Pathologic Correlates of Primary Central Nervous System Lymphoma Defined in an Orthotopic Xenograft Model
15 Mar 2009-Clinical Cancer Research (American Association for Cancer Research)-Vol. 15, Iss: 6, pp 1989-1997
TL;DR: Intracerebral implantation of Raji cells results in a reproducible and invasive xenograft model, which recapitulates the histopathology and molecular features of PCNSL, and is suitable for preclinical testing of novel agents.
Abstract: Purpose: The prospect for advances in the treatment of patients with primary central nervous system lymphoma (PCNSL) is likely dependent on the systematic evaluation of its pathobiology. Animal models of PCNSL are needed to facilitate the analysis of its molecular pathogenesis and for the efficient evaluation of novel therapeutics. Experimental Design: We characterized the molecular pathology of CNS lymphoma tumors generated by the intracerebral implantation of Raji B lymphoma cells in athymic mice. Lymphoma cells were modified for bioluminescence imaging to facilitate monitoring of tumor growth and response to therapy. In parallel, we identified molecular features of lymphoma xenograft histopathology that are evident in human PCNSL specimens. Results: Intracerebral Raji tumors were determined to faithfully reflect the molecular pathogenesis of PCNSL, including the predominant immunophenotypic state of differentiation of lymphoma cells and their reactive microenvironment. We show the expression of interleukin-4 by Raji and other B lymphoma cell lines in vitro and by Raji tumors in vivo and provide evidence for a role of this cytokine in the M2 polarization of lymphoma macrophages both in the murine model and in diagnostic specimens of human PCNSL. Conclusion: Intracerebral implantation of Raji cells results in a reproducible and invasive xenograft model, which recapitulates the histopathology and molecular features of PCNSL, and is suitable for preclinical testing of novel agents. We also show for the first time the feasibility and accuracy of tumor bioluminescence in the monitoring of a highly infiltrative brain tumor.
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TL;DR: Progress in in vitro screening platforms as well as orthotopic and 'orthometastatic' xenograft mouse models has enabled comprehensive characterization of the impact of the tumour microenvironment on therapeutic efficacy, which can hopefully bridge the gap between preclinical studies and clinical trials of anticancer agents.
Abstract: The role of stromal cells and the tumour microenvironment in general in modulating tumour sensitivity is increasingly becoming a key consideration for the development of active anticancer therapeutics. Here, we discuss how these tumour-stromal interactions affect tumour cell signalling, survival, proliferation and drug sensitivity. Particular emphasis is placed on the ability of stromal cells to confer - to tumour cells - resistance or sensitization to different classes of therapeutics, depending on the specific microenvironmental context. The mechanistic understanding of these microenvironmental interactions can influence the evaluation and selection of candidate agents for various cancers, in both the primary site as well as the metastatic setting. Progress in in vitro screening platforms as well as orthotopic and 'orthometastatic' xenograft mouse models has enabled comprehensive characterization of the impact of the tumour microenvironment on therapeutic efficacy. These recent advances can hopefully bridge the gap between preclinical studies and clinical trials of anticancer agents.
395 citations
TL;DR: This review will focus on PCNSL in the immunocompetent host, an uncommon variant of extranodal non-Hodgkin lymphoma that has been challenging to study and an effective standard of care has been difficult to establish.
Abstract: Primary central nervous system (CNS) lymphoma (PCNSL), an uncommon variant of extranodal non-Hodgkin lymphoma (NHL), can affect any part of the neuraxis including the eyes, brain, leptomeninges, or spinal cord. It accounts for approximately 3% of all the primary CNS tumors diagnosed each year in the United States. Congenital or acquired immunodeficiency is the only established risk factor for PCNSL, and individuals with human immunodeficiency virus (HIV) infection are at greater risk for developing this tumor. Infection with HIV likely accounted for the increased incidence in PCNSL observed from 1970 to 2000, but over the last decade the number of cases of PCNSL has stabilized or decreased to about 0.47 cases per 100 000 persons. Owing to the rarity of PCNSL, the disease has been challenging to study and an effective standard of care has been difficult to establish. Unfortunately, although durable remissions may be achieved for some patients with PCNSL, the tumor relapses in most cases. In this review, we will focus on PCNSL in the immunocompetent host.
180 citations
Cites background from "Pathologic Correlates of Primary Ce..."
...Recently, the first mouse model of PCNSL was developed; it could potentially be used to test new therapeutic agents that target these molecular abnormalities including the IL-4 signaling pathway.(4)...
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TL;DR: It is demonstrated for the first time that elevated CXCL13 concentration in cerebrospinal fluid (CSF) is prognostic and that CXC chemokine ligand and IL-10 mediate chemotaxis of lymphoma cells isolated from CNS lymphoma lesions.
178 citations
Cites methods from "Pathologic Correlates of Primary Ce..."
...To enable the generation of adequate numbers of cells for in vitro assays, patient-derived lymphoma cells were implanted into the cerebrum of NOD-SCID g mice whereupon they expanded to form visible tumors within 4 weeks, according to an Institutional Animal Care and Use Committee–approved protocol.(17) At necropsy, intracranial lymphoma xenografts were isolated, and malignant B cells purified by CD191 selection by MACS (Miltenyi Biotech) for in vitro migration assays....
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TL;DR: POM is a promising therapeutic agent for CNS lymphoma with excellent CNS penetration, significant preclinical therapeutic activity, and a major impact on the tumor microenvironment.
Abstract: Primary CNS lymphoma carries a poor prognosis. Novel therapeutic agents are urgently needed. Pomalidomide (POM) is a novel immunomodulatory drug with anti-lymphoma activity. CNS pharmacokinetic analysis was performed in rats to assess the CNS penetration of POM. Preclinical evaluation of POM was performed in two murine models to assess its therapeutic activity against CNS lymphoma. The impact of POM on the CNS lymphoma immune microenvironment was evaluated by immunohistochemistry and immunofluorescence. In vitro cell culture experiments were carried out to further investigate the impact of POM on the biology of macrophages. POM crosses the blood brain barrier with CNS penetration of ~ 39%. Preclinical evaluations showed that it had significant therapeutic activity against CNS lymphoma with significant reduction in tumor growth rate and prolongation of survival, that it had a major impact on the tumor microenvironment with an increase in macrophages and natural killer cells, and that it decreased M2-polarized tumor-associated macrophages and increased M1-polarized macrophages when macrophages were evaluated based on polarization status. In vitro studies using various macrophage models showed that POM converted the polarization status of IL4-stimulated macrophages from M2 to M1, that M2 to M1 conversion by POM in the polarization status of lymphoma-associated macrophages is dependent on the presence of NK cells, that POM induced M2 to M1 conversion in the polarization of macrophages by inactivating STAT6 signaling and activating STAT1 signaling, and that POM functionally increased the phagocytic activity of macrophages. Based on our findings, POM is a promising therapeutic agent for CNS lymphoma with excellent CNS penetration, significant preclinical therapeutic activity, and a major impact on the tumor microenvironment. It can induce significant biological changes in tumor-associated macrophages, which likely play a major role in its therapeutic activity against CNS lymphoma. POM should be further evaluated in clinical trials.
74 citations
Cites background from "Pathologic Correlates of Primary Ce..."
...When they were treated with IL4 followed by POM, they expressed pSTAT1 and iNOS, and did not express pSTAT6 and YM1/FXIIIA, indicating conversion of their polarization from M2 to M1 via activation of STAT1 signalling and inactivation of STAT6 signalling....
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...These cells expressed pSTAT6 and YM1/FXIIIA [12] on treatment with IL4 followed by DMSO, indicating M2 polarization via IL4/STAT6 signalling pathway....
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...In experiments in which IL-4 was used to induce M2 polarization of macrophages, cells were treated with IL-4 (20ng/ml) for 48 hours followed by treatment with POM (10ug/ml) or DMSO control for 48 hours....
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...It has been shown that macrophages in PCNSL are M2 polarized related to upregulation of IL4 in CNS lymphoma [12,13]....
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...POM converted the IL-4-induced M2 polarization of macrophages as indicated by FXIII A and pSTAT6 expression to M1 polarization as indicated by iNOS and pSTAT1 expression....
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TL;DR: The cell lines and murine models used to study lymphomagenesis, the lymphoma microenvironment, and the efficacy of new therapies are summarized to improve understanding of the molecular and cellular mechanisms of tumor genesis and development as well as the fundamental processes governing the interaction of tumors and their host tissues.
Abstract: Human B-cell lymphomas, the fourth most common hematologic malignancy, are currently the subject of extensive research. The limited accessibility of biopsies, the heterogeneity among patients, and the subtypes of lymphomas have necessitated the development of animal models to decipher immune escape mechanisms and design new therapies. Here, we summarize the cell lines and murine models used to study lymphomagenesis, the lymphoma microenvironment, and the efficacy of new therapies. These data allow us to understand the role of the immune system in the fight against tumors. Exploring the advantages and limitations of immunocompetent versus immunodeficient models improves our understanding of the molecular and cellular mechanisms of tumor genesis and development as well as the fundamental processes governing the interaction of tumors and their host tissues. We posit that these basic preclinical investigations will open up new and promising approaches to designing better therapies.
68 citations
References
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TL;DR: The findings suggest that angiogenesis dependent on IL-4 and IL-13 is mainly mediated through a soluble VCAM-1/α4 integrin pathway.
Abstract: IL-13 is a multifunctional lymphokine sharing a number of biological properties with IL-4. We previously observed that IL-4 shows angiogenic activities in vitro as well as in vivo. In this study we examined the effect of IL-13 on angiogenesis in vitro and in vivo and also the underlying mechanisms. Human IL-13 significantly stimulated the formation of tube-like structures in collagen gels by human microvascular endothelial cells and bovine aortic endothelial cells by about 3-fold over the controls in the absence of the cytokines. Administration of murine IL-13 led to neovascularization when implanted in the rat cornea. Coadministration of neutralizing mAb to the IL-4R inhibited both tubular morphogenesis in vitro and activation of STAT6 induced by IL-4 or IL-13. Both IL-4 and IL-13 markedly increased mRNA levels of VCAM-1 in vascular endothelial cells, and the production of the soluble form of VCAM-1 was also stimulated in response to IL-4 or IL-13. Administration of anti-VCAM-1 Ab in vitro blocked tubular morphogenesis induced by IL-4 and IL-13. Angiogenesis induced in vivo in rat cornea by IL-4 and IL-13 was also inhibited by Ab against the rat alpha4 integrin subunit. These findings suggest that angiogenesis dependent on IL-4 and IL-13 is mainly mediated through a soluble VCAM-1/alpha4 integrin pathway.
120 citations
TL;DR: Although some patients had a poor PS and had been heavily pre-treated, temozolomide yielded 26% objective responses and was well tolerated without any major toxicity in the first prospective trial assessing single-agent activity in PCNSL at failure.
117 citations
TL;DR: It is concluded that FXIII-A expression is an intracellular marker for alternatively activated macrophages, while its absence in monocyte-derived macrophage cell line indicates their classically activated state.
Abstract: Factor XIII subunit A of blood coagulation (FXIII-A) is known to be synthesized but not secreted by the monocyte/macrophage cell line. On the basis of its intracellular localization and substrate profile, FXIII-A is thought to be involved in certain intracellular processes. Our present study was designed to monitor the changes in FXIII-A gene expression and protein production in long-term culture of human monocytes during their differentiation into macrophages in the presence of activating agents (interleukin-4, interferon-γ, Mycobacterium bovis BCG) inducing classical and alternative activation pathways. By using quantitative RT-PCR and fluorescent image analysis at the single-cell level we demonstrated that the expression of FXIII-A both at the mRNA as well as at the protein level is inversely regulated during the two activation programmes. Here we conclude that FXIII-A expression is an intracellular marker for alternatively activated macrophages, while its absence in monocyte-derived macrophages indicates their classically activated state.
111 citations
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TL;DR: The incidence of primary CNS lymphoma has increased only slightly in the past 10 years in individuals above the age of 60, and now stands at 0.44/100,000 patient-years as discussed by the authors.
Abstract: Primary CNS lymphoma (PCNSL) affects all age groups with a peak incidence in the fifth to seventh decades in non-AIDS patients. A slight male predominance is observed. The disease represents 2.6% of all primary brain tumors and 2–3% of NHLs [1, 53]. After a threefold rise observed between 1970 and 1990, the incidence of PCNSL has increased only slightly in the past 10 years in individuals above the age of 60, and now stands at 0.44/100,000 patient-years [1, 71]. With all forms of PCNSL, an occult systemic lymphoma is seldom found. Fewer than 10% of PCNSL patients present with brain and systemic lymphoma [85], and a similar percentage develop systemic disease after brain involvement.
102 citations
TL;DR: BLI monitoring can be used as a surrogate for predicting survival benefit from TMZ treatment, permits early determination of relative survival benefit associated with distinct TMZ therapeutic regimens, and offers a means of investigating secondary/salvage therapy efficacy following tumor regrowth from initial therapy.
Abstract: Object Bioluminescence imaging (BLI) offers a rapid and accurate means for longitudinal study of tumor cell growth and response to therapy in rodent models. Because this technology has only recently come into use in the field of small animal imaging, applications in this area have been limited. In the current study we have applied BLI to the analysis of clinically relevant issues involving use of the DNA methylating agent temozolomide (TMZ) in a mouse model. Methods An invasive glioblastoma multiforme xenograft was modified for BLI via transduction with a luciferase-encoding lentivirus. Supratentorial tumors were established in athymic nude mice that were subsequently assigned randomly to control and TMZ treatment groups, and the extent of intracranial tumor was monitored using BLI. Results In an experiment designed to compare the extent of antitumor effect between a single high-dose TMZ treatment and a protracted low-dose TMZ regimen, BLI revealed the protracted regimen as having superior antitumor effec...
79 citations
"Pathologic Correlates of Primary Ce..." refers background or methods in this paper
...The Raji cells were modified to stably express firefly luciferase by means of lentiviral transduction as described (11)....
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...therapeutic response in a variety of cancer xenograft models, including for brain tumors (10, 11)....
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...orthotopic glioma xenografts (11), intracerebral Raji xenografts are moderately sensitive to the alkylating agent temozolomide and exhibit expression of CD20, Bcl-6, MUM-1, IL-4, activated STAT6, Pim-2 kinase, and cathepsin D as well as tumor...
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...We hypothesized that high constitutive expression of the DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) could account for the relative resistance to temozolomide (11)....
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