Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models.

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Lymphoma B-cell responsiveness to CpG-DNA depends on the tumor microenvironment.

BACKGROUND: Toll-like receptor (TLR) agonists have important properties that can be exploited for immunotherapy against tumors. Locally injected immunostimulatory oligodeoxynucleotides containing CpG motifs (CpG-ODNs), which are TLR9 agonists, have shown promise in cancer models. Several studies have demonstrated that these motifs have immunologic effects similar to those of bacterial DNA and can stimulate monocytes, macrophages, dendritic, and B cells, which then produce several proinflammatory cytokines. However, these CpG-ODNs appear to produce opposite effects on tumor B cells. METHODS: In this study, we investigated the direct effects of a murine class B CpG (1826) ODNs on lymphoma B cells in vitro and in vivo, using mouse models of non-Hodgkin B lymphomas developing in immunoprivileged sites, specifically the brain and the eye, and in subcutaneous sites. RESULTS: In vitro, CpG-ODNs produced antiproliferative and proapoptotic effects on lymphoma B cells. In vivo, it had an antitumor effect when injected into tumors in murine models of subcutaneous lymphoma (SCL) and primary cerebral lymphoma (PCL). However, its intravitreal administration into a primary intraocular lymphoma (PIOL) mouse model did not produce an antitumor effect. In vitro experiments using supernatant from mouse PIOL samples demonstrated that the PIOL molecular microenvironment inhibits the antiproliferative effect of CpG-ODNs on lymphoma B-cells. CONCLUSIONS: Responsiveness to CpG stimulation differs in subcutaneous, cerebral, and ocular tumors, according to the tumoral and molecular microenvironment, and this should be considered for further therapeutic approaches.

Cytokine profile in human eyes: contribution of a new cytokine combination for differential diagnosis between intraocular lymphoma or uveitis.

Primary intraocular lymphoma (PIOL), also called primary vitreoretinal lymphomas, often masquerades as uveitis. This misdiagnosis can result in subsequent brain involvement and oculocerebral lymphoma (OCL). In this study, we sought to characterize the helper T-cell type 1 (Th1)/Th2 cytokine profile in vitreous samples from patients with PIOL, OCL, uveitis and controls with non-inflammatory disease. Vitreous and aqueous humor samples from 87 patients with PIOL (n = 30), OCL (n = 12), uveitis (n = 34), and retinal detachment (RD) without hemorrhage (n = 11) were analyzed and their concentrations of interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α were determined by flow cytometric bead arrays (CBA). The IL-10 levels determined by CBA were compared with those by ELISA. IL-10 concentrations measured by CBA and ELISA were highly correlated. IL-2, IL-4, and TNFα were not detected in any sample. The only cytokine detected at a significant level in samples from RD vitreous was IL-6. The IL-10/IL-6 ratio, as previously reported, was slightly higher in PIOL than in uveitis samples, but not for all patients. Cytokine profiles from PIOL and OCL samples did not differ. The combination of the IL-10/IL-6 and IL-10/IFNγ ratios was highly informative for discriminating PIOL/OCL from uveitis samples and for therapeutic follow up of PIOL. This strategy might be very helpful as an initial screening to rule out PIOL in patients thought to have uveitis.

Preclinical study of Ublituximab, a Glycoengineered anti-human CD20 antibody, in murine models of primary cerebral and intraocular B-cell lymphomas.

PURPOSE: Primary cerebral lymphoma (PCL) and primary intraocular lymphoma (PIOL) belong to the systemic diffuse large B-cell lymphoma family and are characterized by the presence of CD20(+) lymphoma B cells in the brain or the eye. These highly aggressive malignancies have a poor prognosis and no specific therapy. The presence of effector immune cells in the damaged brain and vitreous suggests that treatment with anti-human CD20 (hCD20) monoclonal antibodies might be effective. We developed murine models of PCL and PIOL to assess the intracerebral and intraocular antitumor effect of ublituximab, a promising glycoengineered anti-hCD20 mAb with a high affinity for FcγRIIIa (CD16) receptors. METHODS: The murine lymphoma B-cell line A20.IIA-GFP-hCD20 (H-2(d)) was injected into the right cerebral striatum or the vitreous of immunocompetent adult BALB/c mice (H-2(d)). Four to 7 days later, ublituximab was injected intracerebrally or intravitreously into the tumor site. Rituximab was the reference compound. Survival was monitored for injected mice; histopathological and flow cytometric analyses were performed to study tumor growth and T-cell infiltration. RESULTS: Single doses of ublituximab, injected intracerebrally or intravitreously, had a marked antitumor effect, more pronounced than that obtained with the same dose of rituximab in these conditions. The reduction in tumor cells was correlated with an increased proportion of CD8(+) T cells. This efficacy was observed only against lymphoma B cells expressing hCD20. CONCLUSIONS: These in vivo results confirm the potential of the glycoengineered anti-hCD20 mAb ublituximab as an innovative therapeutic approach to treat primary central nervous system lymphoma and other B-cell lymphomas.