Potent antitumor effects of bevacizumab in a microenvironment-dependent human lymphoma mouse model

Abstract

We established a mouse model of microenvironment-dependent human lymphoma, and assessed the therapeutic potential of bevacizumab, an antitumor agent acting on the microenvironment. NOD/Shi-scid, IL-2Rγ^null (NOG) mice were used as recipients of primary tumor cells from a patient with diffuse large B-cell lymphoma (DLBCL), which engraft and proliferate in a microenvironment-dependent manner. The lymphoma cells could be serially transplanted in NOG mice, but could not be maintained in in vitro cultures. Injection of bevacizumab together with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone) significantly increased necrosis and decreased vascularization in the tumor, compared with CHOP alone. Levels of human soluble interleukin-2 receptor (sIL2R) in the serum of bevacizumab+CHOP-treated mice (reflecting the DLBCL tumor burden) were significantly lower than in CHOP recipients. Mice receiving bevacizumab monotherapy also showed significant benefit in terms of tumor necrosis and vascularization, as well as decreased serum sIL2R concentrations. The present DLBCL model reflects the human DLBCL in vivo environment more appropriately than current mouse models using established tumor cell lines. This is the first report to evaluate the efficacy of bevacizumab in such a tumor microenvironment-dependent model. Bevacizumab may be a potential treatment strategy for DLBCL patients.

Citation: Blood Cancer Journal (2012) 2, e67; doi:10.1038/bcj.2012.12& 2012 Macmillan Publishers Limited All rights reserved 2044-5385/12

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ORIGINAL ARTICLE

Potent antitumor effects of bevacizumab in a microenvironment-dependent human lymphoma mouse model

F Mori1, T Ishida1, A Ito1, F Sato2, A Masaki1, H Takino2, M Ri1, S Kusumoto1, H Komatsu1, R Ueda1, H Inagaki2 and S Iida1

We established a mouse model of microenvironment-dependent human lymphoma, and assessed the therapeutic potential of bevacizumab, an antitumor agent acting on the microenvironment. NOD/Shi-scid, IL-2Rgnull (NOG) mice were used as recipients of primary tumor cells from a patient with diffuse large B-cell lymphoma (DLBCL), which engraft and proliferate in a microenvironment-dependent manner. The lymphoma cells could be serially transplanted in NOG mice, but could not be maintained in in vitro cultures. Injection of bevacizumab together with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone) signicantly increased necrosis and decreased vascularization in the tumor, compared with CHOP alone. Levels of human soluble interleukin-2 receptor (sIL2R) in the serum of bevacizumab CHOP-treated mice (reecting the DLBCL tumor

burden) were signicantly lower than in CHOP recipients. Mice receiving bevacizumab monotherapy also showed signicant benet in terms of tumor necrosis and vascularization, as well as decreased serum sIL2R concentrations. The present DLBCL model reects the human DLBCL in vivo environment more appropriately than current mouse models using established tumor cell lines. This is the rst report to evaluate the efcacy of bevacizumab in such a tumor microenvironment-dependent model. Bevacizumab may be a potential treatment strategy for DLBCL patients.

Blood Cancer Journal (2012) 2, e67; doi:http://dx.doi.org/10.1038/bcj.2012.12

Web End =10.1038/bcj.2012.12 ; published online 20 April 2012

Keywords: bevacizumab; NOD/Shi-scid; IL-2Rgnull (NOG) mouse; lymphoma; tumor microenvironment

INTRODUCTIONTumors develop in a complex and dynamic microenvironment.

Surrounding or within the tumor nests, stromal cells, endothelial cells, innate immune cells and other lymphocytes are present that interact with each other and with the tumor cells. A large body of evidence has accumulated in the past decade demonstrating that this complex tumor microenvironment regulates tumor growth, invasion, and metastasis.1 Angiogenesis is one of the most important phenomena within the tumor microenvironment; cancer cells have the ability to recruit and generate new blood vessels through the secretion of angiogenic factors. Tumor angiogenesis ensures that cells in the interior of the tumor receive sufcient nutrients and oxygen to survive. Blocking tumor angiogenesis would therefore severely restrict tumor growth.2 Early experiments using mouse xenografts indicated that antibody-mediated inhibition of vascular endothelial growth factor (VEGF), which promotes the proliferation and migration of vascular endothelial cells and vessel sprouting, could severely inhibit angiogenesis and tumor growth.3 These and other studies led to the development of the anti-VEGF neutralizing antibody bevacizumab for therapeutic use.4 However, a current crucial problem in the research eld of anti-tumor microenvironment agents such as bevacizumab is the lack of suitable small animal models. To the best of our knowledge, all preclinical testing of the antitumor activity of bevacizumab in mice in vivo has been performed using established tumor cell lines, which by denition can be maintained in vitro in culture.

Such tumor cells have thus been selected for survival in the absence of any microenvironment, including the vascular system. Using these established lines in mouse xenograft models therefore seems less relevant for the evaluation of the antitumor activities of anti-angiogenesis agents. Hence, the rst objective of the present study was to overcome this problem. We aimed to establish a mouse model in which primary tumor cells from a patient engraft and proliferate in a microenvironment-dependent manner, using NOD/Shi-scid, IL-2Rgnull (NOG) mice as

recipients.5,6

Bevacizumab is currently approved world-wide for the treatment of several types of solid tumors such as colorectal cancer, breast cancer, non-small cell lung cancer, renal cell cancer and glioblastoma.715 Many aspects of pathological angiogenesis have been extensively studied in many types of solid tumors. However, the precise role of these processes in pathogenesis of hematological malignancies is still under active investigation, and in this context, bevacizumab is not currently approved for the treatment of hematological malignancies in the United States, Europe, or Japan. Thus, the second aim of the present study was to evaluate the therapeutic potential of bevacizumab with or without systemic chemotherapy for hematological neoplasia, using newly established primary tumor cell-bearing NOG mice.We selected diffuse large B-cell lymphoma (DLBCL) as the target disease because this represents the most common type of malignant lymphoma and accounts for B3040% of all cases in adults.16,17

1Department of Medical Oncology and Immunology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-chou, Mizuho-ku, Nagoya, Aichi, Japan and 2Department of Clinical Pathology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-chou, Mizuho-ku, Nagoya, Aichi, Japan. Correspondence: Dr T Ishida, Department of Medical Oncology and Immunology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-chou, Mizuho-ku, Nagoya, Aichi 467-8601, Japan.

E-mail: mailto:[email protected]

Web End [email protected] Received 17 February 2012; revised 29 February 2012; accepted 21 March 2012

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MATERIALS AND METHODSAnimalsNOG mice were purchased from the Central Institute for Experimental

Animals (Kanagawa, Japan) and used at 68 weeks of age. All of the in vivo experiments were performed in accordance with the United Kingdom Coordinating Committee on Cancer Research Guidelines for the Welfare of Animals in Experimental Neoplasia, Second Edition, and were approved by the Ethics Committee of the Center for Experimental Animal Science, Nagoya City University Graduate School of Medical Sciences.

Immunopathological analysisWe assessed the affected lymph nodes of 50 patients with DLBCL by immunopathology. The patients provided written informed consent in accordance with the Declaration of Helsinki, and the present study was approved by the institutional Ethics Committee of Nagoya City University Graduate School of Medical Sciences. Hematoxylin and eosin staining and immunostaining using anti-human CD20 (L26; DAKO, Glostrup, Denmark), CD25, (4C9; Novocastra, Wetzlar, Germany), CD3 (SP7; SPRING BIOSCIENCE, Pleasanton, CA, USA), VEGF-A (sc-152, rabbit polyclonal, Santa Cruz, Heidelberg, Germany), Alpha-Smooth Muscle Actin (a-SMA; 1A4; DAKO), von Willebrand Factor (Rabbit polyclonal, DAKO), CD31 (JC70A, DAKO), CD10 (56C6; Novocastra), BCL-6 (LN22; Novocastra) and MUM1/IRF4 (M-17, Santa Cruz) were performed on formalin-xed, parafn-embedded sections. The presence of Epstein-Barr virus encoded RNA (EBER) was examined by in situ hybridization using EBER Probe (Leica microsystems, Newcastle, UK) on formalin-xed, parafn-embedded sections. DLBCL cases were categorized into germinal center B-cell (GCB) or non-GCB phenotypes using formalin-xed, parafn-embedded sections according to Hans Algorithm.18 VEGF-A expression levels were categorized according to the following formula: 3 positive ifX50%, 2 positive ifo50X30%, 1

positive ifo30X10% and negative ifo10% of the DLBCL tumor cells were stained with the corresponding antibody. Nine 100 high-power elds

(HPF) of hematoxylin and eosin tumor specimens were randomly selected and the area of tumor necrosis (%) was calculated by Image J software19

and then averaged. Nine 100 HPF of von Willebrand Factor-stained

tumor specimens were randomly selected and the numbers of vessels (per mm2) were calculated by Image J software19 and then averaged.

Primary DLBCL cell-bearing mouse modelThe affected lymph node cells from a patient with DLBCL were suspended in RPMI-1640. The tumor cell donor provided written informed consent before sampling in accordance with the Declaration of Helsinki, and the present study was approved by the institutional Ethics Committee of Nagoya City University Graduate School of Medical Sciences. CD3-negative subsets were isolated using anti-human CD3 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and the autoMACS Pro Separator (Miltenyi Biotec) according to the manufacturers instructions. Immunopathological analysis of the patients affected lymph node revealed that the DLBCL type was non-GCB (CD10 , BCL-6 and MUM1/IRF4 ), and VEGF expression

was 1 positive. Six to 8 weeks after intraperitoneal (i.p.) injection, NOG

mice presented with i.p. masses and splenomegaly. Cells from these i.p. masses were suspended in RPMI-1640 and i.p. inoculated into other NOG mice, which then presented with features identical to those of the rst mice.

DLBCL cell linesDB and HT were purchased from DSMZ (Braunschweig, Germany).

KARPAS422, OCI-LY19, Farage, Toledo, Pfeiffer and RL were purchased from ATCC (Manassas, VA, USA).

Quantitative reverse transcription PCRTotal RNA was isolated with RNeasy Mini Kit (QIAGEN, Tokyo, Japan).

Reverse transcription from the RNA to rst strand cDNA was carried out using High Capacity RNA-to-cDNA Kit (Applied Biosystems Inc., Foster City, CA, USA) according to the manufacturers instructions. Human VEGF-A (Hs00900055_m1), VEGF-R1 (Hs00176573_m1), VEGF-R2 (Hs00911700_m1) and b-actin (Hs99999903_m1) mRNA were amplied using TaqMan Gene

Expression Assays with the aid of an Applied Biosystems StepOnePlus according to the manufacturers instructions. The quantitative assessment of the mRNA of interest was done by dividing its level by that of b-actin and expressing the result relative to Human Testis Total RNA (Clontech,

Mountain View, CA, USA) as 1.0. All expressed values were averages of triplicate experiments.

Monoclonal antibodies and ow cytometryThe following monoclonal antibodies (mAbs) were used for ow cytometry: MultiTEST CD3 (clone SK7) FITC/CD16 (B73.1) CD56 (NCAM

16.2) PE/CD45 (2D1,) PerCP/CD19 (SJ25C1) APC Reagent (BD Biosciences, San Jose, CA, USA), PerCP-conjugated anti-human CD45 mAb (2D1, BD Biosciences), APC-conjugated anti-CD19 mAb (HIB19, BD Biosciences), PE-conjugated anti-CD25 mAb (M-A251, BD Biosciences), PE-conjugated VEGF-R1 mAb (49560, BD Biosciences), PE-conjugated VEGF-R2 mAb (89106, R&D Systems Inc., Minneapolis, MN, USA) and the appropriate isotype control mAbs. Whole blood cells from mice were treated with BD FACS lysing solution (BD Biosciences) for lysing red blood cells . Cells were analyzed by a FACSCalibur (BD Biosciences) with the aid of FlowJo software (Tree Star Inc., Ashland, OR, USA).

Cell proliferation assayProliferation of the DLBCL cell lines, which express both VEGF-A and

VEGF-R in the presence of different concentrations of bevacizumab for 48 h, was assessed using the CellTiter 96 Aqueous One Solution cell proliferation assay kit (Promega Corporation, Madison, WI, USA) as described previously.20 Proliferation of the NOG DLBCL cells with or without human interleukin-2 at a nal concentration of 100 IU/ml was also assessed in the same manner.

Primary DLBCL cell-bearing mice treated with CHOP bevacizumab

Tumor cells from the i.p. masses were suspended in RPMI-1640, and1.0 107 were i.p. inoculated into each of 10 NOG mice. The mice were

divided into two groups of ve each for treatment with bevacizumab

CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone) or CHOP alone, 2 days after tumor inoculation. Bevacizumab (10 mg/kg) or control (saline) was i.p. injected into the mice 2, 9, 16, 23, 30, 37 and 44 days after tumor cell inoculations. CHOP was given i.p. 30 days after tumor inoculations at doses as follows: cyclophosphamide, 40 mg/kg; doxorubicin, 3.3 mg/kg; vincristine, 0.5 mg/kg; prednisolone, 0.2 mg/kg.21

Therapeutic efcacies were evaluated 49 days after tumor inoculation. Bevacizumab was purchased from Chugai Pharmaceutical Co., Ltd, Tokyo, Japan; cyclophosphamide and vincristine were purchased from Shionogi Pharmaceutical Co., Ltd, Osaka, Japan; doxorubicin was from Kyowa Hakko Kirin Co., Ltd, Tokyo, Japan and prednisolone was from Nippon Kayaku Co., Ltd, Tokyo Japan.

Primary DLBCL cell-bearing mice treated with bevacizumabA total of 1.0 107 tumor cells were i.p. inoculated into each of 18 NOG

mice, divided into two groups of nine each for bevacizumab or control. Bevacizumab (10 mg/kg) or control (saline) was i.p. injected into the mice after 3, 10, 17, 24, 31, 38 and 45 days, and therapeutic efcacies were evaluated 47 days after tumor inoculations.

Human sIL2R measurementThe concentration of human soluble interleukin-2 receptor (sIL2R) in mouse serum was measured by enzyme-linked immunosorbent assay using the human sIL2R immunoassay kit (R&D Systems, Inc.) according to the manufacturers instructions.

Statistical analysisThe differences between groups regarding the tumor necrosis area, vascular number, percentage of lymphoma cells in mouse spleen cell suspensions and human sIL2R concentrations in mouse serum were examined with the MannWhitney U test. All analyses were performed with SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA). In this study, Po0.05 was considered signicant.

RESULTSVEGF-A expression in DLBCL

VEGF-A expression by DLBCL cells in the lymph node lesions according to GCB or non-GCB phenotypes are shown in Figure 1a.

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Figure 1. VEGF-A expression in DLBCL. (a) VEGF-A expression of DLBCL cells in the lymph node lesions according to GCB or non-GCB phenotypes. VEGF-A expression was categorized based on the percentage of DLBCL cells stained as follows: X50%, 3 positive; 3049%, 2

positive; 1029%, 1 positive; o10%, negative. (b) Cases 1, 2, 3 and 4 are representative of VEGF-A negative, 1 , 2 and 3 positive

categories, respectively. Photomicrographs with hematoxylin and eosin (HE; upper panels) and VEGF-A staining (lower panels) are shown.

Immunopathological features of four cases from each group stratied by VEGF-A expression are shown in Figure 1b. Differences in VEGF-A expression levels between the two DLBCL groups (GCB versus non-GCB) did not achieve signicance (Fishers exact test).

Establishment of the primary DLBCL cell-bearing NOG mouse model

The macroscopic appearance of a primary DLBCL cell-bearing NOG mouse is shown in Figure 2a, demarcating the i.p. mass and splenomegaly by thin white dotted lines. Flow cytometric analysis demonstrated that the mass mainly consisted of human cells expressing CD19 and CD25 (Figure 2b). Immunopathological analysis revealed that it consisted of large atypical cells with irregular and pleomorphic nuclei, and blood vessels. The cells were CD20 , but CD3-negative (Figure 2c). These ndings are

consistent with DLBCL. The cells were in addition positive for CD25 and negative for EBER (data not shown). VEGF expression was 1 positive. The DLBCL cells were also positive for MUM1/

IRF4, but negative for CD10 and BCL-6 (data not shown), and were thus classied as non-GCB phenotype. These immuno-pathological ndings on the NOG DLBCL cells were identical to those of the donor DLBCL.

Blood vessels in the tumor tissue were stained by anti-a-SMA Ab (Figure 2c). Vascular endothelial cells in the tumor tissue were stained by anti-von Willebrand Factor Ab, but not by anti-CD31 mAb (data not shown). These results indicated that blood vessels in the tumor originated from the mouse, because anti-a-SMA and von Willebrand Factor Ab used in the present study recognized the corresponding protein derived from both human and mice, whereas the anti-CD31 mAb recognized the corresponding human but not murine protein (data not shown).

DLBCL cell inltration into spleen, liver and bone marrow was seen both by ow cytometry (Figure 2d, upper panels) and pathological analyses (Figure 2d, lower panels).

The tumor cells recovered from mice receiving the primary lymphoma cells were serially i.p. transplanted into other NOG mice. This procedure of transfer from mouse to mouse was repeated successfully until at least the fth passage. The

macroscopic features of the animals and the immunopathological ndings for the tumor changed little through these serial passages. We could passage tumor cells that had been kept frozen until use, as well as those freshly isolated (data not shown). In contrast, these DLBCL cells could not be maintained in vitro in culture (data not shown).

VEGF-A, VEGF-R1 and -R2 expression in DLBCL cell linesVEGF-A mRNA expression was detected in all eight DLBCL cell lines tested and in NOG DLBCL cells from i.p. masses (Figure 3a, left panel). VEGF-R1 mRNA expression was present only in two (OCILy19 and Toledo) of the DLBCL cell lines and in the NOG DLBCL cells (Figure 3a, right panel). No VEGF-R2 mRNA expression was detected in any of the eight DLBCL cell lines tested, or in the NOG DLBCL cells (data not shown). Flow cytometry demonstrated that VEGF-R1 protein was also expressed in the two lines with mRNA (OCI-Ly19 and Toledo, Figure 3b), consistent with the results from reverse transcription PCR. VEGF-R1 expression in NOG DLBCL cells as assessed by ow cytometry was very weak (Figure 3b) and VEGF-R2 was not expressed at all in any of the DLBCL cell lines tested, or in NOG DLBCL cells (data not shown), also consistent with the reverse transcription PCR results.

Bevacizumab-mediated anti-proliferative activity against DLBCL cells in vitro

Bevacizumab did not directly block the proliferation of OCI-Ly19 and Toledo cells in vitro, despite their expression of both VEGF-A and VEGF-R1 (Figure 3c, upper panels). Neither did it inhibit NOG DLBCL cells, with or without the addition of interleukin-2 (Figure 3c, lower panels).

CHOP bevacizumab has signicantly greater therapeutic efcacy

than CHOP alone in primary DLBCL cell-bearing NOG mice Treatment with CHOP bevacizumab resulted in an increased

percentage of tumor necrosis in the primary DLBCL cell-bearing NOG mice (mean 12.7%, median 11.1%, range 5.218.7%), compared with CHOP alone (mean 1.8%, median 1.5%, range1.02.7%, P 0.0090; Figure 4a, left panel). An example of

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Figure 2. Primary DLBCL cell-bearing NOG mouse model. (a) Macroscopic appearance of a primary DLBCL cell-bearing NOG mouse. The intraperitoneal mass is demarcated by a thin white dotted line. (b) Human CD45 cells in the mass determined by human CD19 and CD25

expression. (c) Immunohistochemical images of the intraperitoneal mass. (d) Human CD45 cells of each organ determined by human CD3

and CD19 expression (upper panels). Photomicrographs with hematoxylin and eosin (HE) staining of each organ (lower panels).

calculating the percentage necrotic area is presented in Figure 4a, right panels. CHOP bevacizumab treatment resulted in decre

ased vasculature in the tumor tissues (41.9, 40.9, 32.551.3/mm2;

(mean, median, range)), compared with CHOP alone (66.3, 71.8,40.779.5/mm2, P 0.0472; Figure 4b, left panel). An example

of this calculation is presented in Figure 4b, right-hand panels.

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Figure 3. VEGF-A, VEGF-R1 and VEGF-R2 expression in DLBCL cell lines. (a) Quantitative reverse transcription (RT)-PCR analysis for VEGF-A and VEGF-R1 in eight DLBCL cell lines, and NOG DLBCL cells from the intraperitoneal mass. (b) Flow cytometry for VEGF-R1 in DLBCL cell lines, and NOG DLBCL cells from the intraperitoneal mass. (c) Bevacizumab has no direct anti-proliferative activity against DLBCL cell lines (OCI-Ly19 and Toledo) expressing both VEGF-A and VEGF-R1 (upper panels), and NOG DLBCL cells (lower panels), in vitro. Each result represents three independent experiments.

As sIL2R appears in the serum, concomitant with its increased expression on cells,22 we measured human sIL2R concentrations as a surrogate marker reecting the tumor burden of the human CD25-expressing DLBCL. Treatment with CHOP bevacizumab

showed signicantly greater therapeutic efcacy as demonstrated by sIL2R concentrations in the primary DLBCL cell-bearing NOG mice (44.6, 46.1, 28.559.2 103 pg/ml), compared with CHOP

alone (83.5, 78.1, 49.5119.3 103 pg/ml, P 0.0283; Figure 4c).

The percentages of DLBCL cells in spleen cell suspensions of CHOP and CHOP bevacizumab-treated mice were 14.1%, 11.4%,

10.220.7%, and 26.1%, 24.6% and 19.034.6%, respectively. This difference was statistically signicant (P 0.0163; Figure 3d,

left panel). An example of the calculation is shown in Figure 4d, right panels.

Macroscopic and microscopic ndings in mice with or without bevacizumab therapy

The appearance of primary DLBCL cell-bearing control mice (treated with saline) or those treated with bevacizumab alone is shown in Figure 5a, upper and lower panels, respectively. Tumor masses are demarcated by thin white dotted lines. Photomicro-graphs of tumor tissue from each mouse are also shown (Figure 5b).

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Figure 4. CHOP bevacizumab has greater therapeutic efcacy than CHOP alone. (a) Area of tumor necrosis (%) of each primary DLBCL-

bearing NOG mouse. The CHOP bevacizumab-treated mice had signicantly greater tumor necrosis than CHOP-treated mice (left panel). An

example of a calculation for tumor necrosis area (%) by means of Image J software is shown (scale bar, 200 mm; right panels). (b) Numbers of vessels (per mm2) of each primary DLBCL-bearing NOG mouse. The CHOP bevacizumab recipients had signicantly fewer than CHOP

recipients (left panel). An example of such a calculation by means of Image J software is shown (scale bar, 200 mm; right panels). (c) Serum

sIL2R concentrations of each primary DLBCL-bearing NOG mouse. The CHOP bevacizumab recipients had signicantly lower levels of sIL2R

than CHOP recipients. (d) Spleen-inltrating tumor cells (%) of each primary DLBCL-bearing NOG mouse. The CHOP bevacizumab recipients

had signicantly lower levels of spleen-inltrating tumor cells than CHOP recipients (left panel). An example of a calculation of spleen-

inltrating tumor cells (%) is shown (right panels).

Bevacizumab therapy alone has signicant therapeutic efcacy in primary DLBCL cell-bearing NOG mice.

Treatment with bevacizumab alone signicantly increased the percentage tumor necrotic area (7.5, 4.8, 2.118.7%) compared with control mice (2.0, 1.7, 0.15.6%, P 0.0070; Figure 6a). This

was also the case when considering vascularization of the tumor tissues (46.2, 43.1, 33.660.0/mm2, compared with 66.7, 64.8,50.199.9/mm2 in controls, P 0.0070; Figure 6b). Treatment with

bevacizumab showed signicantly greater therapeutic efcacy as demonstrated by sIL2R concentrations in the primary DLBCL cell-bearing NOG mice (187.6, 185.2, 5.0350.8 103 pg/ml), compared

with controls (459.6, 482.8, 201.5689.5 103 pg/ml, P 0.0041;

Figure 6c).

The percentages of DLBCL cells in spleen cell suspensions of bevacizumab- and saline-treated mice were 13.1, 14.6, 0.127.5% and 18.7%, 18.8%, 4.031.7%, respectively, but this difference was not statistically signicant (data not shown).

DISCUSSIONIn the present study, we have achieved two goals: rst, to establish a novel mouse model using NOG recipients engrafted with primary DLBCL cells from a patient, in which the tumor cells survive and proliferate in a murine microenvironment-dependent manner; second, to document that bevacizumab possesses signicant therapeutic efcacy in these primary DLBCL cell-bearing mice.

NOG mice have severe, multiple immune dysfunctions, such that human immune cells engrafted into them retain essentially the same functions as in humans.23,24 In the present system,

primary DLBCL cells expressing CD19, CD20 and CD25 formed large i.p. masses, and markedly inltrated into different organs such as spleen, liver and bone marrow. The presented features were very similar to the donor DLBCL patient. The lymphoma cells were positive for VEGF-A and therefore it would be expected that the interaction of VEGF-A produced by tumor cells with VEGF-R2

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Figure 5. Macroscopic and microscopic ndings of mice with or without bevacizumab therapy. (a) Macroscopic appearance of mice treated with saline (control; upper panels) or bevacizumab (lower panels). Tumor masses are demarcated by thin white dotted lines. (b) Photomicrographs with hematoxylin and eosin (HE) staining of saline (control; upper panels) or bevacizumab-treated tumor (lower panels).

on host (mice) endothelial cells should have an important role in tumor angiogenesis, leading to tumor cell survival and proliferation supported by receiving sufcient nutrients and oxygen, as reported by other investigators.2527 To the best of

our knowledge, this is the rst report of primary DLBCL cell-bearing mice, in which the DLBCL cells can be maintained by serial transplantations, but cannot be maintained in vitro in culture. This indicates that the microenvironment is indispensable for tumor survival; thus the present DLBCL model should better reect the human DLBCL in vivo environment, compared with other mouse models using established tumor cell lines. Therefore, this model should provide a powerful tool for understanding the pathogenesis of DLBCL and, furthermore, for the one which can be used not only to evaluate novel cytotoxic anti-DLBCL cell agents, but also antitumor agents targeting the microenvironment, including bevacizumab, more appropriately,

in vivo. The observed signicant antitumor activities of bevacizumab combined with CHOP therapy were expected, because bevacizumab is known only to be of benet to patients with metastatic colorectal, non-small cell lung and metastatic breast cancer, when combined with chemotherapy.810 The effect

observed in mice receiving bevacizumab CHOP, as

demonstrated by the increased tumor necrosis area and reduced vasculature in the tumor tissue, was consistent with the conventional antitumor mechanism of bevacizumab, which neutralizes the human VEGF-A produced by the tumor cells, but not murine VEGF-A.28 It then inhibits the growth of new blood vessels and thus starves tumor cells of necessary nutrients and oxygen.29 This should lead to a reduced tumor burden, as indicated by the sIL2R concentrations measured. It was also reported that lymphoma cell growth was promoted in an autocrine manner via VEGF-A/VEGF-R1 or VEGF-A/VEGF-R2

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Figure 6. Bevacizumab therapy has signicant therapeutic efcacy in the DLBCL mice. (a) Area of tumor necrosis (%) of each primary DLBCL-bearing NOG mouse. The bevacizumab-treated mice had signicantly more tumor necrosis than controls. (b) Vessel numbers (per mm2) of each primary DLBCL-bearing NOG mouse. The bevacizumab recipients had signicantly fewer vessel numbers than controls. (c) Serum sIL2R concentrations of each primary DLBCL-bearing NOG mouse. The bevacizumab recipients had signicantly lower levels of sIL2R than controls.

interactions,25 but the present in vitro data did not support that observation. In the present study, signicant effects of bevacizumab alone were also observed, as demonstrated by the increased area of tumor necrosis and reduced vasculature in the tumor tissue, leading to a degree of antitumor therapeutic efcacy as demonstrated by reduced tumor burden indicated by serum sIL2R concentrations. In contrast to combination therapy, bevacizumab alone was not active when assessed by the percentages of DLBCL cells in the spleen. One possible explanation for this is that the spleen is likely to be more richly vascularized compared with the tumor mass, and thus bevacizumab alone has little starvation effect on the tumor cells therein. It has been reported that VEGF-targeted therapy can normalize the tumor vascular network and that this can lead to a more uniform blood-ow, with subsequent increased delivery of chemotherapeutic agents.3032 Therefore, the tumor cells in spleen might be efciently reduced only when bevacizumab is combined with chemotherapy.

The present study demonstrated the importance of angiogenesis for the pathogenesis of VEGF-expressing DLBCL. Ganjoo et al.33 reported that VEGF expression was detected in 4260% of tumor cells in DLBCL and Gratzinger et al.34 found that 60% of cases showed strong VEGF immunoreactivity, dened as VEGF expression in 430% of the tumor cells. These reports together with our present study indicate that targeting angiogenesis would be a promising strategy for at least a subgroup of DLBCL patients whose tumors depend to a large extent on angiogenesis via VEGF for survival and proliferation. In fact, bevacizumab as a single agent has been reported to have modest clinical activity in patients in the setting of relapsed aggressive non-Hodgkin lymphoma35 and in combination with rituximab-CHOP in rst-line treatment.33 However, a phase III clinical study evaluating the efcacy and safety of bevacizumab together with rituximab plus CHOP in patients with DLBCL (MAIN trial) could not be completed after a safety and efcacy analysis of the rst 720 patients. We believe that this result of the MAIN trial does not necessarily have to lead to the conclusion that bevacizumab is ineffective in DLBCL. Analogously, the epidermal growth factor receptor tyrosine kinase inhibitor, getinib, failed to yield a signicantly improved overall survival in patients with refractory non-small cell lung cancer,36

but did show therapeutic benet in a subgroup of patients with mutated epidermal growth factor receptor.3739 In the case of

mAb targeting the epidermal growth factor receptor, both panitumumab and cetuximab also provide clinical benets only to a subgroup of colorectal cancer patients with wild-type KRAS and BRAF.40 These ndings indicate that we should develop novel treatment strategies based on tumor biology and not on tumor category. DLBCL is a highly heterogeneous category with respect to biology, morphology and clinical presentation,16 as are non-

small cell lung cancer or colorectal cancer. Therefore, further investigations are warranted to determine which subgroups of DLBCL patients will benet from bevacizumab therapy.

In conclusion, using NOG mice as recipients, we have established a novel model in which primary DLBCL cells from a patient engraft and proliferate in a murine microenvironment-dependent manner. The present DLBCL model should more truly reproduce the human DLBCL in vivo environment, compared with any other current models, which use established tumor cell lines. This is the rst report to evaluate the efcacy of bevacizumab in such a tumor microenvironment-dependent model. Bevacizumab therapy could be a potential treatment strategy for that subgroup of DLBCL depending to a large extent on angiogenesis via VEGF for tumor survival and proliferation.

CONFLICT OF INTEREST

The authors declare no conict of interest.

ACKNOWLEDGEMENTS

We thank Ms Chiori Fukuyama for her excellent technical assistance. Grants-in-aid for (a) Young Scientists (no. 22689029: T Ishida) and (b) Scientic Research (no. 22300333: T Ishida and R Ueda), and Scientic Support Programs for Cancer Research (no. 221S0001, T Ishida) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Grants-in-aid for National Cancer Center Research and Development Fund (no. 21-6-3: T Ishida); and Health and Labour Sciences Research Grants (H22-Clinical Cancer Research-general-028: T Ishida and H23-Third Term Comprehensive Control Research for Cancer-general-011: T Ishida and H Inagaki) from Ministry of Health, Labour and Welfare, Japan.

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