A novel orally available inhibitor of focal adhesion signaling increases survival in a xenograft model of diffuse large B-cell lymphoma with central nervous system involvement

Abstract

Central nervous system dissemination is a relatively uncommon but almost always fatal complication in diffuse large B-cell lymphoma patients. Optimal therapy for central nervous involvement in this malignancy has not been established. In this paper, we aimed to evaluate the therapeutic effect of E7123, a celecoxib derivative that inhibits focal adhesion signaling, in a novel xenograft model of diffuse large B-cell lymphoma with central nervous system involvement. Cells obtained after disaggregation of HT subcutaneous tumors (HT-SC cells) were intravenously injected in NOD/SCID mice. These mice received oral vehicle or 75 mg/kg of E7123 daily until they were euthanized for weight loss or signs of sickness. The antitumor effect of E7123 was validated in an independent experiment using a bioluminescent mouse model. Intravenously injected HT-SC cells showed higher take rate and higher central nervous system tropism (associated with increased expression of β1-integrin and p130Cas proteins) than HT cells. The oral administration of E7123 significantly increased survival time in 2 independent experiments using mice injected with unmodified or bioluminescent HT-SC cells. We have developed a new xenograft model of diffuse large B-cell lymphoma with central nervous system involvement that can be used in the pre-clinical evaluation of new drugs for this malignancy. E7123 is a new, well-tolerated and orally available therapeutic agent that merits further investigation since it may improve current management of diffuse large B-cell lymphoma patients with central nervous system involvement.

Figure 1.

Survival in HT-SC mice is significantly lower than in control HT mice. (A) Kaplan-Meier analysis shows a significant reduction in survival time in HT-SC (n=7) as compared to control HT mice (n=10). P value for the log rank test was <0.01. (B) Macroscopic evidence for central nervous system involvement of DLBCL in NOD/SCID mice injected with HT-SC cells via the tail vein. *The remaining mice (n=8) were euthanized 95 days after injection without any sign of disease.

Figure 2.

Histological and immunohistochemical phenotype of the DLBCL model with CNS involvement. H&E staining of representative brains in HT-SC mice. Lymphoma cells infiltrated the leptomeninges in most mice (A-D), reaching the parenchyma in isolated cases (D). Original magnification ×40 and corresponding magnification ×400 (E-H). Immunohistochemical analyses of CD20 (I), CD10 (J) and Ki67 (K) in representative sections of HT-SC infiltrated brains. Original magnification ×400. Slides were viewed with an Olympus BX51 microscope. Images were acquired using an Olympus DP72 digital camera and processed with the Olympus Cell^D Imaging 3.3 software.

Figure 3.

Expression of p130Cas and β1-integrin in HT and HT-SC cells. (A) Immunoblotting of FAK (Tyr 397), FAK, β1-integrin, p130Cas, Pyk2, Lyn, AKT (Thr 308) and AKT in protein extracts of control HT or HT-SC cells one day (HT-SC1) or seven days (HT-SC7) after disaggregation of subcutaneous tumors. WSU cell line lysate was used as positive control. HT-SC protein extracts showed higher expression levels of β1-integrin and p130cas than HT control cells. Equal loading was checked by immunobotting with β-actin. (B) Immunostaining analysis of p130Cas and β1-integrin in control HT cells, HT subcutaneous tumor, HT-SC cells (original magnification ×400) and HT-SC infiltrated brain sections (original magnification ×100, inset ×400). Slides were viewed with an Olympus BX51 microscope. Images were acquired using an Olympus DP72 digital camera and processed with the Olympus Cell^D Imaging 3.3 software.

Figure 4.

E7123 antitumor effect in an independent experiment using a bioluminescent model of DLBCL with CNS involvement. (A) Representative BLI images showing lymphoma HT-Luc-Sc cell growth evolution in the CNS in vehicle (PEG:FBS) or treated groups (75 mg/kg E7123). Vehicle-treated and E7123-treated mice were sacrificed on Days 56 and 92 post-injection of cells, respectively. Arbitrary color bars illustrate relative light intensity levels from firefly luciferase, ranging from low (blue) to high (red). Corresponding grayscale photographs and bioluminescent images were superimposed for each animal. (B) BLI signal quantification curves in the CNS for vehicle and E7123-treated mice. At week 6 after tumor cell injection, one vehicle-treated mouse could not be evaluated for BLI because it showed signs of sickness that precluded the use of anesthesia. BLI signal was quantified and expressed as Photon Counts (PHC). Curves represent the kinetics of BLI intensity mean for each group. Error bars represent the standard error. (C) Survival time in HT-Luc-SC mice treated with E7123 (75 mg/kg) was significantly higher than in control mice (PEG:FBS). P value for the log rank test was <0.01. Kaplan-Meier analysis showed the significant increase in survival time in treated mice (n=8) as compared to control mice (n=7).