Development of transplantable B-cell lymphomas in the MHC-defined miniature swine model

Abstract

Background: Establishment of transplantable tumors in clinically relevant large animals allows translational studies of novel cancer therapeutics.

Methods: Here we describe the establishment, characterization, and serial transplantation of a naturally occurring B-cell lymphoma derived from a unique, highly inbred sub-line of Massachusetts General Hospital (MGH) major histocompatibility complex (MHC)-defined miniature swine.

Results: The lymphoblastic cell line (LCL) originated from peripheral blood of a 2.5 year old female swine leukocyte antigen (SLA)^dd-inbred miniature swine breeder demonstrating clinical signs of malignancy. Flow cytometric phenotypic analysis of subclones derived from the original cell line revealed surface markers commonly expressed in a B-cell lineage neoplasm. A subclone of the original LCL was transplanted into mildly-conditioned histocompatible miniature swine and immunocompromised NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl /SzJ (NSG) mice. Tissue and blood samples harvested 2 weeks following subcutaneous and intravenous injection in a highly inbred SLA^dd pig were cultured for tumor growth and phenotypic analysis before serial transfer into NSG mice. Evidence of tumor growth in vivo was found in all tumor cell recipients. In vitro growth characteristics and surface phenotype were comparable between the original and serially transplanted tumor cell lines.

Conclusions: These results indicate the feasibility of developing a large-animal transplantable tumor model using cells derived from spontaneously occurring hematologic malignancies within the highly inbred miniature swine herd.

Keywords: B-cell lymphoma, preclinical large animal model; Serial transplantation.

Fig. 1

Schematic of experimental design. Flow chart including primary cell line harvest, scale up, and transfer within miniature swine and NSG mice

Fig. 2

Growth characteristics of the swine derived tumor cell lines. A LCL-21353.1B2 culture image taken after third passage. B LCL-21353.2F8 culture image taken after third passage. C 21353.2F8-23482 culture image taken after second passage. D Growth curve comparing the three cell lines. All microscopy was performed using Nikon Eclipse TE2000-U microscope under 20× magnification at room temperature. Images processed using Adobe Photoshop 6.0 (Adobe Systems)

Fig. 3

Flowcytometric analysis of tumor cell lines. a Descriptive list of staining characteristics for the three swine derived tumor cell lines. No change from negative control (−), shift between 10¹ and 10² (+), shift between 10² and 10³ (++), shift greater than 10³ (+++). b Histograms depicting surface staining of the three tumor cell lines for characteristic markers of a B-cell lineage neoplasm. c Histograms depicting intracellular staining of mu heavy and lambda light chain. Gray filled histograms with dashed outline represent LCL-21353.1B2, white filled histograms with solid outline represent LCL-21353.2F8, and white filled histograms with dotted outline represent LCL-21353.2F8-23482. Non-viable cells were removed using 7-AAD, gate placed on lymphocytes population identified in the forward/side-scatter prior to analysis

Fig. 4

Histologic analysis of tumor injected swine 23482. A Gross image of subcutaneous tumor injection site harvested at necropsy (11 days post-injection). B H&E staining, C CD45 staining, D CD79a staining, E lambda light chain staining of punch biopsy taken from the subcutaneous site 7 days after injection. All images were taken at 20× magnification

Fig. 5

Histologic analysis of Group 1 mice (recipients of LCL-21353.2F8). A Image of intact vasculature found on the abdominal tumor mass from mouse 2588. B Measurement of the isolated mass from mouse 2588. C H&E, D CD45, and E CD79a immunostaining performed on an abdominal tumor mass from mouse 2589 using porcine specific antibodies. All images were taken at 20× magnification. Staining was performed on masses harvested 70 days after tumor injections

Fig. 6

Flow cytometric evidence of LCL-21353.2F8-23482 in NSG spleen. a Representative phenotyping of a small abdominal tumor mass harvested from mouse 2560 which received LCL-21353.2F8 tumor injections. b Presence of tumor cells found in the spleen of mouse 44, despite the lack of solid mass formation in all Group 2 mice. Non-viable cells were removed using 7-AAD, gate placed on large lymphocytes in the forward/side-scatter shown above