2.1.

Cell Lines and Culture

Two established human ovarian cancer cell lines (SKOV3 and SHIN3) were used in this study. SKOV3 overexpresses HER2/neu,⁹ but both express D-galR.¹⁰ SHIN3 cells were transfected with a plasmid expressing red fluorescent protein (RFP) to create a red fluorescent phenotype (SHIN3/RFP) that permitted optical identification of SHIN3-derived tumor implants, as previously described.¹¹ All cell lines were grown in RPMI 1640 medium (Invitrogen Corporation, Carlsbad, CA) containing 10% fetal bovine serum [(FBS), Invitrogen Corporation], 0.03% L-glutamine at $37°\mathrm{C}$ , $100\text{units}∕\mathrm{mL}$ penicillin, and $100\mu \mathrm{g}∕\mathrm{mL}$ streptomycin in 5% $\mathrm{C}{\mathrm{O}}_2$ .

2.2.

Synthesis of Rhodamine Green-Conjugated GSA

GSA was purchased from Sigma Chemical (St. Louis, MO), amino-reactive Rhodamine Green (RhodG-NHS) was purchased from Invitrogen Corporation. At room temperature, $1\mathrm{mg}$ $\left(13\mathrm{nmol}\right)$ of GSA in $0.1\mathrm{M}$ ${\mathrm{Na}}_2\mathrm{H}\mathrm{P}{\mathrm{O}}_4$ was incubated with $130\mathrm{nmol}$ of RhodG-NHS, at pH 8.5 for $30\min$ . The mixture was purified with a gel filtration column (Sephadex G50 column, PD-10; GE Healthcare, Piscataway, NJ) and the GSA binding fraction $(2.7–4.5\mathrm{mL})$ was eluted by $0.066\mathrm{M}$ PBS at pH 7.4 and collected in a test tube. GSA-RhodG was kept at $4°\mathrm{C}$ in the refrigerator as stock solutions. The protein concentrations of GSA-RhodG samples were determined with the Coomassie Plus protein assay kit (Pierce Biotechnology, Rockford, IL) by measuring the absorption at $595\mathrm{nm}$ with a ultraviolet-visible (UV-vis) system (8453 Value UV-Visible Value System, Agilent Technologies, Santa Clara, CA) using standard solutions of known concentrations of GSA $(100\mu \mathrm{g}∕\mathrm{mL})$ . The concentration of RhodG was then measured by absorption at $503\mathrm{nm}$ with the UV-vis system to confirm the number of fluorophore molecules conjugated to each GSA molecule. The number of fluorophore molecules per GSA was adjusted to approximately 3–3.5.

2.3.

Synthesis of Rhodamine Green and Alexa680-Conjugated Trastuzumab

Trastuzumab (Herceptin®), an FDA-approved humanized anti-HER2 antibody, which has a complimentary determination region against HER2 grafted on a human IgG1 framework, was purchased from Genentech Inc. (San Francisco, CA). RhodG-NHS and Alexa680-NHS were purchased from Invitrogen Corporation. At room temperature, $1\mathrm{mg}$ $\left(6.8\mathrm{nmol}\right)$ of trastuzumab in $0.1\mathrm{M}$ ${\mathrm{Na}}_2\mathrm{H}\mathrm{P}{\mathrm{O}}_4$ was incubated with $68\mathrm{nmol}$ of RhodG-NHS or Alexa680-NHS, at pH 8.5 for $30\min$ . The mixture was purified with a gel filtration column (Sephadex G50 column, PD-10; GE Healthcare, Piscataway, NJ) and the antibody binding fraction $(2.7–4.5\mathrm{mL})$ was eluted by $0.066\mathrm{M}$ PBS at pH 7.4 and collected in a test tube. Trastuzumab-RhodG and trastuzumab-A680 were kept at $4°\mathrm{C}$ as stock solutions. The protein concentrations of samples were determined with the Coomassie Plus protein assay kit (Pierce Biotechnology, Rockford, IL) by measuring the absorption at $595\mathrm{nm}$ with a UV-vis system (8453 Value UV-Visible Value System, Agilent Technologies) using standard solutions of known concentrations of trastuzumab $(200\mu \mathrm{g}∕\mathrm{mL})$ . The concentration of RhodG and Alexa680 was then measured by absorption at 503 and $679\mathrm{nm}$ , respectively, with the UV-vis system to confirm the number of fluorophore molecules conjugated to each trastuzumab molecule. The number of fluorophore molecules per trastuzumab was adjusted to approximately 3–3.5.

2.4.

In Vitro Fluorescence Microscopy

Fluorescence microscopy was performed with an Olympus BX61 microscope (Olympus America, Inc., Melville, NY) equipped with the following filters: for the blue light filter, a bandpass filter from $470\text{to}490\mathrm{nm}$ and a bandpass filter from $515\text{to}550\mathrm{nm}$ were used for excitation and emission light, respectively; for the green light filter, the values were from $530\text{to}585\mathrm{nm}$ and from $605\text{to}680\mathrm{nm}$ , respectively, and for the red light filter, the values were from $590\text{to}650\mathrm{nm}$ and from $665\text{to}740\mathrm{nm}$ , respectively. Transmitted light differential interference contrast (DIC) images were also acquired.

Either SHIN3 cells $(1\times {10}^4)$ or SKOV3 cells $(1\times {10}^4)$ were plated on a cover glass–bottomed culture well and incubated at $37°\mathrm{C}$ in an atmosphere of 5% $\mathrm{C}{\mathrm{O}}_2$ for $16\mathrm{h}$ . GSA-RhodG $(3\mu \mathrm{g}∕\mathrm{mL})$ , trastuzumab-Alexa680 $(30\mu \mathrm{g}∕\mathrm{mL})$ , or both were added to the culture media. The cells were incubated and removed at the following time points: 1, 8, $24\mathrm{h}$ . Following removal, the cells were washed once with PBS, followed by fluorescence microscopy with the blue and red filter sets. To better localize the optical probes intracellularly, we also performed a colocalization study, adding $75\mathrm{nM}$ of the lysosomal marker (LysoTracker Red DND-99, Molecular Probe Inc., Eugene, OR) $1\mathrm{h}$ prior to imaging SKOV3 cells treated with either GSA-RhodG or trastuzumab-RhodG. LysoTracker selectively accumulates in the acidic compartments of the cell (i.e., the lysosome) and emits red fluorescence.¹² Fluorescence microscopy was performed with the blue, green, and red light filters at $1\text{-}\mathrm{h}$ and $8\text{-}\mathrm{h}$ time points.

To demonstrate dual targeting in vitro, a coculture of SKOV3 and SHIN3/RFP cells was created. Both SHIN3-RFP cells $(1\times {10}^4)$ and SKOV3 cells $(1\times {10}^4)$ were plated on a cover glass–bottomed culture well and incubated at $37°\mathrm{C}$ in a 5% $\mathrm{C}{\mathrm{O}}_2$ atmosphere for $16\mathrm{h}$ . Both GSA-RhodG $(3\mu \mathrm{g}∕\mathrm{mL})$ and trastuzumab-Alexa680 $(30\mu \mathrm{g}∕\mathrm{mL})$ were added to the culture media. The cells were incubated and removed at 1 and $8\mathrm{h}$ . Following removal, cells were washed once with PBS and fluorescence microscopy was immediately performed with the blue, green, and red light filters.

2.5.

Tumor Model

All procedures were approved by the National Cancer Institute Animal Care and Use Committee. The tumor implants were established by intraperitoneal injection of $2\times {10}^6$ cells suspended in $200\mu \mathrm{L}$ of PBS in female nude mice (National Cancer Institute Animal Production Facility, Frederick, MD). Experiments with tumor-bearing mice were performed at $14\text{days}$ for the SHIN3 mouse model and $35\text{days}$ for the SKOV3 mouse model. Also, to establish a coincident tumor model, another group of mice received the intraperitoneal injection of SKOV3 cells ( $2\times {10}^6$ cells) followed seven days later by an intraperitoneal injection of SHIN3/RFP cells ( $2\times {10}^6$ cells). Experiments with SKOV3 and SHIN3/RFP tumor-bearing mice were performed at $30\text{days}$ after injection of the SKOV3 cells.

2.6.

In Vivo Spectral Fluorescence Imaging

After establishing the intraperitoneal dissemination model, groups of mice (four per group) with SHIN3, SKOV3, or the coincident model of SHIN3/RFP and SKOV3 were injected i.p. with $50\mu \mathrm{g}$ of trastuzumab-Alexa680 diluted with $300\mu \mathrm{L}$ PBS, $24\mathrm{h}$ prior to imaging, followed by $25\mu \mathrm{g}$ of GSA-RhodG diluted with $300\mu \mathrm{L}$ PBS, $4\mathrm{h}$ prior to imaging. Mice were sacrificed with carbon dioxide, and then the abdominal cavities were exposed and the peritoneal membranes were spread on nonfluorescent black plates. Spectral fluorescence images were obtained using the Maestro In Vivo imaging system (CRi, Inc., Woburn, MA). Magnified multicolor spectral fluorescence images of the peritoneal membranes were obtained with a multiexcitation acquisition¹³ using the blue-filter setting (excitation filter: $445–490\mathrm{nm}$ , emission filter: $515\mathrm{nm}$ long pass) and the red-filter setting ( $615–655\mathrm{nm}$ , $700\mathrm{nm}$ long pass) for the single-injected model, and the blue-filter, green-filter ( $503–555\mathrm{nm}$ , $580\mathrm{nm}$ long pass), and red-filter settings for the coincident tumor (SKOV3 and SHIN3/RFP) model. The tunable filter was automatically stepped in $10\text{-}\mathrm{nm}$ increments from $500\text{to}800\mathrm{nm}$ for the blue-filter settings and from $650\text{to}950\mathrm{nm}$ for red-filter settings, while the camera sequentially captured images at each wavelength interval. The spectral fluorescence images consisting of autofluorescence spectra, RhodG, RFP, and Alexa680 were unmixed, using commercial software (Maestro software, CRi).

All spectral fluorescence images of tumor-bearing mice with a single tumor type underwent a region-of-interest (ROI)–based analysis. First, all nodules, whose long axis was $>0.5\mathrm{mm}$ , were identified on either unmixed RhodG or Alexa680 spectral images. Then, single maximum-sized ROIs were placed over those nodules using Image J software (http://rsbweb.nih.gov/ij/), and the average fluorescence intensity of each ROI was calculated. A “positive” nodule was defined as having an average fluorescence intensity of $⩾25$ arbitrary units on unmixed images, whereas a “negative” nodule was defined as having an average fluorescence intensity of $<25$ arbitrary units. The number of positive nodules for both RhodG and Alexa680 and the number of positive nodules for either RhodG or Alexa680 were recorded.

2.7.

In Situ Fluorescence Microscopy

After the surgical procedure described in the previous section, the abdominal contents were isolated and spread on a glass slide, and then subjected to in situ fluorescence microscopy using an Olympus BX61 microscope (Olympus America, Inc., Melville, NY). DIC imaging was used to guide spectral fluorescence imaging, which was performed either with a two-filter set for the single-tumor model or a three-filter set for the coincident tumor model. Only nodules sized from $0.5\text{to}2.5\mathrm{mm}$ on DIC images were included in this analysis.

3.1.

In Vitro Fluorescent Microscopic Study

Serial observations of SHIN3 and SKOV3 cells incubated with GSA-RhodG, trastuzumab-Alexa680, or the both were done using fluorescence microscopy (Fig. 1, 2, 3, 4 ). Fluorescence microscopic images of SHIN3 cells demonstrated binding of GSA-RhodG to SHIN3 cells denoted by small, peripheral fluorescent dots within cells at $1\mathrm{h}$ postincubation [Fig. 1a]. After $8\mathrm{h}$ , the bright fluorescent dots had coalesced and remained present in the cytoplasm at the $24\text{-}\mathrm{h}$ time point. Additionally, GSA-RhodG demonstrated a similar phenomenon when incubated with SKOV3 cells. Incubation of the two cell types individually with trastuzumab-Alexa680 failed to reveal binding of this probe to SHIN3 cell at any of the time points [Fig. 1a], whereas SKOV3 cells demonstrated cell-surface binding of trastuzumab-Alexa680 at $1\mathrm{h}$ [Fig. 1b]. The specific binding of each reagent was demonstrated with experiments with the similar setting but with blocking by addition of 100-fold excess nonlabeled reagent as shown in Fig. 5 . At $8\mathrm{h}$ the probe was internalized, as evidenced by the presence of numerous intracellular fluorescent foci homogenously distributed in the cytoplasm, and remained present when imaged at the $24\text{-}\mathrm{h}$ time point. These studies revealed that binding of GSA-RhodG to SKOV3 cells and SHIN3 cells was comparable while trastuzumab-Alexa680 bound only to SKOV3 cells. Additionally, even in the coculture model of SKOV3 and SHIN3/RFP cells, SKOV3 cells were depicted by both GSA-RhodG and trastuzumab-Alexa680, while SHIN3/RFP cells were depicted only by GSA-RhodG [Fig. 1c].

Fig. 1

Fluorescence microscopic images of SHIN3, SKOV3, and coculture of SHIN3/RFP and SKOV3 treated with the both GSA-RhodG $3\mu \mathrm{g}∕\mathrm{mL}$ and trastuzumab (Tras)-Alexa680 $30\mu \mathrm{g}∕\mathrm{mL}$ . Camera exposure times were $200\mathrm{ms}$ for GSA-RhodG and $1\mathrm{s}$ for RFP and trastuzumab-Alexa680. (a) Fluorescence microscopic images of SHIN3 at $1\mathrm{h}$ shows binding of GSA-RhodG to SHIN3 cells as peripheral small fluorescent dots. After $8\mathrm{h}$ , the small dots spread homogenously throughout the cytoplasm and persisted at least $24\mathrm{h}$ . 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 rimlike fluorescence on the cell surface at $1\mathrm{h}$ . With time, the complexes are internalized into the cell and can be identified as small fluorescent dots at $8\mathrm{h}$ , which persist at least $24\mathrm{h}$ . Also, GSA-RhodG binds to SKOV3 cells with the similar binding pattern to SHIN3 cells. (c) In the coculture 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 dualtargeting of SKOV3 is feasible even when two cell lines are cocultured.

Fig. 2

Fluorescence microscopic images of SHIN3 treated with either GSA-RhodG $(3\mu \mathrm{g}∕\mathrm{mL})$ or trastuzumab (Tras)-Alexa680 $(30\mu \mathrm{g}∕\mathrm{mL})$ . Exposure times were $200\mathrm{ms}$ for GSA-RhodG, and $1\mathrm{s}$ for trastuzumab-Alexa680. SHIN3 cells are depicted by GSA-RhodG, while are not depicted by trastuzumab-Alexa680. Binding patterns of GSA-RhodG are the same as those treated by the both GSA-RhodG and trastuzumab-Alexa680, as shown in Fig. 1a.

Fig. 3

Fluorescence microscopic images of SKOV3 treated with either GSA-RhodG $(3\mu \mathrm{g}∕\mathrm{mL})$ or trastuzumab (Tras)-Alexa680 $(30\mu \mathrm{g}∕\mathrm{mL})$ . Camera exposure times were $200\mathrm{ms}$ for GSA-RhodG, and $1\mathrm{s}$ for trastuzumab-Alexa680. SKOV3 cells are depicted by the both GSA-RhodG and trastuzumab-Alexa680. Binding patterns of each probe are the same as those incubated with both GSA-RhodG and trastuzumab-Alexa680, as shown in Fig. 1b.

Fig. 4

In order to demonstrate the homogeneous targeting of cells with each reagent, low magnification microscopic images obtained $8\mathrm{hrs}$ after incubation are shown in comparison to the high magnification images shown in Fig. 1 (white squares in the DIC images).

Fig. 5

In order to demonstrate the specific binding of each reagent, a blocking experiment was performed by adding 100-fold molar excess of non-labeled reagents $30\min$ prior to the start of incubation with each fluorescence-labeled reagents. The results were analyzed with both flow-cytometry and fluorescence microscopy. For both GSA and trastuzumab, the fluorescence signals from the target cells were significantly reduced by blocking with excess non-labeled reagents demonstrating that the binding of both GSA and trastuzumab to respective target cells was specific.

Fluorescence microscopy of SKOV3 cells exposed to either GSA-RhodG or trastuzumab-RhodG and incubated with Lysotracker was performed to localize the optical probes within the cell (Fig. 6 ). At $1\mathrm{h}$ postincubation, trastuzumab-RhodG bound only to the cell surface, and did not colocalize with the Lysotracker [Fig. 6a]. However, by $8\mathrm{h}$ postincubation, trastuzumab-RhodG had been internalized and could be identified as small dots, which colocalized to the lysosome. These results indicate that trastuzumab-RhodG binds to HER2/neu receptor expressed on the cell surface and then the complex is internalized forming an endosome, later fusing with the lysosome to form an endolysosome structure. A similar phenomenon was seen with incubation with GSA-RhodG at $8\mathrm{h}$ postincubation, as small fluorescent dots were identified and corresponded to lysosomes labeled by Lysotracker [Fig. 6b].

Fig. 6

Fluorescence microscopic images of SKOV3 treated with LysoTracker $(75\mathrm{nM}\mathrm{)}$ and either GSA-RhodG $(3\mu \mathrm{g}∕\mathrm{mL})$ or trastuzumab (Tras)-RhodG $(3\mu \mathrm{g}∕\mathrm{mL})$ . Camera exposure times were $200\mathrm{ms}$ for GSA-RhodG, and $1\mathrm{s}$ for trastuzumab-RhodG and Lysotracker. (a) At $1\mathrm{hr}$ post incubation with trastuzumab-RhodG, trastuzumab-RhodG binds only to the cell surface, and does not co-localize to the lysosome as labeled by Lysotracker. However, at $8\mathrm{hrs}$ post incubation, Trastzumab-RhodG is internalized and distributed to the lysosomes which are identified as small dots co-localizing with Lysotracker. (b) With GSA-RhodG, small fluorescent dots can be identified at $8\mathrm{hrs}$ post incubation and correspond to the lysosome as labeled by Lysotracker. These small fluorescent dots were also identified at $1\mathrm{hr}$ , however, it was far less intense than those at $8\mathrm{hrs}$ , and not sufficient for co-localization study with LysoTracker.

3.2.

In Vivo Multicolor Spectral Fluorescence Imaging with Single-Tumor Model

Seventy-one tumor nodules $(>0.5\mathrm{mm})$ could be identified on unmixed spectral images in four mice with SHIN3 tumors, while 69 tumor nodules $(>0.5\mathrm{mm})$ could be identified on unmixed spectral images in four mice with SKOV3 tumors. The mean size of the nodules was $1.23\pm 0.52\mathrm{mm}$ $(\text{mean}\pm \mathrm{sd})$ in the SHIN3 group, and $1.08\pm 0.56\mathrm{mm}$ in SKOV3 group. In the SHIN3 group, all 71 tumor nodules had emission spectra corresponding only to GSA-RhodG ( $71∕71$ : 100%) and no tumor nodules had an emission spectra corresponding to trastuzumab-Alexa680 [Fig. 7 and 8a ]. In the SKOV3 group, 64 nodules were successfully targeted with both targeting probes, visualized by the presence of a distinct spectra pattern comprised of both GSA-RhodG and Tratuzumab-Alexa680 spectra ( $64∕69$ : 94.2%) [Fig. 8b]. However, five nodules failed to demonstrate the distinct emission spectra; three nodules were positive only for GSA-RhodG, while two nodules were positive only for tratuzumab-Alexa680.

Fig. 7

In order to demonstrate that free dyes alone could not target cells, incubation studies with target cells were performed using same amount of targeted or free dyes. The results were analyzed with flow cytometry and fluorescence microscopy. Neither free RhodG (a) nor Alexa680 (b) dyes showed significant accumulation within SHIN3 (a) and SKOV3 (b) cells respectively. Moreover, free Alexa680 dyes did not show specific accumulation in the SKOV3 tumors in mice (c).

Fig. 8

In vivo multicolor 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 $5\mathrm{mm}$ . (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 images, 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.

3.3.

In Situ Fluorescence Microscopy Single-Tumor Model

Fifty-nine tumor nodules $(0.5–1.5\mathrm{mm})$ could be identified on DIC images in four mice with SHIN3 tumors, while 36 tumor nodules $(0.5–1.5\mathrm{mm})$ could be identified on DIC images in four mice with SKOV3 tumors. Nodule size averaged $1.18\pm 0.47\mathrm{mm}$ $(\text{mean}\pm \mathrm{sd})$ in the SHIN3 group, and $0.98\pm 0.40\mathrm{mm}$ in the SKOV3 group. In the SHIN3 group, all 59 tumor nodules were depicted with GSA-RhodG and no signal corresponding to trastuzumab-Alexa680 was observed ( $59∕59$ ; 100%) [Fig. 9a ]. In the SKOV3 group, all 36 nodules could be depicted with both GSA-RhodG and trastuzumab-Alexa680 ( $36∕36$ ; 100%) [Fig. 9b].

Fig. 9

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 $1\mathrm{s}$ for all fluorescence images. Black scale bars on DIC images indicate $0.5\mathrm{mm}$ . (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.

3.4.

In Vivo Multicolor Spectral Fluorescence Imaging and In Situ Fluorescence Microscopy in a Combined SKOV3 and SHIN3/RFP Tumor Model

After establishing a reproducible mixed-tumor model composed of SKOV3 and SHIN3/RFP, we injected both GSA-RhodG and trastuzumab-Alexa680 into mixed-tumor–bearing mice to determine the capability of multiple targeting to distinguish SKOV3 containing tumors from SHIN3 containing tumors in vivo and in fusion tumors composed of both cell lines in the same animal. In vivo multicolor spectral fluorescence imaging showed that tumor nodules from SKOV3 lesions were easily identified by detection of Alexa680 and RhodG, but not the RFP spectrum that was the intrinsic marker for SHIN3 [Fig. 10a ]. SHIN3/RFP lesions demonstrated RhodG and RFP spectra, but no Alexa680 spectrum. Furthermore, in situ fluorescence microscopy confirmed these results [Fig. 10b].

Fig. 10

In vivo multicolor 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 $1\mathrm{s}$ for all fluorescence microscopic images. (a) In vivo multicolor 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 multicolor spectral fluorescence images.