Semiquantitative assessment of the microdistribution of fluorescence-labeled monoclonal antibody in small peritoneal disseminations of ovarian cancer

Abstract

Uniform antibody microdistribution throughout tumor nodules is crucial for antibody-targeted therapy, because non-uniform microdistribution leads to suboptimal therapeutic effect, a commonly observed limitation of therapeutic antibodies. Herein, we evaluated the microdistribution of different doses of intraperitoneally injected fluorescence-labeled full-antibody trastuzumab (15, 50, and 150 microg) and its Fab fragment (trastuzumab-Fab: 15 and 50 microg) in a mouse model of ovarian cancer with peritoneal disseminated tumor. A semiquantitative approach (central/peripheral accumulation ratio; C/P ratio) was developed using in situ fluorescence microscopy. Furthermore, we compared the microdistribution of intact trastuzumab with a mixed injection of trastuzumab and trastuzumab-Fab or serial injections of trastuzumab using in situ multicolor fluorescence microscopy. Fluorescence images after the administration of 15 or 50 microg trastuzumab and 15 microg trastuzumab-Fab demonstrated antibody accumulation in the tumor periphery, whereas administration of 150 microg trastuzumab and 50 microg trastuzumab-Fab showed relatively uniform accumulation throughout the tumor nodule. Using serial injections (19-h interval) of trastuzumab-rhodamine green and carboxytetramethylrhodamine (TAMRA), it was observed that the latterly injected trastuzumab-TAMRA was distributed more centrally than trastuzumab-rhodamine green injected first, whereas no difference was observed in the control mixed-injection group. Moreover, the mixed injection of trastuzumab and trastuzumab-Fab showed that trastuzumab-Fab distributed more centrally than the same amount of co-injected trastuzumab. Our results suggest that the strategies of increasing dose and using Fab fragments can be used to achieve a uniform antibody distribution within peritoneal disseminated nodules after intraperitoneal injection. Furthermore, serial-injection and mixed-injection strategies can modify antibody microdistribution within tumors and have the potential for preferential delivery of anticancer drugs to either the tumor periphery or its center.

Figure 1

Image analysis procedures for central/peripheral accumulation ratio (C/P ratio) calculations. (a) A linear region of interest (ROI) is placed on fluorescence images of each tumor nodule along its long axis based on the transmitted light differential interference contrast (DIC) images. (b) Linear ROI are divided evenly into three portions and the signal intensities of each pixel are measured. Mean signal intensities are calculated in each portion (P1, C, and P2). Finally, a C/P ratio is calculated using the formula described in the Materials and Methods section.

Figure 2

In situ fluorescence microscopic images of the (a) trastuzumab‐injected and (b) trastuzumab‐Fab‐injected groups, and (c) central/peripheral accumulation ratios (C/P ratios) (means and SD) in each group. Fluorescence images of the 15‐μg trastuzumab, 50‐μg trastuzumab, and 15‐μg trastuzumab‐Fab groups show peripheral dominant antibody accumulation, whereas the 150‐μg trastuzumab and 50‐μg trastuzumab‐Fab groups show relatively homogenous accumulation throughout tumors. C/P ratios demonstrate that the 150‐μg trastuzumab group has the highest among all groups. The 50‐μg trastuzumab‐Fab group also shows a higher C/P ratio than the other three groups (15‐μg trastuzumab, 50‐μg trastuzumab, and 15‐μg trastuzumab‐Fab groups), and these three groups show no significant difference. Scale bars on DIC images = 500 μm. *P < 0.05, **P < 0.001, ***P < 0.0001.

Figure 3

In situ fluorescence microscopic images of the (a) serial‐injection and (b) mixed‐injection groups of trastuzumab‐rhodamine green (RhodG) and ‐carboxytetramethylrhodamine (TAMRA). Scatter plots of central/peripheral accumulation ratios (C/P ratios) in each group are also shown: (c) interval‐injection group; (d) non‐interval‐injection group. The C/P ratios of each set of tumor nodules are connected in scatter plots. A composite image of the serial‐injection group clearly shows that the secondarily injected trastuzumab‐RhodG (green) distributes more centrally than trastuzumab‐TAMRA (red), which was injected first. C/P ratios demonstrate that the second injection of trastuzumab‐RhodG demonstrates a statistically higher C/P ratio than trastuzumab‐TAMRA, which was injected first. In contrast, the mixed‐injection group shows the same accumulation patterns between the two trastuzumabs in both fluorescence images and C/P ratio analysis. Scale bars on DIC images = 500 μm. ***P < 0.0001, NS, not significant.

Figure 4

(a) In situ fluorescence microscopic images and (b) scatter plot of central/peripheral accumulation ratios (C/P ratios) in mixed injections of trastuzumab and trastuzumab‐Fab. The C/P ratios of the same tumor nodules are connected in the scatter plots. Both fluorescence images and C/P ratio analysis show that trastuzumab‐Fab distributed more centrally than co‐injected trastuzumab. Comparison of C/P ratios of trastuzumab‐Fab with or without co‐injection of trastuzumab (c, also see Fig. 2) shows that co‐injection of trastuzumab alters the trastuzumab‐Fab accumulation so that it has a more central distribution pattern. Scale bars on DIC images = 500 μm. ***P < 0.0001.