Eradication of tumor colonization and invasion by a B cell-specific immunotoxin in a murine model for human primary intraocular lymphoma

Abstract

Human primary intraocular lymphoma (PIOL) is predominantly a B cell-originated malignant disease with no appropriate animal models and effective therapies available. This study aimed to establish a mouse model to closely mimic human B-cell PIOL and to test the therapeutic potential of a recently developed immunotoxin targeting human B-cell lymphomas. Human B-cell lymphoma cells were intravitreally injected into severe combined immunodeficient mice. The resemblance of this tumor model to human PIOL was examined by fundoscopy, histopathology, immunohistochemistry, and evaluated for molecular markers. The therapeutic effectiveness of immunotoxin HA22 was tested by injecting the drug intravitreally. Results showed that the murine model resembles human PIOL closely. Pathologic examination revealed that the tumor cells initially colonized on the retinal surface, followed by infiltrating through the retinal layers, expanding preferentially in the subretinal space, and eventually penetrating through the retinal pigment epithelium into the choroid. Several putative molecular markers for human PIOL were expressed in vivo in this model. Tumor metastasis into the central nervous system was also observed. A single intravitreal injection of immunotoxin HA22 after the establishment of the PIOL resulted in complete regression of the tumor. This is the first report of a murine model that closely mimics human B-cell PIOL. This model may be a valuable tool in understanding the molecular pathogenesis of human PIOL and for the evaluation of new therapeutic approaches. The results of B cell-specific immunotoxin therapy may have clinical implications in treating human PIOL.

Figure 1

Human B-cell lymphoma cell line CA46 expresses CD22, CXCR4, CXCR5, and IL-10. CA46 cells were cultured and maintained in vitro as described in Materials and Methods. A, cultured cells were analyzed for surface expression of CD22 (top), CXCR4 and CXCR5 (middle), or intracellular staining for IL-10 (bottom) by flow cytometry. X axis, relative fluorescence intensity (expression level). Y-axis, relative cell numbers. B, total RNA was isolated from cultured cells and mRNA levels of IL-10 and CXCR4 were measured by a quantitative real-time PCR as described. The levels of mRNA were expressed as the fold increase of that measured in RNA isolated from CA46 cells over that of the control RNA (mouse universal RNA). The calculation of the fold increase was detailed in Materials and Methods.

Figure 2

Histopathology of murine intraocular B lymphoma. Cultured CA46 cells were intravitreally injected into SCID mice at different doses. Eye tissues were collected at various time points postinjection, sectioned, and stained for H&E. A, typical early phase (day 10 postintravitreous injection of 20,000 CA46 cells per eye) histopathology of the mouse intraocular lymphoma. Note the tumor cells in the vitreous beginning to colonize, invading into the retina and optic nerve head (i, arrows). Most of the retinal tissue remains intact. ii and iii, the same picture at higher magnifications. L, lens; V, vitreous; R, retina; ON, optic nerve. B, late-phase (day 21 postintravitreous injection of 20,000 CA46 cells per eye) histopathology of the mouse intraocular lymphoma. a, tumor cells infiltrating the retinal layers causing massive tissue damage. b and c, magnified views of the retina showing the preferential expansion of tumor cells (arrows) into the subretinal space (SRS). Note that the tumor cells invading the retina lie between the retinal photoreceptors and retinal pigment epithelium (RPE; b). d, tumor colonization and invasion in the optic nerve head. Arrows, tumor cells. C, metastasis of tumor cells into CNS. CA46 cells (200,000 per eye per injection) were inoculated into the eyes of SCID mice. Brain tissues were collected on day 35 and processed for histopathology. The tumor cells (arrows) invaded into brain tissues, accumulated around the meningeal and parenchymal brain, and were mixed with a few inflammatory cells.

Figure 3

Molecular mimicry of mouse intraocular B-cell lymphoma model to human PIOL. CA46 cells were intravitreally injected into SCID mice (20,000 per eye per injection), animals were sacrificed on day 21 postinjection, and eyes were collected and snap-frozen on dry ice. The frozen eye tissues were sectioned and stained for tumor cells. The tumor cells were microdissected out from the eye section, total RNA was isolated, and real-time PCR was done as described in Materials and Methods. A, in vivo expression of both IL-10 (top) and CXCR4 (bottom) in the tumor cells. B, immunohistochemistry shows in vivo expression of CD22, CXCR4, and CXCR5 on the tumor cells. Left, expression levels of CD22, CXCR4, and CXCR5 in the in vitro cultured tumor cells. Right, in vivo expression levels in the mouse tissue after injection on day 21 (red, positive staining). All molecular markers are expressed in vivo and the expression of CXCR5 seems to be up-regulated in vivo (see Results and Discussion for details). Overlay pictures showing both DAPI staining (blue, nuclei) and the antibody staining (red, CD22, or CXCR4 or CXCR5) can be seen in Supplementary Fig. S1.

Figure 4

Therapeutic effect of intravitreous injection of an immunotoxin targeting on surface CD22. Murine intraocular B-cell lymphoma was induced (20,000 tumor cells per injection per eye) and treated with either control vehicle [PBS (pH 7.3)] or immunotoxin HA22 at various dosages on day 12 posttumor injection as described in the text; eye tissues were collected on day 21 and processed for histopathology.A, histopathology of ocular tissue in mice treated with control vehicle [PBS (pH 7.3)]. There is widespread colonization and invasion of tumor cells (arrows) and massive retina damage.B, mice treated with HA22 at 200 ng/eye/injection. There was no tumor colonization and invasion into the retina tissue but only inflammatory cells, suggesting a clearing process of tumors by the immunotoxin. Note also that there is no visible retinal damage by therapy (bottom).C, two representative animals treated with higher dose of HA22 (2,000 ng/mL). Again, no tumor was observed after immunotoxin treatment.Top, representative of some of the mice that had substantial retinal damage after high-dose HA22 treatment;bottom, some of the mice that showed relative milder retinal damage. There was no therapeutic effect when a control immunotoxin, erb-38, was used (see Supplementary Fig. S2).