exist.
Conceived and designed the experiments: PCH RB HM. Performed the experiments: PCH PØS HE KAB HM. Analyzed the data: PCH PØS HE HM. Contributed reagents/materials/analysis tools: KAB RB HM. Wrote the paper: PCH HM.
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
When evaluating therapeutic approaches to be implemented in clinical oncology, using animal models with high relevance to human tumors is essential. We have previously established and characterized a patient biopsy xenograft model of glioblastoma multiforme in T cell-compromised nude rats, which has been applied in several studies of basic- and translational neuro-oncology [
Here, we assessed xenograft engraftment rates, host survival, dominant leukocyte populations and cytokine responses in an effort to establish an animal model for human GBMs in immunocompetent Rowett rats. We show that human GBM tissue serially passaged in nude rat brains may engraft in immunocompetent littermates in contrast to spheroids made directly from patient biopsies. We investigated some possible adaptation mechanisms that may have facilitated the tolerance of human tumor xenografts in fully immunocompetent rats.
Primary GBM biopsies were obtained at the Department of Neurosurgery, Haukeland University Hospital, Bergen. All patients gave a written informed consent for tumor biopsy collection and signed a declaration permitting the use of their biopsy specimens for research. The study was approved by the Norwegian Regional Research Ethics Committee (Rek-Vest, approval number 013.09). All animal protocols were approved by authorities in an AAALAC-accredited animal facility at the Haukeland University Hospital and were in accordance with the national regulations of Norway. Case approval numbers were 08/38978-2008120 and 08/110915-2008350.
Spheroid cultures were established as previously described [
Rowett nude rats were maintained in our facility by breeding homozygous males (
Implantation of tumor spheroids were performed as described [
Tumor growth was visualized using a Bruker Pharmascan 7 Tesla MR scanner (Bruker Biopsin MRI GmbH, Ettlingen, Germany) using T2-, and occasionally T1 (with gadolinium contrast agent) sequences as previously described [
Assessment of engraftment was done by evaluating MR images and histology. The animals were divided into two groups:
tumor rejection/immune system activation: no engraftment or the evidence of rejection on MRI, and proof of rejection on histology (various-sized lesions with significant leukocytic infiltration into the tumor bed concomitant with the presence of perivascular leukocytes in the normal brain). tumor engraftment/tolerance: progressive xenograft growth on MRI (comparable to nude rats), full-sized tumors at sacrifice without leukocyte infiltration into the tumor bed, no perivascular leukocytes in the normal brain tissue.
Fluorescent double staining was performed using combinations of human specific rabbit anti-nestin (AB5922, Chemicon, 1:200), rat-specific CD45 (#554875, BD, 1:200) antibodies and appropriate secondary reagents.
Immunostaining against rat-specific CD markers was performed on frozen brain sections from saline-perfused animals. After fixation and blocking, the sections were incubated for 1 hour with mouse anti-rat antibodies (all purchased from BD Pharmingen, Franklin Lakes, NJ), used at the following dilutions: CD4 (554841, OX-38, 1:100), CD8a (554854, 1:600), CD45 (554875, OX-1, 1:500), CD161a (NKR-P1A, 555006, 1:400), Dendritic Cell Antigen (555010, OX-62, related to CD103), 1:400; CD68 (ED1, 550305, 1:100). The sections were developed using horse-anti mouse biotinylated secondary antibodies and subsequently avidin-biotin complexes, using 3–3`-diaminobenzidine as substrate.
Dilutions of primary antibodies were: anti-Granzyme B (Abcam, Ab4059), 1:75; anti-Foxp3 (Biolegend, clone 150D), 1:50; anti- AIF-1 (LifeSpan Biosciences, Nordic Biosite, Oslo, Norway), 1:100, anti-Cathepsin S (sc-6503, Santa Cruz Biotechnology, Heidelberg, Germany), 1:100. The sections were developed using the DAKO Envision system with diamidobenzidine as substrate.
After capturing eight to ten x400 fields per case for each of the antigens, the images were saved on a Nikon light microscope and associated software (Nikon, Tokyo, Japan). The numbers of positive cells or the area fraction occupied by positive cells per high power field were counted.
Blood was harvested through the saphenous vein from twelve immunocompetent rats implanted with P3 high generation spheroids. The blood was collected into heparinized Microvette tubes (Sarstedt, Numbrecht, Germany). Blood samples were centrifuged at 1000G for 10 minutes. Isolated sera were stored at -80°C until analysis, performed within 4 months from the day of storage. Animals were followed by biweekly MRIs. Rats with large tumors were followed closely and sacrificed at the earliest onset of symptoms, whereas symptom-free rats were taken on day 90 p.i. Tumor histology was assessed by microscopy.
Serum samples were run on a fluorescence-based rat-specific cytokine 11-plex for quantification (Millipore, Oslo, Norway). The following cytokines were included: GM-CSF, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17, IL-18, IFN-γ and TNF-α. Serum samples were taken on days 0, 4, 7, 11, 18, 43, and 53 p.i.
Brain sections were cut in a cryotome under sterile conditions for RNA isolation. The first and last section was stained with haematoxylin and eosin to confirm the presence of tumor in the sections that were analysed. Human-specific primers were designed using the Primer Blast Program (NCBI). Lack of cross-hybridization with the rat-specific form was confirmed with Primer Blast and run on cross-reaction control samples. For assessment of human transcripts, the human Cytokines & Chemokines RT² Profiler PCR Array representing 84 key molecules (Qiagen, Oslo, Norway) was run on samples from xenografts of the same original patient biopsy.
Groups were analyzed for normal distribution by the Shapiro-Wilk Test significance (SPSS). For immunopositive cell counts and comparison of cytokine concentration values, groups were compared using the Mann–Whitney
We transplanted biopsy spheroids derived from six patients diagnosed with primary GBM to the right hemisphere of immunocompetent and nude rats. Three specimens were generated directly from patient biopsies (primary spheroids), one specimen was passaged once in a nude rat (low generation) and two specimens were high generation spheroids that have undergone multiple transplantation cycles in nude rats (for an overview, see
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.
(A, top row) Serial MRI sections (from left to right) show a lesion that appeared ten weeks p.i. of low generation spheroids. The xenograft presented with a diffuse, weakly hyperintense signal on T2-weighted images without a clear demarcation toward the brain parenchyma (upper panel). Four weeks later, there is a reduction in the volume of the hyperintense area, and the lesion now shows a demarcated border toward the brain. Micrograph: Arrowheads point to perivascular (top) and peritumoral (bottom) leukocytic infiltrates in the brain. (B) Serial MRI slices representative of progressive tumor growth. The upper panel shows slices of the lesion fourteen weeks post implantation. The lower panel shows expansion of the tumor four weeks later. Control injections with PBS only did not produce any MRI signal apart from an outline of the needle track (hypo-intense) on early scans. Micrograph: No infiltration of leukocytes in the brain or around the tumor. Scale bars: 50 μm. C. Immunofluorescent micrographs show xenograft rejection (a to f) and tolerance (g, h). GBM cells are marked by human-specific nestin (red) and host cells by rat leukocytic common antigen (CD45, green). (a,b) Early phase of rejection. The tumor bed (T) is surrounded by a band of host leukocytes. CD45+ cells are observed in the meninges and perivascularly (arrowheads) in the brain. c.c.: corpus callosum. (c,d) Later stage of rejection. The tumor (asterisk) is infiltrated by host leukocytes. In the surrounding brain (B), numerous microvessels have perivascular cuffs indicating recruitment of leukocytes from the circulation (arrowheads). (e,f) Complete rejection. In the tumor bed, only islands of tumor cell foci remain (arrowheads). (g,h) Tolerance. A full-sized, vascularized tumor. Leukocytes are mainly seen around necrotic areas (N) and around tumor blood vessels. Infiltration into the tumor bed is limited. Original magnification of the figures; a, e, c, g: x50; b, d, h: x100, f: x200.
Primary/low generation xenografts (%) | High generation xenografts (%) | ||
---|---|---|---|
Number of xenografts that appeared on initial MRI scans | 6/21 (28.6) | 14/24 (58) | 0.0715 |
Number of xenografts rejected after appearing on MRI scans | 6/6 (100) | 6/14 (42.9) | |
Number of xenografts that engrafted | 0/21 (0) | 8/24 (33.3) |
Next, we evaluated if one successful engraftment event in an immunocompetent rat brain led to complete adaptation to the immunocompetent host. We euthanized nude- and immunocompetent rats that developed full-blown brain tumors, established spheroids in short-term culture and implanted them into new immunocompetent recipients (
Co-staining of tumor cells (human-specific nestin) and leukocytes (rat-specific leukocyte common antigen or CD45) revealed the pattern of host cell infiltration in the xenografts (
In the tumor bed of low- and high generation xenografts which underwent rejection (
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.
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
We assessed the contribution of regulatory T-cells by anti-Forkhead box P3 (Foxp3) immunostaining. Positive cells were found mostly around tumor blood vessels (
(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
We assessed the contribution of cytotoxic effector cells by staining for Granzyme B, which identified pro-apoptotic granules in activated killer cells. In xenografts with on-going rejection, cytotoxic cells were in close contact with glioma cells in the tumor bed (
To assess the systemic responses to GBM xenografts growing in immunocompetent rats, we analysed sera from a group of animals (n = 12) implanted with high generation P3 spheroids. We compared serum cytokine concentration values for animals with radiological and histological proof of rejection versus tolerance. Of the panel of cytokines evaluated (see
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
An adaptation process by tumor cells to the host during repeated
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
To further evaluate the adaptation process, we performed human-specific gene expression arrays encompassing a broad panel of chemokines, cytokines and growth factors on corresponding low- and high generation xenografts of P3 and P8. Transcripts that were up- or down-regulated with at least a three-fold difference between low- and high generation xenografts are presented (
Here, we have shown that human GBM cells passaged intracerebrally in nude rats are able to engraft in the brain of immunocompetent littermates, whereas primary biopsy spheroids are subject to chronic cellular graft rejection. Most likely, a concerted action of ED1+ monocytes, presumably acting as antigen presenting-cells and of primed T cells is necessary for rejection of human GBM xenografts in the rat CNS, since 1) primary GBM xenograft tissue consistently engrafts in T cell compromised nude rats (here, and [
Although xenograft rejection in general is a function of the innate immune response [
Serum levels of IL-1α, IL-18 and TNF-α correlated with GBM xenograft rejection, reaching statistical significance on day 53 p.i. Several of these cytokines were similarly up-regulated in the brain of rats transplanted with fetal porcine neurons, indicating their importance in CNS xenograft rejection [
To delineate the differences between low- and high generation xenografts that may have affected engraftment, we have validated candidate GBM-derived factors with roles in immunological responses. Significant increases in TGF-β2 transcript levels, and decreases in CXCL10 and CXCL12 were observed in corresponding low- and high generation tumors used in this study. TGF-β is an essential glioma-derived cytokine that specifically represses the proliferation of antigen-specific CD4+ T cells [
Bone morphogenetic proteins are members of the TGF superfamily and are important regulators of neural differentiation. In our xenograft model, BMP-2 and 7 were differentially regulated. BMPs and their receptors are expressed by GBMs, and treatment of glioma cells with additional BMPs reduces proliferation and induces astroglial differentiation [
In conclusion, we have shown that tolerance to human GBM xenografts in the xenogeneic brain is enabled by a combination of several factors, such as 1) inadequate infiltration of the xenograft tissue and the brain by leukocytes, 2) attenuated systemic levels of pro-inflammatory cytokines, 3) increases in the proportion of Foxp3+ regulatory T cells and reduction in the absolute numbers of Granzyme B+ effector cells and 4) an increase in the levels of tumor-produced immunosuppressive TGF-β2 and decreased levels of leukocyte-attracting chemokines. Our data suggest an adaptation of human GBM cells after serial passaging in nude rats to the xenogeneic environment. Such “adapted” tumor cells are able to engraft in the brain of immunocompetent rats without signs of an inflammatory response. The presented work also shows the feasibility of growing human GBMs in fully immunocompetent animals, which may be further evaluated in a therapeutic context.
(A, B) Survival curves of immunocompetent rats implanted with spheroids derived from xenografts growing in immunocompetent (sph. from
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Panels show typical distribution of NKR-P1-positive and dendritic cell antigen-positive leukocyte subsets in the tumor and brain tissue of immunocompetent rats implanted with GBM xenografts. 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. DCA-dendritic cell antigen (OX-62, related to CD103), NKR-P1 (CD161a).
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We thank Ingrid Gavlen, Bodil Hansen, Christine Eriksen, Tove Johansen, Dagny Ann Sandnes and Marianne Eidsheim for technical assistance. We acknowledge MIC Bergen access to microscopic imaging and MRI facilities. The Vivarium (University of Bergen) is acknowledged for animal provision and care.