Comparison between Fractionated Dose and Single Dose of Cu-64 Trastuzumab Therapy in the NCI-N87 Gastric Cancer Mouse Model

Abstract

For optimum radioimmunotherapy (RIT), deep penetration and uniform distribution into the tumor core is important. The solid tumor microenvironment, consisting of a highly fibrotic or desmoplastic tumor, abnormal tumor vasculature, high fluid pressure, and the absence of fluid lymphatics, limits the distribution of monoclonal antibodies mAbs to the tumor core. To investigate the optimal rationale for therapeutic mAbs administration and the microdistribution of mAbs, single and serial fractional dosage regimens of Cu-64-trastuzumab (TRZ) with paclitaxel were evaluated. Groups of nude mice were inoculated with gastric cancer cell line NCI-N87 tumor cells. When the tumor size reached 200 ± 20 mm³, the mice were divided into two groups for injection of Alexa-647-TRZ. One group (n = 5) was injected with 15 mg/kg in a single dose (SD), and the other group (n = 5) with two doses of 7.5 mg/kg (fractionated dose (FD)). In both cases, the injections were done intravenously in combination with intraperitoneal paclitaxel either as a SD of 70 mg/kg or fractionated into two doses of 40 and 30 mg/kg. Tumors were harvested, flash frozen, and sectioned (8 µm) five days after Alexa-647-TRZ injection. Rhodamine lectin (rhodamine-labeled Ricinus communis agglutinin I, 1 mg in 0.2 mL of phosphate-buffered saline (PBS)) was intravenously injected to delineate the functional vessel for a wait time of 5 min before animal euthanization. Microscopic images were acquired with an IN Cell Analyzer. The amount of TRZ that penetrated the tumor surface and the tumor vessel was calculated by area under the curve (AUC) analysis. For RIT efficacy (n = 21), Cu-64-TRZ was injected following the same dose schedule to observe tumor volume and survival ratio for 30 days. The SD and FD regimens of Alexa-647-TRZ were observed to have no significant difference in penetration of mAbs from the tumor edge and vessel, nor was the total accumulation across the whole tumor tissue significantly different. Additionally, the SD and FD regimens of Cu-64-TRZ were not proven to be significantly efficacious. Our study reveals that SD and FD in a treatment design with Cu-64-TRZ and paclitaxel shows no significant difference in therapeutic efficacy on tumor growth inhibition in vivo in mice bearing human gastric cancer xenografts overexpressing HER2 antigen.

Keywords: Herceptin; fractional dose (FD); gastric cancer; penetration; radioimmunotherapy; single dose (SD); therapeutics; trastuzumab.

Figure 1

Schematic design of combination therapy by evaluating drug delivery and efficacy. (a) Single dose (SD) and fractionated dose (FD) of combination therapy were injected into NCI-N87 xenograft-bearing mice, and penetration of mAb was evaluated from the vessel, near the edge, and total accumulation across the tumor. (b) Experimental schedule for Alexa-647-congugated TRZ and Cu-64-labeled-TRZ. (c) SD and FD dosage regimens. Cu-64-TRZ, Cu-64-Trastuzumab; Alexa-647-TRZ, Alexa-647-Trastuzumab; PTX, paclitaxel. TRX, trastuzumab.

Figure 1

Schematic design of combination therapy by evaluating drug delivery and efficacy. (a) Single dose (SD) and fractionated dose (FD) of combination therapy were injected into NCI-N87 xenograft-bearing mice, and penetration of mAb was evaluated from the vessel, near the edge, and total accumulation across the tumor. (b) Experimental schedule for Alexa-647-congugated TRZ and Cu-64-labeled-TRZ. (c) SD and FD dosage regimens. Cu-64-TRZ, Cu-64-Trastuzumab; Alexa-647-TRZ, Alexa-647-Trastuzumab; PTX, paclitaxel. TRX, trastuzumab.

Figure 2

Representative images of SD and FD. (a) DAPI (4′,6-diamidino-2-phenylindole) in blue, TRZ (trastuzumab) intensity in green, functional vessel in red, merged images, and zoomed merged area near vessel. (b) Histogram plot shows line profiling comprising mean and standard deviation as error bars (above) of SD (single dose) and FD (fractional dose) with respective AUC values.

Figure 3

Representative images of SD (single dose) and FD (fractional dose). (a) DAPI in blue, TRZ (trastuzumab) intensity in green, functional vessel in red, merged images, and zoomed merged area near tumor edge. (b) Histogram plot shows line profiling comprising mean and standard deviation as error bars (above) of SD and FD with respective AUC values.

Figure 4

Representative images of SD (single dose) and FD (fractional dose). (a) DAPI in blue, TRZ (trastuzumab) intensity in green, functional vessel in red, merged images showing accumulation of TRZ across the whole tumor. (b) Segmented TRZ image and polyline traced across the tumor surface. For intensity quantification, colored images from ZEN blue were traced with polyline, and (c) mAb accumulation per tumor area was plotted as a bar graph. Data shown as means and standard deviation as error bar. n.s., nonsignificant.

Figure 4

Representative images of SD (single dose) and FD (fractional dose). (a) DAPI in blue, TRZ (trastuzumab) intensity in green, functional vessel in red, merged images showing accumulation of TRZ across the whole tumor. (b) Segmented TRZ image and polyline traced across the tumor surface. For intensity quantification, colored images from ZEN blue were traced with polyline, and (c) mAb accumulation per tumor area was plotted as a bar graph. Data shown as means and standard deviation as error bar. n.s., nonsignificant.

Figure 5

(a) Plot of tumor data showing means of tumor volume for each group with standard deviations as error bars (above). (b) Kaplan–Meier curves for survival data of FD (fractional dose) and SD (single dose) were recorded up to 30 days. The data are statistically nonsignificant.

Figure 5

(a) Plot of tumor data showing means of tumor volume for each group with standard deviations as error bars (above). (b) Kaplan–Meier curves for survival data of FD (fractional dose) and SD (single dose) were recorded up to 30 days. The data are statistically nonsignificant.