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 over-express D-galactose receptor (D-GalR), but only SKOV3 over-expresses 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, multi-targeted multi-color optical imaging enabled specific in vivo diagnosis of tumors expressing distinct patterns of receptors, leading to improved diagnostic accuracy.

Introduction

Optical imaging probes with targeting ligands directed at cell surface markers overexpressed on cancers have the potential to detect and characterize tumors in vivo with high sensitivity and specificity ^1-4. Such probes could help direct surgery ⁵ or guide non-conventional treatments such as photodynamic therapy ⁶. However, malignant tumors are notorious for their heterogenous and diverse expression of cell surface markers ⁷; the degree and variety of target molecule expression may vary between and within tumor foci. Moreover, there is inevitably some low level expression in normal tissue. However, by utilizing a strategy that employs more than one type of targeting ligand in a single imaging session, it should be possible to detect more tumor volume with great specificity than can be accomplished with a probe directed at a single target. Thus, if the simultaneous diagnosis of several targets on a tumor cell were possible in vivo, it could improve both tumor detection and characterization potentially leading to more effective targeted therapy.

Herein, we describe the simultaneous use of two optical probes, each targeting a different cell surface marker and each conjugated to a different optical fluorophore. We employed two human ovarian cancer cell lines, SHIN3 ⁸ and SKOV3⁹, which produce peritoneal dissemination when introduced into mice via intraperitoneal injection. While both cell lines express D-galactose receptor (D-galR)¹⁰, only SKOV3 also over-expresses HER2/neu⁹. We chose these receptor systems to demonstrate simultaneous multi-target molecular imaging because both of them are feasible for clinical application. D-galR is expressed by various ovarian cancer cells¹⁰, therefore, it is a good target for developing fluorescence-enhanced surgical guidance for disseminated peritoneal ovarian cancer. HER2/neu is a well-established cancer cell surface marker expressed on various cancers including breast, colon, and ovarian cancers. A humanized monoclonal antibody, trastuzumab, has been approved for molecular-specific target cancer reagent by FDA for patients with breast cancers that overexpress HER2/neu and is used commonly in clinical practice. We employed galactosyl serum albumin (GSA) conjugated Rhodamine Green (RhodG) to bind D-galR-expressing cells and trastuzumab conjugated Alexa680 to bind HER2/neu-expressing cells ^{2, 3}. Given that SKOV3 expresses both targets, we predicted these cells would bind both GSA-RhodG and trastuzumab-Alexa680 while SHIN3 tumors, expressing only D-galR would only bind GSA-RhodG. Using this strategy we demonstrate multiple targeting to a single tumor type, and characterize the distribution of the surface marker expression in vivo.

Materials and Methods

Cell lines and culture

Two established human ovarian cancer cell lines (SKOV3 and SHIN3) were used in this study. SKOV3 over-expresses HER2/neu⁹, 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, Carlsbad, CA), 0.03% L-glutamine at 37 °C, 100 units/mL penicillin, and 100 μg/mL streptomycin in 5% CO₂.

Synthesis of Rhodamine Green-conjugated galactosyl serum albumin

Galactosyl serum albumin (GSA) was purchased from Sigma Chemical (St. Louis, MO), amino-reactive Rhodamine Green (RhodG-NHS) was purchased from Invitrogen Corporation (Carlsbad, CA). At room temperature, 1 mg (13 nmol) of GSA in 0.1M Na₂HPO₄ was incubated with 130 nmol of RhodG-NHS, at pH 8.5 for 30 minutes. 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 mL-4.5 mL) was eluted by 0.066 M PBS at pH 7.4 and collected in a test tube. GSA-RhodG was kept at 4°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 595nm with a UV-Vis system (8453 Value UV-Visible Value System; Agilent Technologies, Santa Clara, CA) using standard solutions of known concentrations of GSA (100 μg/mL). The concentration of RhodG was then measured by absorption at 503 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 to 3.5.

Synthesis of Rhodamine Green and Alexa680-conjugated Trastuzumab

Trastuzumab (Herceptin®), an FDA-approved humanized anti-HER2 antibody, which has a complimentary determination region (CDR) against HER2 grafted on a human IgG1 framework, was purchased from Genentech Inc. (South San Francisco, CA). RhodG-NHS and Alexa680-NHS were purchased from Invitrogen Corporation (Carlsbad, CA). At room temperature, 1 mg (6.8 nmol) of trastuzumab in 0.1M Na₂HPO₄ was incubated with 68 nmol of RhodG-NHS or Alexa680-NHS, at pH 8.5 for 30 minutes. 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 mL-4.5 mL) was eluted by 0.066 M PBS at pH 7.4 and collected in a test tube. Trastuzumab-RhodG and trastuzumab-A680 were kept at 4°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 nm with a UV-Vis system (8453 Value UV-Visible Value System; Agilent Technologies, Santa Clara, CA) using standard solutions of known concentrations of trastuzumab (200 μg/mL). The concentration of RhodG and Alexa680 was then measured by absorption at 503, and 679 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 to 3.5.

In vitro fluorescence microscopy

Fluorescence microscopy was performed with an Olympus BX51 microscope (Olympus America, Inc., Melville, NY) equipped with the following filters: for the blue light filter, a band pass filter from 470 to 490 nm and a band pass filter from 515 to 550 nm were used for excitation and emission light, respectively; for the green light filter, the values were from 530 to 585 nm and from 605 to 680 nm, respectively, and for the red light filter, the values were from 590 to 650 nm and from 665 to 740 nm, respectively. Transmitted light differential interference contrast (DIC) images were also acquired.

Either SHIN3 cells (1×10⁴) or SKOV3 cells (1×10⁴) were plated on a cover glass-bottomed culture well and incubated at 37°C, in an atmosphere of 5% CO₂ for 16 hrs. GSA-RhodG (3 μg/mL), trastuzumab-Alexa680 (30 μg/mL), or both were added to the culture media. The cells were incubated and removed at the following time points: 1, 8, 24 hrs. 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 nM of the lysosomal marker (LysoTracker Red DND-99, Molecular Probe Inc., Eugene, OR) 1 hr 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 1hr and 8hr time points.

To demonstrate dual targeting in vitro, a co-culture of SKOV3 and SHIN3/RFP cells was created. Both SHIN3-RFP cells (1×10⁴) and SKOV3 cells (1×10⁴) were plated on a cover glass-bottomed culture well and incubated at 37°C, in a 5% CO₂ atmosphere for 16 hrs. Both GSA-RhodG (3 μg/mL) and trastuzumab-Alexa680 (30 μg/mL) were added to the culture media. The cells were incubated and removed at 1 and 8 hrs. Following removal, cells were washed once with PBS and fluorescence microscopy was immediately performed with the blue, green, and red light filters.

Tumor model

All procedures were carried out in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), the National Research Council, and were approved by the National Cancer Institute Animal Care and Use Committee. The tumor implants were established by intraperitoneal injection of 2 × 10⁶ cells suspended in 200 μL of PBS in female nude mice (National Cancer Institute Animal Production Facility, Frederick, MD). Experiments with tumor bearing mice were performed at 14 days for SHIN3 mouse model, and 35 days for SKOV3 mouse model. Also, to establish a coincident tumor model, another group of mice received the intraperitoneal injection of SKOV3 cells (2 × 10⁶ cells) followed 7 days later by an intraperitoneal injection of SHIN3/RFP cells (2 × 10⁶ cells). Experiments with SKOV3 and SHIN3/RFP tumor-bearing mice were performed at 30 days after injection of the SKOV3 cells.

In vivo spectral fluorescence imaging

After establishing the intraperitoneal dissemination model, groups of mice (4 per group) with SHIN3, SKOV3, or the coincident model of SHIN3/RFP and SKOV3, were injected i.p. with 50 μg of trastuzumab-Alexa680 diluted with 300 μL PBS 24 hrs prior to imaging followed by 25 μg of GSA-RhodG diluted with 300 μL PBS 4 hrs prior to imaging. Mice were sacrificed with carbon dioxide, and then the abdominal cavities were exposed and the peritoneal membranes were spread on non-fluorescent black plates. Spectral fluorescence images were obtained using the Maestro In-Vivo imaging system (CRi, Inc., Woburn, MA). Magnified multi-color spectral fluorescence images of the peritoneal membranes were obtained with a multi-excitation acquisition ¹³ using the blue-filter setting (excitation filter: 445 to 490 nm, emission filter: 515 nm long pass) and the red-filter setting (615 to 655 nm, 700 nm long pass) for the single-injected model, and the blue-filter, green-filter (503 to 555 nm, 580 nm long pass), and red-filter setting for the coincident tumor (SKOV3 and SHIN3/RFP) model. The tunable filter was automatically stepped in 10 nm increments from 500 to 800 nm for the blue filter settings and from 650 to 950 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 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 ≥25 arbitrary units on unmixed images, whereas a “negative” nodule was defined as having an average fluorescence intensity <25 arbitrary units. The number of nodules positive for both RhodG and Alexa680 and the number of nodules positive for either RhodG or Alexa680 were recorded.

In situ fluorescence microscopy

After the surgical procedure described above, the abdominal contents were isolated and spread on a glass-slide, and then subjected to in situ fluorescence microscopy using an Olympus BX51 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 single tumor model or a three filter set for coincident tumor model. Only nodules sized from 0.5 to 2.5 mm on DIC images were included in this analysis.

Results

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, Supplemental Figs.1-2). Fluorescence microscopic images of SHIN3 cells demonstrated binding of GSA-RhodG to SHIN3 cells denoted by small, peripheral fluorescent dots within cells at 1hr post-incubation (Fig.1A). After 8 hrs, the bright fluorescent dots had coalesced and remained present in the cytoplasm at the 24 hr 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 1hr (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 non-labeled reagent as shown in Supplemental Fig.3. At 8 hrs 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 hr 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 co-culture 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 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.

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 (Supplemental Fig.4). At 1 hr post incubation, trastuzumab-RhodG bound only to the cell surface, and did not co-localize with the Lysotracker (Supplemental Fig.4A). However, by 8 hrs post incubation, trastuzumab-RhodG had been internalized and could be identified as small dots, which co-localized 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 hrs post incubation, as small fluorescent dots were identified and corresponded to lysosomes labeled by Lysotracker (Supplemental Fig.4B).

In vivo multi-color spectral fluorescence imaging with single tumor model

Seventy-one tumor nodules (>0.5mm) could be identified on unmixed spectral images in 4 mice with SHIN3 tumors, while 69 tumor nodules (>0.5mm) could be identified on unmixed spectral images in 4 mice with SKOV3 tumors. The mean size of the nodules was 1.23 ± 0.52 mm (mean ±SD) in the SHIN3 group, and 1.08 ± 0.56 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.2A). 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.2B). However, 5 nodules failed to demonstrate the distinct emission spectra; 3 nodules were positive only for GSA-RhodG, while 2 nodules were positive only for Tratuzumab-Alexa680.

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.

In situ fluorescence microscopy single tumor model

Fifty-nine tumor nodules (0.5 to 1.5mm) could be identified on DIC images in 4 mice with SHIN3 tumors, while 36 tumor nodules (0.5-1.5mm) could be identified on DIC images in 4 mice with SKOV3 tumors. Nodule size averaged 1.18 ± 0.47 mm (mean ± SD) in the SHIN3 group, and 0.98 ± 0.40 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.3A). In the SKOV3 group, all 36 nodules could be depicted with both GSA-RhodG and trastuzumab-Alexa680 (36/36; 100%) (Fig.3B).

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.

In vivo multi-color 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 multi-color 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.4A). SHIN3/RFP lesions demonstrated RhodG and RFP spectra, but no Alexa680 spectrum. Furthermore, in situ fluorescence microscopy confirmed these results (Fig.4B).

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.

Discussion

In this study, we demonstrated the ability of two distinct optical probes, GSA-RhodG and trastuzumab-Alexa680, targeting different cell-surface markers, D-galR and HER2/neu, respectively, to distinguish two different cell lines of human ovarian cancer in vivo. Thus, SKOV3 cells, which express both D-galR and HER2/neu, can be distinguished from SHIN3 cells, which express only D-galR, even in tumor nodules that are composed of both cell types. While such distinctions have been made in vitro, only recently has multi-targeted in vivo imaging become feasible. This is made possible by multi-color spectral fluorescence imaging and fluorescence microscopy which permit the spectral separation of RhodG and Alexa680 by using different excitation and emission filter sets as well as by using multiplexed targeted optical probes.

Muti-color fluorescence imaging has been performed mostly in visible light range with fluorescent proteins¹⁴. With injectable fluorescent probes, a wider variety of wave lengths can be used for imaging. However, the only reason why Alexa680, a near infrared fluorophore, was used was that it provided adequate spectral separation from RFP, which was the marker of SHIN3 cells. For surface applications surgery assistance or endoscopy, visible fluorescence is advantageous compared to near infrared fluorescence. Because we used RFP as a validation tool, RhodG was a natural choice as an exogenous fluorophore since there was sufficient spectral separation between these two fluorophores. TAMRA, another desirable fluorophore has an emission spectra that overlaps with RFP. However, in clinical applications, where RFP is not present, the combination of TAMRA and RhodG might be ideal for dual targeting.

Although single target probes have demonstrated remarkably high specificity and sensitivity, false positives persist ³. One advantage to using multiplexed targeted optical probes for tumor detection is that this approach reduces the number of false positives by increasing the specificity for tumor detection compared to a single target probe approach. Another advantage of this strategy is that it enables the detection and characterization of different types of tumor lesions as shown in Fig.4, in which all tumor nodules were detected by GSA-RhodG (a broadly sensitive probe), but only HER2/neu expressing cells were detected with trastuzumab-Alexa680 (a highly specific probe). This strategy is akin to “in vivo immunohistochemistry” commonly used in modern pathology labs. Moreover, by changing the combination of targeted optical probes, it might be possible to distinguish a tumor lesion from surrounding inflammatory changes that are frequently found in clinical specimens (e.g. lung cancer associated with pneumonia), if probes specific for inflammatory markers were developed for optical imaging. Therefore, if combined with new technical advances in real-time fluorescence cameras^{5, 16}, a multiplex targeting approach could enable more detailed identification of tumors in real time, and be useful during surgery or endoscopy.

In this study, there were 5 false negative nodules that were not detected with in vivo multi-color spectral fluorescence imaging. Although these tumor nodules yielded signal, it was too weak and below the assigned cut-off value such that these tumors were not diagnosed correctly with the in vivo camera. It is likely that the delivery of the imaging agent and/or excitation light to these tumors was insufficient due to the particular anatomic location or shape of these specific tumors. This problem would be less likely to occur during fluorescence microscopy because of the restricted and flat field of view and homogeneously strong lighting achievable with microscopy. With this in mind, it would be more likely to detect a lesion with fluorescence microscopy than with in vivo imaging, leading to false negative results.

In conclusion, a method for in vivo simultaneous multi-targeted optical imaging of different receptors was successfully established. This method may have implications for improving the diagnostic accuracy and therapeutic efficacy in cancer diagnosis and treatment.

Supplementary Material

Supplementary Fig. 1 Fluorescence microscopic images of SHIN3 treated with either GSA-RhodG (3 μg/mL) or trastuzumab (Tras)-Alexa680 (30 μg/mL). Exposure times were 200 ms for GSA-RhodG, and 1 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.

Supplementary Fig. 2 Fluorescence microscopic images of SKOV3 treated with either GSA-RhodG (3 μg/mL) or trastuzumab (Tras)-Alexa680 (30 μg/mL). Camera exposure times were 200 ms for GSA-RhodG, and 1 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.

Supplementary Fig. 3. In order to demonstrate the homogeneous targeting of cells with each reagent, low magnification microscopic images obtained 8 hrs after incubation are shown in comparison to the high magnification images shown in Fig. 1 (white squares in the DIC images).

Supplementary Fig. 4. 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.

Supplementary Fig. 5 Fluorescence microscopic images of SKOV3 treated with LysoTracker (75 nM) and either GSA-RhodG (3 μg/mL) or trastuzumab (Tras)-RhodG (30 μg/mL). Camera exposure times were 200 ms for GSA-RhodG, and 1 s for trastuzumab-RhodG and Lysotracker. (A) At 1 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 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 hrs post incubation and correspond to the lysosome as labeled by Lysotracker. These small fluorescent dots were also identified at 1 hr, however, it was far less intense than those at 8hrs, and not sufficient for co-localization study with LysoTracker.

Supplementary Fig. 6. 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).