Multicolor in vivo targeted imaging to guide real-time surgery of HER2-positive micrometastases in a two-tumor coincident model of ovarian cancer

Abstract

One of the primary goals of oncological molecular imaging is to accurately identify and characterize malignant tissues in vivo. Currently, molecular imaging relies on targeting a single molecule that while overexpressed in malignancy, is often also expressed at lower levels in normal tissue, resulting in reduced tumor to background ratios. One approach to increasing the specificity of molecular imaging in cancer is to use multiple probes each with distinct fluorescence to target several surface antigens simultaneously, in order to identify tissue expression profiles, rather than relying on the expression of a single target. This next step forward in molecular imaging will rely on characterization of tissue based on fluorescence and therefore will require the ability to simultaneously identify several optical probes each attached to different targeting ligands. We created a novel 'coincident' ovarian cancer mouse model by coinjecting each animal with two distinct cell lines, HER2+/red fluorescent protein (RFP)- SKOV3 and HER2-/RFP+ SHIN3-RFP, in order to establish a model of disease in which animals simultaneously bore tumors with two distinct phenotypes (HER2+/RFP-, HER2-/RFP+), which could be utilized for multicolor imaging. The HER2 receptor of the SKOV3 cell line was targeted with a trastuzumab-rhodamine green conjugate to create green tumor implants, whereas the RFP plasmid of the SHIN3 cells created red tumor implants. We demonstrate that real-time in vivo multicolor imaging is feasible and that fluorescence characteristics can then serve to guide the surgical removal of disease.

Figure 1

In vitro validation of targeting two ovarian cancer cell lines with different fluorescence signals. (a) Flow cytometry data confirming the presence of cell populations with distinct fluorescence, SHIN3‐red fluorescent protein (RFP) and SKOV3. (b) Fluorescence microscopy utilizing filters specific for red and green wavelengths confirmed the presence of two distinct cell populations. The red fluorescence is secondary to RFP expression from the SHIN3‐RFP cell line, whereas the green fluorescence is due to trastuzumab–rhodamine green (RhodG) binding epidermal growth factor receptor 2 receptors of the SKOV3 cell line. DIC, differential interference contrast.

Figure 2

Establishment of a coincident ovarian cancer model in mice for multicolor optical molecular imaging. Fluorescence microscopy verifying the presence of both SHIN3‐red fluorescent protein (RFP)‐derived and SKOV3‐derived peritoneal tumors within the mesentery of the coincident tumor model mice treated with intraperitoneal trastuzumab–rhodamine green (RhodG). Fluorescence microscopy permitted visualization of red fluorescence signal from the SHIN3‐RFP tumors as well as green fluorescence from trastuzumab–RhodG binding to epidermal growth factor receptor 2 receptors of SKOV3 tumor implants and therefore confirmed the ability of the dual‐injection method to establish a coincident tumor model. DIC, differential interference contrast.

Figure 3

(a) Loops of bowel with mesenteric tumor implants were imaged with real‐time multicolor imaging (left) and multispectral imaging (right). SHIN3‐red fluorescent protein (RFP) tumor implants demonstrated red fluorescence (arrows), whereas SKOV3 tumor implants demonstrated green fluorescence (arrowheads) from trastuzumab–rhodamine green (RhodG) binding to epidermal growth factor receptor 2 receptors. Asterisks (*) indicate the intestinal loop. (b) Multispectral images of mesenteric tumor implants resected using real‐time multicolor imaging as a guide. Using real‐time multicolor imaging, nodules were surgically removed and organized according to fluorescence pattern. All nodules demonstrating any green fluorescence (with or without red fluorescence) as seen with real‐time multicolor imaging were placed in the right column. All nodules demonstrating only red fluorescence were placed in the left column. Multispectral imaging was then used to evaluate the accuracy of tissue fluorescence characterization obtained with real‐time multicolor imaging (also see Table 1).

Figure 4

Multispectral images of in vivo mesenteric tumor implants. Multispectral images of in vivo mesenteric tumor implants, which were resected with real‐time multicolor image‐guided surgery from the abdominal space in five coincident tumor‐bearing living mice (see Supporting video S2). Wavelength‐specific images unmixed for red fluorescent protein (RFP) and trastuzumab–rhodamine green (RhodG) demonstrated that mesenteric tumor implants from individual mice consisted of both SHIN3‐RFP and SKOV3 bound to trastuzumab–RhodG (see also Table 2). HER2, epidermal growth factor receptor 2.