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Engraftment of Human Glioblastoma Cells in Immunocompetent Rats through Acquired Immunosuppression.

Huszthy PC, Sakariassen PØ, Espedal H, Brokstad KA, Bjerkvig R, Miletic H - PLoS ONE (2015)

Bottom Line: 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-1α, IL-18 and TNF-α [corrected].We observed that in nude rat brains, an adaptation to the host occurs after several in vivo 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.

View Article: PubMed Central - PubMed

Affiliation: K.G. Jebsen Brain Tumour Research Centre, Department of Biomedicine, University of Bergen, Bergen, Norway; Centre for Immune Regulation, Department of Immunology, University of Oslo/the National Hospital, Oslo, Norway.

ABSTRACT
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-1α, IL-18 and TNF-α [corrected]. We observed that in nude rat brains, an adaptation to the host occurs after several in vivo 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 in vivo 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.

No MeSH data available.


Related in: MedlinePlus

GBM xenograft rejection in immunocompetent rats evidenced by MRI and immunohistology.(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.
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pone.0136089.g002: GBM xenograft rejection in immunocompetent rats evidenced by MRI and immunohistology.(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.

Mentions: 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 Fig 1). High generation xenografts P3 and P8 (denoting Patient tumor 3 and 8) have been characterized and published [1, 6]. We assessed initial engraftment using T2-weighted MRI. The xenografts appeared as weakly hyperintense, diffuse lesions discernible from three weeks post-implantation. Visible lesions appeared in all nude rats and grew progressively. On the other hand, initial lesions appeared in only 20 of 45 (44%) of immunocompetent animals, when evaluated starting three weeks p.i. We followed the animals by bi-weekly MRIs and noted that all primary- and low generation xenografts were rejected, whereas one out of three high generation tumors engrafted successfully (P = 0.016; Table 1). Rejection was evident when the slightly hyperintense area associated with xenograft tissue gradually decreased on successive time points (Fig 2A). Progressive growth was associated with a successive increase of the tumor size (Fig 2B) until the animals reached the humane endpoint. Longitudinal follow-up suggested that xenograft rejection was complete, i.e. a subpopulation of cells did not escape and regrow, at least within the first five months. Histological analysis of xenografts that underwent rejection revealed accumulation of leukocytes at the tumor border and around brain blood vessels (Fig 2A). In case of tolerance, leukocytes did not infiltrate the xenografts or enter the normal brain tissue (Fig 2B).


Engraftment of Human Glioblastoma Cells in Immunocompetent Rats through Acquired Immunosuppression.

Huszthy PC, Sakariassen PØ, Espedal H, Brokstad KA, Bjerkvig R, Miletic H - PLoS ONE (2015)

GBM xenograft rejection in immunocompetent rats evidenced by MRI and immunohistology.(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.
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pone.0136089.g002: GBM xenograft rejection in immunocompetent rats evidenced by MRI and immunohistology.(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.
Mentions: 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 Fig 1). High generation xenografts P3 and P8 (denoting Patient tumor 3 and 8) have been characterized and published [1, 6]. We assessed initial engraftment using T2-weighted MRI. The xenografts appeared as weakly hyperintense, diffuse lesions discernible from three weeks post-implantation. Visible lesions appeared in all nude rats and grew progressively. On the other hand, initial lesions appeared in only 20 of 45 (44%) of immunocompetent animals, when evaluated starting three weeks p.i. We followed the animals by bi-weekly MRIs and noted that all primary- and low generation xenografts were rejected, whereas one out of three high generation tumors engrafted successfully (P = 0.016; Table 1). Rejection was evident when the slightly hyperintense area associated with xenograft tissue gradually decreased on successive time points (Fig 2A). Progressive growth was associated with a successive increase of the tumor size (Fig 2B) until the animals reached the humane endpoint. Longitudinal follow-up suggested that xenograft rejection was complete, i.e. a subpopulation of cells did not escape and regrow, at least within the first five months. Histological analysis of xenografts that underwent rejection revealed accumulation of leukocytes at the tumor border and around brain blood vessels (Fig 2A). In case of tolerance, leukocytes did not infiltrate the xenografts or enter the normal brain tissue (Fig 2B).

Bottom Line: 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-1α, IL-18 and TNF-α [corrected].We observed that in nude rat brains, an adaptation to the host occurs after several in vivo 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.

View Article: PubMed Central - PubMed

Affiliation: K.G. Jebsen Brain Tumour Research Centre, Department of Biomedicine, University of Bergen, Bergen, Norway; Centre for Immune Regulation, Department of Immunology, University of Oslo/the National Hospital, Oslo, Norway.

ABSTRACT
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-1α, IL-18 and TNF-α [corrected]. We observed that in nude rat brains, an adaptation to the host occurs after several in vivo 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 in vivo 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.

No MeSH data available.


Related in: MedlinePlus