There are numerous types of B-cell lymphoma including Burkitt lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma/mucosa-associated lymphoid tissue lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma and other less frequent subtypes. However, the molecular pathogenesis of B-cell lymphoma remain largely unknown. Here we chose three different B-cell lymphoma subtype cell lines to establish mouse models, which could be useful to determine the efficacy and mechanisms of novel therapies.

Firstly, we injected subcutaneously lymphoma cells into the right axillary region of BALB/c (nu/nu) nude mice. DOHH2 F0, Ly8 F0, Raji F0 appeared 7, 7, 21 days later. When tumors growed to 1.5cm at length, successful passage of mouse models was established by injecting cell suspension obtained from mincing and grinding tumors through a 70-μm cell strainer. The cell suspension was cultured at 37°C in a humidified atmosphere containing 5% CO2. The cells were cryopreserved in cryopreservation-medium (90% FBS, 10% DMSO), stored in liquid nitrogen, thawed again (with a viability of more than 70%) and successfully reconstituted. The above cell lines mouse models have been passaged to the 16th, 18th, 13th generation.

For histological examination, an autopsy was performed on each passage of nude mice, and immunohistochmistry was use to determine the expression of surface markers. The tumor tissues from DOHH2 F0, DOHH2 F6, DOHH2 F12 were positive for CD20, cytoplasmic CD3, CD10, CD5, CD21, Ki67, CyclinD1, Bcl-2, Bcl-6. EBV was also detected in situ hybridization. Grade 3B Follicular lymphoma was identified in this B-cell lymphoma and was consistent with the primary origination of DOHH2. Immunohistochemical staining of Ly8 F0, Ly8 F6, Ly8 F12 showed the large atypical cells were positive for CD20, CD3, CD10, Bcl-2, mum-1, no reactivity to antibodies for bcl-6 and negative to EBER. Ki-67 values were graded as 30%. The pathologic expression in Raji F0, Raji F6, Raji F12 is like the following: CD20+/CD3+-/Ki67+-/CD10+/Bcl2+-/ Bcl6- /Mum1+/EBER++.

Flow cytometry analysis was performed on cell suspensions cultured from each passage tumor tissues. DOHH2 F0, DOHH2 F6,DOHH2 F12 cells were CD19+/CD20-/CD10+/CD5-/CD38+/HLA-DR+/ CD22+ /CD23+/Ki-67+CD71+, and the kappa light chain was expressed monoclonally. Similar to DOHH2 cells, the serial Ly8 cells were CD19+ /CD20-/CD10+/CD5-/CD38+/HLA-DR+/CD22+/CD23+Ki-67+CD71+/EBER-, and the kappa light chain was expressed monoclonally. As Burkitt lymphoma, Raji and its serial cell clones were CD19+/CD20+/CD10+/CD5-/CD38+/HLA-DR+/CD22+/Ki-67+/CD71+/EBER++.

In conclusions, we firstly and systematically established mouse models of B-cell lymphoma and take into passage successfully.

Figure 1
Figure 1.
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Disclosures

No relevant conflicts of interest to declare.

Topics:
antibodies, atmosphere, unit of pressure, atypical, b-cell lymphomas, burkitt's lymphoma, carbon dioxide, cd20 antigens, cryopreservation, diffuse large b-cell lymphoma, dimethyl sulfoxide

Author notes

*

Asterisk with author names denotes non-ASH members.

© 2017 by The American Society of Hematology
2017
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