Development and Analysis of Novel Intravascular Large B-Cell Lymphoma NOD/Shi-Scid IL2Rγ^null Mouse Xenograft Model

Background: Intravascular large B-cell lymphoma (IVLBCL) is a distinct disease entity of extranodal large B-cell lymphoma according to the current WHO classification. The peculiar characteristics of the disease of selective growth of tumor cells in the lumina of small vessels make an accurate diagnosis difficult, and the delay of timely diagnosis results in the poor prognosis. Recent retrospective analyses revealed that the prognosis of the disease was improved in the rituximab era (Shimada et al. J Clin. Oncol. 2008 and Lancet Oncol. 2009) and central nervous system recurrence was still important complication (Shimada et al. Cancer Sci. 2010). Meanwhile, the underlying biology of the disease, that is why the tumor cells lodge in the lumina of vessels, remains unknown due to the difficulty of obtaining sufficient patient tumor samples.

Purpose: To uncover the underlying mechanisms of the disease, we developed and analyzed the NOD/Shi-scid IL2Rγ^null (NOG) mouse xenograft models established from primary IVLBCL patient samples.

Material and methods: Patient bone marrow samples were obtained from 4 IVLBCL patients with written informed consent and injected into NOG mice intravenously. The successes of the engraftment and surface antigens of tumor cells were analyzed by the flow cytometry. Pathological specimens of the mouse models were evaluated by hematoxylin-eosin and immunohistochemical stainings. Global gene expression profiling and genomic wide analyses by array comparative genomic hybridization (CGH) were performed and genomic copy number abnormalities were confirmed by RQ-PCR and FISH analyses.

Result: Patient tumor cellswere successfully engrafted in all 4 xenograft models.The serial passages of the xenograft tumor cells were also successful. Pathologically, the tumor cells were distributed into systemic organs including liver, bone marrow, spleen, kidney, adrenal gland and lung except for testis. The peculiar characteristics of the growth of tumor cells in the lumina of vessels or the sinusoid in the organs were retained. The surface antigens analyses relevant to the lymphocyte migration revealed the expression of L-selectin (CD62L) was low in all 4 IVLBCL models and the expressions of LFA-1 and VLA-4 were various. The expression of CXCR4 and CXCR5 were observed in all 4 models. The time lapse engraftment analyses revealed that tumor cells initially engrafted in the sinusoid and the lumina of vessels in the liver a week after inoculation and then engrafted in the various organs including bone marrow, lung, spleen and adrenal gland two weeks later after inoculation, which indicated that the sinusoid in the liver might be advantageous for the engraftment of the tumor cells compared to other organs. Intriguingly, tumor cells from bone marrow, spleen and liver in the first transplanted mice distributed into the various organs in the secondary transplanted mice in the serial tumor passage, meanwhile tumor cells from adrenal gland in the first transplanted mice mainly distributed into adrenal gland in the secondary transplanted mice, which indicated that tumor cells might be selected at the engraftment of the specific organ. In terms of the dependency on vascular endothelial cells, the survival of tumor cells were dependent on the human umbilical vein endothelial cells (HUVEC) in in vitro co-culture assay (co-culture vs mono-culture, 84% vs 9%, p=0.0067). Gene expression profiling and array CGH analyses revealed the similar gene expression pattern relevant to the adhesion molecule and the consistent loss of chromosome 6 and 9 in all 4 xenograft models. Genomic copy number analysis targeted the gene located on chromosome 6 and 9 confirmed the loss of copy number in 3 of 4 IVLBCL models. FISH analyses are now under investigation.

Discussion and Conclusions: The characteristics of IVLBCL tumor cells from patient bone marrow samples were retained in our xenograft mouse models. Moreover we could expand the tumor cells in the mouse models and then investigate the biological characteristics of this disease entity. Further investigations including global gene mutation analysis to uncover the mechanism of the disease should be required.