Multi-targeted multi-color in vivo optical imaging in a model of disseminated peritoneal ovarian cancer

Abstract

Commonly used in flow cytometry, multiplexed optical probes can diagnose multiple types of cell surface marker, potentially leading to improved diagnosis accuracy in vivo. Herein, we demonstrate the targeting of two different tumor markers in models of disseminated ovarian cancer. Two ovarian cancer cell lines (SKOV3 and SHIN3) were employed; both overexpress D-galactose receptor (D-galR), but only SKOV3 overexpresses HER2/neu. Additionally, fusion tumors composed of SKOV3 and SHIN3/RFP were evaluated. Both galactosyl serum albumin-rhodamine green (GSA-RhodG), which binds D-galR, and trastuzumab-Alexa680, which binds HER2/neu, were administered to tumor-bearing mice for in vivo fluorescence imaging and in situ fluorescence microscopy. In vivo fluorescence imaging depicted 64 of 69 SKOV3 tumors (94.2%) based on their dual spectra corresponding to both RhodG and Alexa680, while all 71 SHIN3 tumors (100%) were detected based on their single spectrum corresponding only to RhodG. All 59 SHIN3 and 36 SKOV3 tumors were correctly diagnosed with in situ microscopy. Additionally, in the mixed tumor model, all tumors could be depicted using the RhodG spectrum, but only SKOV3 components also showed the Alexa680 spectrum. In conclusion, multitargeted multicolor optical imaging enabled specific in vivo diagnosis of tumors expressing distinct patterns of receptors, leading to improved diagnostic accuracy.

Fig. 1

Fluorescence microscopic images of SHIN3, SKOV3, and co-culture of SHIN3/RFP and SKOV3 treated with the both GSA-RhodG (3 μg/mL) and trastuzumab (Tras)-Alexa680 (30 μg/mL). Camera exposure times were 200 ms for GSA-RhodG, and 1 s for RFP and trastuzumab-Alexa680. (A) Fluorescence microscopic images of SHIN3 at 1 hr shows binding of GSA-RhodG to SHIN3 cells as peripheral small fluorescent dots. After 8 hrs, the small dots spread homogenously throughout the cytoplasm and persisted at least 24 hrs. In contrast, trastuzumab-Alexa680 does not bind to SHIN3 cells at any time points. (B) SKOV3 cells show the binding of trastuzumab-Alexa680 to the HER2/neu receptor as rim-like fluorescence on the cell surface at 1hr. With time, the complexes are internalized into the cell, and can be identified as small fluorescent dots at 8 hrs which persist at least 24 hrs. Also, GSA-RhodG binds to SKOV3 cells with the similar binding pattern to SHIN3 cells. (C) In the co-culture of SHIN3/RFP and SKOV3, SKOV3 cells are depicted by both GSA-RhodG and trastuzumab-Alexa680, while SHIN3/RFP cells are depicted by only GSA-RhodG. This reveals that in vitro dual-targeting of SKOV3 is feasible even when two cell lines are co-cultured.

Fig. 2

In vivo multi-color spectral fluorescence images of single types of cells (either SHIN3 or SKOV3) exposed to both GSA-RhodG and trastuzumab (Tras)-Alexa680. White scale bars on GSA-RhodG spectral images indicate 5 mm. (A) In the SHIN3 group, the nodules depicted by GSA-RhodG spectral images do not appear on the trastuzumab-Alexa680 spectral images. (B) In the SKOV3 group, most of the nodules depicted on GSA-RhodG spectral images are also depicted on the trastuzumab-Alexa680 spectral images. However, in the upper image, the nodules depicted on GSA-RhodG spectral images are not shown on the trastuzumab-Alexa680 spectral images (arrows), resulting in examples of failure to label both targets of SKOV3.

Fig. 3

In situ fluorescence microscopic images with single-injected tumor model of SHIN3 and SKOV3 treated with the both GSA-RhodG and trastuzumab (Tras)-Alexa680. Camera exposure times were 1 s for all fluorescence images. Black scale bars on DIC images indicate 0.5 mm. (A) Tumor nodules derived from SHIS3 cells are depicted by GSA-RhodG, while these are not depicted by trastuzumab-Alexa680. (B) Tumor nodules derived from SKOV3 cells are depicted by both GSA-RhodG and trastuzumab-Alexa680.

Fig. 4

In vivo multi-color spectral fluorescence images and in situ fluorescence microscopy with the coincident tumor model of SKOV3 and SHIN3/RFP exposed to both GSA-RhodG and trastuzumab (Tras)-Alexa680. Camera exposure times were 1 s for all fluorescence microscopic images. (A) In vivo multi-color spectral fluorescence images shows that all tumor nodules are depicted on GSA-RhodG spectral images, while only SKOV3 lesions, which do not have RFP labeling, are depicted on trastuzumab-Alexa680 spectral images. Thus, SKOV3 lesions are easily identified on the composite image even when they are mixed with other lesions. (B) In situ fluorescence microscopic images completely correspond to the result of in vivo multi-color spectral fluorescence images.