Fluorescent nanoparticle uptake for brain tumor visualization

Abstract

Accurate delineation of tumor margins is vital to the successful surgical resection of brain tumors. We have previously developed a multimodal nanoparticle CLIO-Cy5.5, which is detectable by both magnetic resonance imaging and fluorescence, to assist in intraoperatively visualizing tumor boundaries. Here we examined the accuracy of tumor margin determination of orthotopic tumors implanted in hosts with differing immune responses to the tumor. Using a nonuser-based signal intensity method applied to fluorescent micrographs of 9L gliosarcoma green fluorescent protein (GFP) tumors, mean overestimations of 2 and 24 microm were obtained using Cy5.5 fluorescence, compared to the true tumor margin determined by GFP fluorescence, in nude mice and rats, respectively. To resolve which cells internalized the nanoparticle and to quantitate degree of uptake, tumors were disaggregated and cells were analyzed by flow cytometry and fluorescence microscopy. Nanoparticle uptake was seen in both CD11b+ cells (representing activated microglia and macrophages) and tumor cells in both animal models by both methods. CD11b+ cells were predominantly found at the tumor margin in both hosts, but were more pronounced at the margin in the rat model. Additional metastatic (CT26 colon) and primary (Gli36 glioma) brain tumor models likewise demonstrated that the nanoparticle was internalized both by tumor cells and by host cells. Together, these observations suggest that fluorescent nanoparticles provide an accurate method of tumor margin estimation based on a combination of tumor cell and host cell uptake for primary and metastatic tumors in animal model systems and offer potential for clinical translation.

Figure 1

CLIO-Cy5.5 nanoparticle as a preoperative MRI and as an intraoperative optical contrast agent. (A) A T₁-weighted image 10 minutes after gadolinium injection and 24 hours after CLIO-Cy5.5 injection, and an identically located T₂-weighted image in the same rat taken 24 hours after CLIO-Cy5.5 injection but before gadolinium injection. (B) Magnification of tumor region shown in (A), with arrows highlighting tumor borders. The CLIO-contrasted tumor is mottled, with a dark clearly defined margin on T₂-weighted imaging. (C). Optical images of mouse brain and rat brain after craniotomy. From left to right, the images are white light, GFP fluorescence, and CLIO-Cy5.5 fluorescence. Bar = 5 mm.

Figure 2

Flow cytometry of CLIO-Cy5.5 in cells from the 9L gliosarcoma. (A) A tumor-bearing animal was sacrificed 24 hours after injection with CLIO-Cy5.5, and the tumor was dissected and disaggregated. A dot plot showing three distinct cell populations is shown in the upper right panel. Populations were as follows: CD11b⁺/GFP^- (I); CD11b^-/GFP^-, DN, (II); and CD11b^-/GFP⁺ (III). The uptake of CLIO-Cy5.5 as Cy5.5 fluorescence was analyzed in each of the three populations that are evident on the dot plot. CD11b⁺ (I) and GFP⁺ (III) cells internalized the nanoparticle as illustrated by the shift of the peaks. However, the DN population did not take up CLIO-Cy5.5. (B) The percentages of cells (left) and the relative median fluorescence (CLIO-Cy5.5 uptake; right) are given for each population of cells in both animal models.

Figure 3

CLIO-Cy5.5 uptake in cells from the 9L gliosarcoma. Activated microglia/macrophages were detected with R-phycoerythrin-labeled anti-CD11b (blue) in the rat host. Cells were disaggregated from intact tumors 24 hours after intravenous administration of CLIO-Cy5.5. For a CD11b⁺ disaggregated cell, CD11b fluorescence (blue; A), CLIO-Cy5.5 fluorescence (red; B), and a merged image (C) are shown. For a GFP⁺ disaggregated cell, GFP fluorescence (green; D), CLIO-Cy5.5 fluorescence (E), and a merged image (F) are shown. The nanoparticle appears to be in cytoplasmic vesicles within disaggregated cells and is in both tumor and CD11b⁺ cells. Bar = 25 µm.

Figure 4

Tumor border determination using CLIO-Cy5.5. (A) Tumor border determined using signal intensity measurements. The border was determined using Eq. 1 (9L gliosarcoma/nude mouse host) based on GFP fluorescence (green boundary) or CLIO-Cy5.5 fluorescence (red boundary). GFP fluorescence is shown in gray. (B) Accuracy of tumor border by CLIO-Cy5.5 fluorescence. The Cy5.5 border extension beyond the GFP border is defined as overestimation; the reverse is defined as underestimation. The majority of measurements between the two borders were close to zero for both rat and nude mouse models. (C) Accuracy of border determination by CLIO-Cy5.5 fluorescence. The standard deviation (SD), standard error of the mean (SEM), maximum overestimation, and maximum underestimation are given. (D) Etiology of slight differences in margin determination between models. Fluorescent microscopy micrograph of a 20-µm-thick brain slice labeled with anti-CD11b antibody for microglia/macrophage staining (blue); tumor cells are in green, CLIO-Cy5.5 is in red, and normal brain is in black. (D) Inset: Accumulation of CLIO-Cy5.5 inside CD11b⁺ cells (microglia/macrophages). The nanoparticle localizes at the periphery of the tumor inside microglia/macrophages and in the tumor inside tumor cells and microglia/macrophages. The slight overestimation in rat brains corresponds to the nanoparticle localized around the tumor inside microglia/macrophages.

Figure 5

Distribution of microglia/macrophages and astrocytes with the 9L gliosarcoma. (A) Fluorescence from anti-CD11b antibody stain (microglia/macrophages) in the rat. (B) Fluorescence from anti-CD11b antibody stain in the nude mouse. CD11b⁺ cells form a ring-like pattern around the tumor that is more pronounced in the rat than in the nude mouse. (C) Tumor margin in the rat host after staining with anti-GFAP, a marker of astrocytes. Astrocytes are in blue, CLIO-Cy5.5is in red, and tumor GFP is in green. Astrocytes lack CLIO-Cy5.5 and are not present in the tumor. (D) Tumor GFP fluorescence at low magnification. (E) Anti-GFAP fluorescence of astrocytes at low magnification. The antibody was Cy3-labeled. (F) Color merge of (D) and (E). Astrocytes do not centrally infiltrate brain tumor and do not internalize CLIO-Cy5.5. Bar = 200 µm.

Figure 6

CLIO-Cy5.5 uptake by tumor and host cells in primary and metastatic tumor models. (A) Nonfluorescent CT26 colon carcinoma implanted in GFP-expressing mouse, injected with CLIO-Cy5.5, and imaged. CLIO-Cy5.5 (red) is found in GFP (green)-expressing cells (host) and in regions of no GFP (tumor cells). (B) Gli36 GFP tumor (green) shows CLIO-Cy5.5 fluorescence (red) associated with GFP, demonstrating tumoral uptake. CLIO-Cy5.5 nanoparticles that do not colocalize with tumor cells are in CD11b⁺ cells. (C) The center of the 9L tumor expressing GFP. Tumor cells are in green, CLIO-Cy5.5 is in red, and microglia/macrophages are in blue. The arrow points to the colocalization of the CLIO-Cy5.5 with microglia/macrophages, and arrowheads point to colocalization with tumor cells.