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Surface-enhanced resonance Raman scatteringnanostars for high-precision cancer imagingStefan Harmsen,1* Ruimin Huang,1,2* Matthew A. Wall,1,3 Hazem Karabeber,1 Jason M. Samii,1

Massimiliano Spaliviero,4 Julie R. White,5,6 Sébastien Monette,5,6 Rachael O’Connor,7

Kenneth L. Pitter,6 Stephen A. Sastra,8,9 Michael Saborowski,6 Eric C. Holland,10 Samuel Singer,7

Kenneth P. Olive,8,9 Scott W. Lowe,6,11 Ronald G. Blasberg,1,2,12,13,14 Moritz F. Kircher1,2,12,15†

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The inability to visualize the true extent of cancers represents a significant challenge in many areas of oncology. Themargins of most cancer types are not well demarcated because the cancer diffusely infiltrates the surroundingtissues. Furthermore, cancers may be multifocal and characterized by the presence of microscopic satellite lesions.Such microscopic foci represent a major reason for persistence of cancer, local recurrences, and metastatic spread,and are usually impossible to visualize with currently available imaging technologies. An imaging method to revealthe true extent of tumors is desired clinically and surgically. We show the precise visualization of tumor margins,microscopic tumor invasion, and multifocal locoregional tumor spread using a new generation of surface-enhancedresonance Raman scattering (SERRS) nanoparticles, which are termed SERRS nanostars. The SERRS nanostars featurea star-shaped gold core, a Raman reporter resonant in the near-infrared spectrum, and a primer-free silicationmethod. In genetically engineered mouse models of pancreatic cancer, breast cancer, prostate cancer, and sarcoma,and in one human sarcoma xenograft model, SERRS nanostars enabled accurate detection of macroscopic malignantlesions, as well as microscopic disease, without the need for a targeting moiety. Moreover, the sensitivity (1.5 fM limitof detection) of SERRS nanostars allowed imaging of premalignant lesions of pancreatic and prostatic neoplasias.High sensitivity and broad applicability, in conjunction with their inert gold-silica composition, render SERRSnanostars a promising imaging agent for more precise cancer imaging and resection.

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INTRODUCTION

The accurate determination of cancer spread is critical for diagnosis,staging, treatment, and follow-up of oncologic patients. Imagingmethods, and in particular those based on molecular techniques, havethe potential to address this crucial aspect of cancer management in anoninvasive, nondestructive fashion (1). Current clinically available mo-lecular imaging methods mostly use positron emission tomography(PET) or magnetic resonance imaging (MRI) (1). Several new molec-ular imaging methods are being explored, including ultrasound withmolecularly targeted contrast agents (2, 3), hyperpolarized MRI (1, 2),photoacoustic imaging (4), and fluorescence imaging (5, 6). However,no single molecular imaging method to date has been able to fulfill all

1Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY 10065,USA. 2Brain Tumor Center, Memorial Sloan Kettering Cancer Center, New York, NY 10065,USA. 3Department of Chemistry, Hunter College, City University of New York, New York, NY10065, USA. 4Urology Service, Department of Surgery, Sidney Kimmel Center for Prostateand Urologic Cancers, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.5Tri-Institutional Laboratory of Comparative Pathology, Memorial Sloan Kettering CancerCenter, The Rockefeller University, and Weill Cornell Medical College, New York, NY 10065,USA. 6Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center,New York, NY 10065, USA. 7Department of Surgery, Memorial Sloan Kettering CancerCenter, New York, NY 10065, USA. 8Department of Medicine, Herbert Irving Compre-hensive Cancer Center, Columbia University Medical Center, New York, NY 10032, USA.9Department of Pathology and Cell Biology, Herbert Irving Comprehensive Cancer Center,Columbia University Medical Center, New York, NY 10032, USA. 10Human Biology Divisionand Solid Tumor Translational Research, Fred Hutchinson Cancer Research Center, AlvordBrain Tumor Center, University of Washington, Seattle, WA 98109, USA. 11Howard HughesMedical Institute, New York, NY 10065, USA. 12Center for Molecular Imaging and Nano-technology (CMINT), Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.13Department of Neurology, Memorial Sloan Kettering Cancer Center, New York, NY 10065,USA. 14Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering CancerCenter, New York, NY 10065, USA. 15Department of Radiology, Weill Cornell MedicalCollege, New York, NY 10065, USA.*These authors contributed equally to this work.†Corresponding author. E-mail: kircherm@mskcc.org

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of the following criteria that, in combination, would be consideredtransformative in the field of oncology: (i) both high sensitivity andhigh specificity for cancer; (ii) high spatial resolution, allowing identi-fication of microscopic tumor clusters; and (iii) universality (oneprobe that could be used for all cancer types).

Fluorescence imaging has so far been a leading modality with re-gard to combining high sensitivity and high spatial resolution. How-ever, natural emission of light by biological structures (autofluorescence)can result in false positive signals; rapid photochemical destruction(photobleaching) of fluorescent molecules limits study duration; andphoton scattering and limited depth penetration further reduce itsutility in clinical settings. The utility of some other emerging methodsis limited by factors such as the destruction of the contrast agent dur-ing imaging (ultrasound microbubbles) (2, 3) or limited spatial reso-lution (hyperpolarized MRI) (1, 2).

Raman imaging using surface-enhanced Raman scattering (SERS)nanoparticles has shown promise in animals in overcoming these lim-itations (7, 8). Raman imaging is an optical imaging modality based onthe inelastic scattering of photons upon interaction with matter. Suit-able molecules (Raman reporters) of different composition generateunique, fingerprint-like Raman spectra. Although the Raman effect isintrinsically relatively weak (only 1 in ~107 scattered photons is Raman-shifted), the Raman cross section of a molecule is greatly enhanced whenthese molecules are brought in close proximity to metal nanoparticlesurfaces through a phenomenon known as SERS; in this instance, en-hancement factors of 107 to 1010 have been reported (9).

SERS thus allows for the realization of highly sensitive nanoparticle-based Raman imaging probes that are brighter and more stable thancurrent fluorescent agents (10) and can be detected with higher certain-ty due to their molecular Raman “fingerprints” (8). Without a prioritargeting, nonresonant SERS nanoparticles have been shown to enable

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visualization of only one type of primary tumor in vivo after intra-venous injection (7); this limitation was likely due to the fact thatnanoparticle accumulation varies widely in different tumor types andthat the signal strength of previous SERS probes was not sufficientlyhigh to visualize those tumor types with lower nanoparticle accumu-lation. We hypothesized that the design of a new generation of Ramannanoparticles with markedly improved signal intensity could expand

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their use to many other tumor types and en-able the visualization of the full extent of tu-mors both macro- and microscopically.

Theoretical considerations have suggestedthat orders-of-magnitude-higher SERS signalscan be achieved when the metal-molecule sys-tem of the nanoprobe is in resonance with theincident detection laser (11). With biologicalapplications in mind, we therefore designed,synthesized, and tested a new surface-enhancedresonance Raman scattering (SERRS) nanoprobethat is resonant in the near-infrared (NIR) win-dow, where optical penetration in tissue is maxi-mized. Our SERRS nanoprobe has the followingfeatures: (i) a 75-nm star-shaped gold core dem-onstrating a localized surface plasmon resonance(LSPR) in the NIR window, (ii) a Raman reportermolecule that is in resonance with the detectionlaser (785 nm), and (iii) a biocompatible en-capsulation method that allows efficient loadingof the resonant Raman reporter molecule at thegold surface. This SERRS nanoparticle—termedSERRS nanostar—has a detection limit that is~400-fold lower than that of previous genera-tions of nonresonant Raman nanoparticles (7).

Here, we report that SERRS nanostars en-abled the visualization of the full tumor extentin state-of-the-art transgenic mouse models ofbreast cancer, sarcoma, pancreatic ductal adeno-carcinoma (PDAC), and prostate cancer, with-out requiring a dedicated targeting moiety. Inother words, a “one-probe-fits-all” concept. At avery low injected dose, the SERRS nanostarsenabled Raman imaging of the margins ofthe primary cancerous lesion, microscopic tu-mor foci invading into the surrounding tissues,regional micrometastases, and even precancer-ous lesions. The inert composition of the SERRSnanostars and ongoing development of moreadvanced Raman detection systems (12–14)should help facilitate eventual clinical translationof this versatile nanoparticle-based molecularimaging probe.

RESULTS

Design, synthesis, characterization, andbiodistribution of SERRS nanostarsThe 75-nm star-shaped gold core was synthesizedby rapidly reducing gold chloride with ascorbic

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acid. The gold nanostars were coated with silica in the presence of theresonant Raman reporter IR-780 perchlorate without the need for anysurface primers. Our SERRS nanostar synthesis produced narrowlydispersed nanoprobes with a hydrodynamic diameter of ~140 nm andan LSPR red-shifted toward the NIR window (Fig. 1, A and B, fig. S1,A and B, and table S1). SERRS nanostars generated photostable SERRSsignal with a limit of detection of 1.5 fM in solution (Fig. 1, C and D). In

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and a population of SERRS nanostars are shown. (B) SERRS nanostar size distribution as determinedby nanoparticle tracking analysis. (C) Raman spectra showing photostability of 1 nM SERRS nanostarsduring continuous irradiation (100 mW laser power) for 30 min. Spectra were acquired at 5-minintervals (50-mW laser power, 1-s acquisition time, 5× objective). cps, counts/s. (D) Limit of detectionof SERRS nanostars in solution was 1.5 fM at 100 mW, 1.5-s acquisition time, 5× objective. Data arerepresentative of three separate experiments. (E and F) Serum stability of the SERRS signal intensity(E) and hydrodynamic diameter (F) of 1.0 nM PEGylated SERRS nanostars during incubation in 50%mouse serum. Data are means ± SEM (n = 3).

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absolute terms, this corresponds to 12 zeptomoles at100-mW laser power, 1.5-s acquisition time, usinga 5× objective.

SERRS nanostars showed a high degree of batch-to-batch consistency with minimal batch-to-batchvariation in SERRS signal intensity (±4.5% coeffi-cient of variation) and size distribution (±2.8% co-efficient of variation) (fig. S1, C and D). Functionalgroups were introduced to the SERRS nanostar sil-ica surface via condensationof functional silane pre-cursors [mercaptopropyltrimethoxysilane (MPTMS)or aminopropyltrimethoxysilane] with silanol groupson the silica surface. Conjugation of 2 kDa poly(ethylene glycol) (PEG)–maleimide onto the silicasurface of the sulfhydryl-modified SERRS nanostarsproduced PEGylated SERRS nanostars with highstability in serum at 37°C (≥72 hours) in terms ofboth SERRS signal intensity (3.2% decrease in signalintensity) (Fig. 1E) andhydrodynamic diameter (0.4%increase in size) (Fig. 1F).

We then evaluated the biodistribution of PEG-ylated SERRS nanostars (hereinafter referred to onlyas SERRS nanostars) in vivo in wild-type C57BL/6mice (n = 5). The mice were injected with SERRSnanostars (30 fmol/g) intravenously, sacrificed af-ter 16 hours, and homogenized tissues were ana-lyzed by Raman imaging (fig. S2A). In addition tothe expected accumulation of nanoparticles in or-gans of the reticuloendothelial system, such as theliver and spleen, the gallbladder also demonstratedsubstantial SERRS nanostar signal, indicating clear-ance of the nanoparticles from the liver into the bile.

For further confirmation of biliary clearance, weexamined the entire gastrointestinal tracts of aseparate set of C57BL/6 mice (n = 5) 16 hours afterinjectionwith SERRSnanostars (30 fmol/g). SERRSnanostar signal could be detected starting from thesecond portion of the duodenum (where bile is de-livered from the liver into the bowel via the commonbile duct) and throughout the intraluminal compart-ment of the more distal small bowel and colon (fig.S2B). SERRS nanostar signal from other organs wasnegligible.

Delineation of primary tumors usingSERRS nanostarsTo visualize macroscopic primary tumors, detect mi-croscopic, infiltrative tumor margins and satellitemetastases, and detect premalignant lesions, weperformed Raman imaging of SERRS nanostarsin murine cancer models. These models were se-lected a priori because they are especially relevantto humans given their high incidence (breast andprostate), mortality and/or morbidity (pancreas andbreast), or recurrence rate (sarcoma and breast).We chose mouse models that are genetically engi-neered to recapitulate human biology as closely aspossible: the KPC PDAC model (n = 5) (15); the

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Fig. 2. Imaging of breast cancer in the MMTV-PyMT mouse model. Images are representa-tive of n = 6mice. (A and B) Two adjacent tumors developed in the upper and lower right thoracic

mammary glands. Gray dashed box in photograph indicates areas scanned with Raman imaging.After imaging, the first tumor was resected along the white dotted line (A). Anti-PEG IHC stainingshows presence of SERRS nanostars in the tumor. The second tumor was then also resected alongthe white dotted line (B). (C) Gray dashed box in photograph indicates resection bed after removalof tumors in (A) and (B). Staining for PyMT indicated residual microscopic tumor (arrow). Ramansignal intensity is displayed in counts/s. Insets in the a-PEG images are 2× magnification views.

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Hi-Myc prostate cancer model (n = 5) (16); the Ink4a/Arf−/− fibro-sarcomamodel (n = 4) (17); the mouse mammary tumor virus–polyomavirus middle T antigen (MMTV-PyMT) breast cancer model (n = 6)(18); and the implanted human dedifferentiated liposarcoma (DDLS)model (n = 7) (19). All mice were injected with the same dose ofSERRS nanostars (30 fmol/g) intravenously via tail vein. Raman im-aging and tumor resections were performed 16 to 18 hours later to allowsufficient time for the nanoparticles to clear from the vasculature andaccumulate in the tumor tissue.

Raman images of primary tumors in all five models were acquiredto evaluate SERRS nanostar accumulation. The tissues were then ex-amined histopathologically and compared with the Raman images.Raman imaging accurately delineated the macroscopic extent of allexamined tumor types (Fig. 2, A and B; Fig. 3, A to C; Fig. 4A; Fig. 5,A and B; and Fig. 6). This was further corroborated by histologicalcorrelation of positive anti-PEG immunohistochemistry (IHC) (indi-cating the presence of SERRS nanostars) in addition to staining forrespective tumor markers.

Detection of infiltrative margins and microscopic satellitemetastases using SERRS nanostarsAfter surgical resection of the bulk tumors in the MMTV-PyMTbreast cancer, DDLS, and Ink4a/Arf −/− fibrosarcoma models, we eval-uated the ability of Raman imaging to detect SERRS nanostars in theinfiltrative tumor margins and microscopic satellite metastases. Care-

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ful correlation of the Raman and histological images was performed toidentify microscopic disease in the tissues surrounding the primarytumors. In a representative example of the mouse MMTV-PyMT breastcancer model, the two primary tumors were resected by a surgeon onthe basis of white light illumination only, being blinded to the Ramansignal (Fig. 2, A and B). No residual tumor tissue could be identifiedwith white light imaging (Fig. 2C). However, when a Raman image ofthe resection bed was acquired (Fig. 2C, upper panel), multiple sub-millimeter foci were identified that were positive for the Raman spec-tral fingerprint of the SERRS nanostars. Hematoxylin and eosin (H&E)staining and overexpression of PyMT (Fig. 2C, lower panel) confirmedthe presence of microscopic tumor cell deposits in these SERRSnanostar–positive foci.

In the Ink4a/Arf −/− fibrosarcoma mouse model, we found areas inthe skin overlying the bulk tumor containing SERRS nanostars (Fig.3B), which were confirmed by IHC to be fibrosarcomatous skin infil-trations (Fig. 3D). In the human DDLS sarcoma model, the tumor bedwas scanned after primary tumor resection was performed using whitelight illumination only. Residual lesions harboring SERRS nanostarswere consistently found by Raman imaging in the resection beds ofall sarcomas and were confirmed to represent infiltrating microscopictumor by IHC (Fig. 4B). Notably, in some of the mice, such lesions weredetected by Raman imaging at a substantial distance (5 to 10 mm) fromthe bulk liposarcomatous mass. In the example shown in Fig. 4C,Raman imaging detected five microscopic SERRS nanostar foci (as

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the overlying skin (gray box 2) had been lifted off. Raman images of eachboxed area were acquired, focusing on the bulk tumor (box 1) and the skin

mice. (A) White dashed box in photograph highlights the primary tumoron the right shoulder of an Ink4a/Arf −/− fibrosarcoma-bearing mouse afterhair removal. Despite the red discoloration, the skin overlying the tumor isintact. Images were obtained before surgical exposure of the tumor. (B)Photograph on the upper left shows the bulk tumor (black box 1) after

overlying the tumor (box 2), respectively. (C and D) Histological analysis ofthe resected bulk tumor (C) and the skin overlying the tumor (D) at differ-ent magnifications of indicated regions (boxes). Antibody against themarker Ki-67 (a-MKI67) indicated cell proliferation and a-PEG stained forSERRS nanostars. Raman signal intensity is displayed in counts/s.

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small as 100 mm) about 10 mm from the bulk tumor margin, all of whichwere confirmed by IHC for human-specific vimentin to be microme-tastatic satellite lesions. The residual tumor and the micrometastases

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would have been missed by the surgeonwithout the SERRS nanostar Raman imageguidance.

Detection of premalignant lesionsusing SERRS nanostarsNext, we aimed at investigating the abilityof SERRS nanostar–based Raman imagingto detect premalignant cells. To this end,we selected the KPC and Hi-Myc mousemodels because they demonstrate multi-focal, heterogeneous tumor developmentclosely resembling the hallmarks of humanpancreatic and prostate cancer develop-ment, respectively (15, 16). In KPC mice,Raman imaging of the exposed pancreaswas performed in situ. This not only deli-neated the bulk tumor but also detectedsmaller, submillimeter SERRS nanostar–positive foci in the body and tail of the pan-creas (Fig. 5, A and B). Histological exam-ination of the bulk tumor (Fig. 5B, arrow 1)demonstrated that the SERRSnanostars ac-cumulated both in the tumor stroma andwithin epithelial tumor cells. Examinationof the small scattered foci (arrow 2) dem-onstrated the presence of pancreatic intra-epithelial neoplasia (PanIN), a precursorof invasive pancreatic cancer, confirmedby histology (Fig. 5C). These premalignantlesions are known to develop and ultimatelyprogress to overt PDAC with 100% pene-trance (15). Notably, these lesions were alsodetected in situ in a simulated intraoperativescenario without Raman signal interferencefrom liver or spleen (Fig. 5A).

In 10-month-old Hi-Myc mice, SERRSnanostar–positive foci were found inmulti-ple areas of the prostate, each correlatingwith neoplasias of different stage, grade,and cell differentiation. In a representa-tive example, the Raman-positive focus 1was resected first (Fig. 6A, arrow 1, alongthe dashed line). A second Raman scanwas then obtained, and a larger partial pros-tatectomy was performed to resect otherSERRSnanostar–positive areas (Fig. 6A, ar-rows 2 and 3, dashed line). After the secondresection, a third Raman scan demonstratedresidual signal within the resection bed,which was also removed (Fig. 6A, arrow 4,dashed line). IHC of all three specimensshowed expression of Myc, androgen re-ceptor, and a-PEG, the latter confirmingthe presence of SERRS nanostars. High-

grade prostatic intraepithelial neoplasia (arrow 1 in Fig. 6A), a pre-malignant stage, was identified in the tissue of the first resection (Fig. 6B).Invasive prostatic carcinoma with squamous differentiation (arrow 2)

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Fig. 4. Microscopic infiltration at tumormargins and regional satellite metastases in the human DDLSmouse model. Images are representative of n = 7 mice. (A) SERRS nanostars were detected by Raman im-

aging of the bulk tumor. IHC staining for human vimentin indicated the presence of tumor cells; anti-PEG IHCstaining indicated the presence of SERRS nanostars. (B) Raman image of the resection bed acquired aftersurgical excision of the bulk tumor in (A); resection was guided by white light only. IHC images on the farright are magnified views of the areas indicated with arrows 1 and 2. (C) In a different mouse bearing aliposarcoma, multiple small foci of Raman signal (arrows 1 to 5) were found ~10 mm away from the marginsof the bulk tumor. As confirmed by IHC, each of these five SERRS nanostar–positive foci correlated with aseparate tumor cell cluster (vimentin+) as small as 100 mm (micrometastases). Images on far right are mag-nified views of the metastases labeled 4 and 5. Raman signal intensity is displayed in counts/s.

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and invasive prostatic carcinoma with mucous cell differentiation(arrow 3) were identified in the tissue obtained from the second resec-tion (Fig. 6B). Invasive adenocarcinoma (arrow 4) was identified in theprostatic tissue obtained from the third resection (Fig. 6B).

Macropinocytosis and cellular uptake of SERRS nanostarsRecent studies have shown that macropinocytosis, a rapid multistependocytic process capable of bulk endocytosis, is selectively up-regulatedby tumor cells (20). Furthermore, macropinocytosis has also been im-plicated in the active cellular uptake of nanoparticles with similar sizeand composition as the SERRS nanostars (21). We therefore investi-gated the role of macropinocytosis as an underlying mechanisminvolved in the active uptake of SERRS nanostars by cancer cells.

To faithfully recapitulate the uptake in vitro, we studied SERRSnanostar uptake in cell lines derived from tumors from the samemousemodels used for the in vivo studies (Figs. 2 to 6). These cell lines con-stituted theMMTV-PyMTmurine breast cancer–derived AT-3 cell line(22), the KPC-derived murine PDAC cell line PCC-9 with mutated p53and Kras (23), the Hi-Myc–derived murine prostate cancer cell lineMyc-CaP (24), and the human DDLS8817 liposarcoma cell line. The

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cell lines were pretreated with four different smallmolecule inhibitors: the Na+/K+ exchange inhib-itor 5-(N-ethyl-N-isopropyl)amiloride (EIPA); thephosphatidyl-4,5-bisphosphate 3-kinase (PI3K) in-hibitors NVP-BEZ235 and wortmannin; and theactin polymerization inhibitor cytochalasin D (21).After 30 min, the cells were incubated with SERRSnanostars, and Raman images were obtained.

Pretreatment with the different inhibitors mark-edly decreased the uptake of SERRS nanostars byall cell lines (~70 to 90% reduction; Fig. 7). BecausePI3K underlies other forms of endocytosis as well(for example, clathrin-mediated endocytosis), PI3Kinhibition alone is not sufficient to identify macro-pinocytosis. However, the ability of EIPA and cyto-chalasin D to significantly decrease cellular SERRSnanostar uptake does distinguish macropinocytosisfrom phagocytosis and other endocytic processes asthe mechanism underlying cellular SERRS nanostaraccumulation (25, 26).

DISCUSSION

Here, we report the development of SERRS nano-stars that, because of their low limit of detection(1.5 fM using parameters amenable to in vivo im-aging), enabled the visualization of different cancertypes, from premalignant to highly invasive, withmicroscopic precision. The Raman signal intensityof the SERRS nanostars is a ~400-fold improvementover that of other recently reported SERS nanopar-ticles (7). This increase was achieved by the applica-tion of (i) a star-shaped gold core that red-shiftedthe LSPR to the NIR region, (ii) a Raman reporterthat is in resonancewith theNIRdetection laser, and(iii) a new silica-based encapsulationmethod, whichallowed the incorporation of ionic NIR dyes (resonant

Raman reporters) in the absence of widely used stabilizing agents, such aspolyvinylpyrrolidone, cetyltrimethylammonium bromide, sodium dode-cylsulfate, and albumin (7, 27–29). As a result, the surface of the gold corewasnot compromised by primers or stabilizing agents that could replacethe Raman reporter at the gold-silica interface. In addition, because thespectral fingerprint of the SERRSnanostars is unique to theRaman reporterand is nonexistent in biological tissues, the detection of this spectralfingerprint unequivocally corresponds to the presence of SERRS nanostars.This is in contrast to other optical methods, such as fluorescence-basedimaging, which usually relies on detection of one broad-based emissionpeak and where tissue autofluorescence can lead to false-positivesignals (30).

The femtomolar sensitivity and high signal specificity of SERRSnanostars enabled delineation of not only primary tumors but alsoresidual tumor cells after radical resection of the bulk tumors and mi-croscopic locoregional metastatic tumor deposits as small as 100 mmin diameter, independent of the cancer stage, type, and subtype. It isthought that these microscopic tumor deposits cause local recurrenceafter seemingly adequate surgical treatment (31). Identification of suchmicroscopic lesions by SERRS nanostars could enable more complete

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in a mouse with a PDAC in the head of the pancreas (outlined with white dotted line).Corresponding Raman images, showing SERRS nanostar signal in the macroscopically visibletumor in the head as well as small scattered foci of SERRS signal in other normal-appearingregions of the pancreas, are also shown. (B) Photographic and high-resolution Raman imagesof the excised pancreas from (A). (C) H&E staining of the whole pancreas, including PDAC (arrow 1)and PanIN (arrow 2). Histology and keratin 19 (KRT19) staining in regions 1 and 2 confirmedlesions. Insets are 4× magnification views. Raman signal intensity is displayed in counts/s.

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Fig. 6. Imagingdifferent stages andgrades of prostatic neoplasiawith- each resection. (B) Histological staining for the tumor marker MYC, androgen

in the same prostate in the Hi-Myc mouse model. Images are repre-sentative of n = 5 mice. (A) Sequential resection of the prostatic tumors withcorresponding Raman images. White dotted lines indicate the margins of

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receptor (AR), and PEG (indicating the presence of SERRS nanostars; insets are2×magnification views) of the respective resected tumors in (A). Raman signalintensity is displayed in counts/s. PIN, prostatic intraepithelial neoplasia.

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resection, particularly in the treatment of cancers such as sarcomasand breast cancer, where the recurrence rate is high even after widesurgical excision (32, 33).

Because the accumulation of SERRS nanostars in the lesions did notrequire specific targeting moieties on the nanoparticle surface, we con-clude that their uptake depends on a property of cancer that is notunique to a specific type, subtype, or stage. It is already known thatnanoparticles within a certain size range and surface charge accumu-late specifically in cancer tissue, but not in normal tissues (34). Thisphenomenon, termed the enhanced permeability and retention (EPR)effect, is a passive targeting mechanism for nanoparticles with similarcompositions and dimensions as SERRS nanostars and may explainthe accumulation of these probes in cancerous tissues. The EPR effecthas been reported to exist in nearly every cancer type in animal models(34). Sarcomas have been reported to exhibit a lower EPR effect thancarcinomas, making them theoretically even more difficult to detectwith nanoparticle imaging agents (35). We therefore deliberately choseto also test SERRS nanostars in sarcomas to evaluate nanoparticleuptake in the most challenging scenario. Despite the expected low ac-cumulation of nanoparticles in such tumors, the high sensitivity of theSERRS nanostars enabled robust visualization of both spontaneousand human-implanted sarcoma types (Fig. 4).

A cancer type that is notoriously difficult to image as a result ofpoor vascularization and the abundant presence of poorly perfusedfibrotic stroma is PDAC (36). In the KPC mouse model, which close-ly recapitulates human PDAC, SERRS nanostars accumulated in thetumor stroma and within tumor cells throughout the primary tumor,whereas no SERRS nanostars were found in the surrounding normalacinar tissue. The SERRS nanostars were also able to visualize PanINin vivo. Direct comparisons between Raman imaging and histology(IHC) demonstrated the accumulation of SERRS nanostars bothin the stroma surrounding the PanIN and within PanIN-associatedepithelial cells. The presence of SERRS nanostars in PanINs may belinked to tumor angiogenesis at the very early stages of neoplasia (37).

The accumulation of SERRS nanostars in both premalignant andcancer cells suggests an active cellular uptake mechanism selectively

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exploited by both premalignant and malignant cells. Macropinocytosishas recently gained the attention of both the nanotechnology and thecancer research communities. It is an endocytic process that rapidlyinternalizes macromolecules in bulk from the extracellular milieu. Ithas been shown that the driving oncogene in pancreatic cancer, mu-tant Kras, stimulates macropinocytosis to obtain amino acids from theinternalized proteins (for example, serum albumin) to support cancercell metabolism (20). Because oncogenic mutation is a very early eventin cell transformation and shared by many cancers, we reasoned thatmacropinocytosis might be an underlying mechanism for activeSERRS nanoprobe internalization by cancer cells. Indeed, we foundthat macropinocytosis inhibitors markedly reduced the intracellularuptake of SERRS nanoparticles in vitro.

The inert materials used in SERRS nanostars make them biocom-patible. Gold-silica SERS nanoparticles similar in size and compositionto the SERRS nanostars have been evaluated extensively with regard totheir in vivo biocompatibility, with no significant adverse effects ob-served (38). Other gold-silica nanoparticles developed for therapeuticpurposes are already being tested in clinical trials (39). Moreover, the(at least partial) biliary excretion we observed for the SERRS nanostarsin mice decreases their potential for toxicity, as does the fact that theydo not require a surface-targeting moiety, which reduces concerns forpotentially immunogenic surface moieties needed for targeted probes.

Our study and the Raman imaging technology reported herein stillhave several limitations. First, Raman spectroscopy is not inherently adeep tissue imaging method, and therefore, whole-body imagingmethods, such as computed tomography, MRI, and PET, will continueto be essential for the initial staging examinations in most cancer pa-tients. Second, the extent of the EPR effect and of macropinocytosismay vary substantially between mouse models and human patients,and clinical trials will be needed to assess how well SERRS nanostarsperform in the clinical setting. Third, more advanced Raman imagingsystems, such as clinical wide-field scanners, will have to be developedto enable Raman-guided cancer resection in patients.

Collectively, our data demonstrate that SERRS nanostars representa new class of molecular imaging agent that enables delineation ofprimary tumors, microscopic locoregional tumor deposits as smallas 100 mm, and premalignant lesions.We show in relevant animalmod-els that this is feasible regardless of the cancer stage, type, and subtypeand without the need for a priori active targeting. Our data in mice arepromising, because Raman imaging was performed in vivo underconditions that are close to those required for clinical translation, suchas relatively low laser power (10 to 100 mW) and near-real-time imag-ing conditions. New Raman imaging devices are currently being devel-oped that also enable endoscopic detection (14, 40), detection in deepertissues (41), and even tomographic Raman imaging (13) (fig. S3). We en-vision that these advances will result in a broadened clinical applicationof Raman imaging in oncology, ranging from much-improved image-guided tumor resections inopenorminimally invasive approaches, to ear-ly cancer detection using Raman endoscopes, to noninvasive imaging ofdeeper tissues.

MATERIALS AND METHODS

Study designThe objectives of this study were to design, synthesize, and characterizea new generation of SERRS nanoparticles and to test their ability to

Fig. 7. Macropinocytosis is a major contributor to SERRS nanostaruptake by tumor cells. Four small molecule inhibitors—EIPA, NVP-BEZ235,

wortmannin, and cytochalasin D (Cyto D)—were applied in vitro to tumor celllines established from primary spontaneous tumors of theMMTV-PyMT [AT-3],KPC [PCC-9], andHi-Myc [Myc-CaP] transgenicmice andDDLS8817 liposarcomacells. Raman images of the cells were acquired, and the Raman signal from theaccumulated SERRS nanostars was quantified and normalized to the cell num-ber. Data aremeans± SDnormalized todimethyl sulfoxide (DMSO) vehicle con-trol (defined as 100%) and are representative of three separate experiments.*P < 0.05 versus the DMSO control; unpaired t-test.

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accurately detect and delineate both premalignant and malignant lesionsafter intravenous administration in multiple murine cancer models. Allanimal experiments were approved by the Institutional Animal Careand Use Committees of Memorial Sloan Kettering Cancer Center(MSKCC). Sample sizes (at least four mice per group) were chosenon the basis of previously published work (7). All Raman scans wereacquired 16 to 18 hours after intravenous SERRS nanostar injection,using 10 to 100 mW laser power, a 1.0 to 1.5 s acquisition time, anda 5× objective. Raman images were generated by applying a directclassical least square (DCLS) algorithm, which linearly matches thepredefined Raman spectrum of the SERRS nanostars with the Ramanspectra of the scanned tissues. The scanned tissues were processed forhistological examination and independently interpreted by two veteri-nary pathologists who were blinded to the Raman data (J.R.W. and S.M.).The mechanism of intracellular uptake of SERRS nanostars was assessedin cell lines derived from tumors of the mouse models used in thisstudy. The cell lines were incubated with typical macropinocytosis in-hibitors before addition of the SERRS nanostars. The difference inSERRS nanostar internalization was normalized to the vehicle controlfor each separate cell line and plotted accordingly. The difference inuptake between vehicle- and inhibitor-treated cells was determined inthree separate experiments in triplicate without excluding any samples.

ChemicalsGold chloride trihydrate (HAuCl4·3H2O), ascorbic acid, tetraethylorthosilicate (TEOS), IR-780 perchlorate, ammonium hydroxide 28%(v/v), MPTMS, 2-(N-morpholino)ethanesulfonic acid (MES), methoxy-polyethylene glycol [mPEG;Mn (number-average molecular weight), 2kDa]–maleimide, ethanol, and N,N-dimethylformamide (DMF)were purchased from Sigma-Aldrich.

Gold nanostar synthesis and encapsulationGold nanostars were synthesized by rapidly adding 10 ml of 20 mMHAuCl4 to 990ml of 40mMascorbic acid at 4°C. The nanostars werecollected by centrifugation (10 min, 4000g, 4°C) and dialyzed (3.5 kDamolecular weight cutoff; Slide-A-Lyzer G2, Thermo Fisher ScientificInc.) against 18.2-megohm·cm water. Dialyzed gold nanostars (~75 nm)were directly coated with dye-embedded silica via a modified Stöbermethod without the need for surface priming. In brief, 1.0 ml of 3.0 nMgold nanostars in water was added to 8.5 ml of ethanol to which 15 mlof 25 mM resonant IR-780 perchlorate in DMF, 320 ml of TEOS, and130 ml of 28% ammonium hydroxide were added and allowed to reactfor 25 min. The as-synthesized SERRS nanostars were isolated by cen-trifugation (3500g, 10 min) and washed with ethanol.

SERRS nanostar surface modificationTo enable PEGylation, sulfhydryl groups were introduced on the silicasurface byheating the SERRSnanostars (3 nMconcentration) for 1hourat 72°C in ethanol containing 1% (v/v) MPTMS. The surface-modifiedSERRS nanostars were thoroughly washed with ethanol and water torid the surfacemodifier and redispersed in 10mMMES buffer (pH 7.1).An equal volume of 10mMMES buffer (pH 7.1) containing 2% (w/v)methoxy-terminated (m)PEG2000-maleimide was added to 3.0 nM as-synthesized SERRS nanostars. The mPEG2000-maleimide was allowedto react with the sulfhydryl-modified silica surface for 2 hours underambient conditions. The PEGylated SERRS nanostars were thoroughlywashed with water, redispersed in filter-sterilized 10 mM MES buffer(pH 7.3), and stored at 4°C before injection.

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In vivo Raman imaging of SERRS nanostarsMouse models of human cancer were generated as described in Sup-plementary Materials. The mice were injected with SERRS nanostars(30 fmol/g) via tail vein. Raman imaging was performed after 16 to18 hours. The MMTV-PyMT, DDLS, Ink4a/Arf −/−, KPC, and Hi-Mycmice were anesthetized by intraperitoneal injection of a ketamine(15 mg/ml) and xylazine (1.5 mg/ml) cocktail (10 ml/g) and scannedin vivo. All Raman scans were performed on an inVia Raman micro-scope (Renishaw) equipped with a 300 mW 785 nm diode laser and a1 inch charge-coupled device detector with a spectral resolution of1.07 cm−1. The SERRS spectra were collected through a 5× objective(Leica). Laser output at the microscope objective was measured with ahandheld laser power meter (Edmund Optics Inc.) and determined tobe 100 mW when the laser was running at 100% laser power at themicroscope objective. Typically, in vivo and ex vivo Raman scans wereperformed at 10 to 100 mW laser power, 1.5-s acquisition time, inStreamLine high-speed acquisition mode. The Raman maps were gen-erated and analyzed by applying a DCLS algorithm (WiRE 3.4software, Renishaw). Counts per second represent the intensity ofthe 950 cm-1 peak of SERRS nanostars.

Immunohistochemical stainingThe tissues from the imaging studies were collected and fixed in 4%paraformaldehyde at 4°C overnight, and subsequently processed to beembedded in paraffin. The DISCOVERY XT biomarker platform(Ventana) was used to stain the tissue sections (5 mm). Heat-inducedepitope retrieval was performed using citrate buffer (pH 6.0). The primaryantibodies were diluted as follows: anti-PyMT antibody (1:800, ab15085,Abcam); anti-PEG antibody (1:100, PEG-B-47, ab51257, Abcam); anti–Ki-67 antibody (1:250, VP-RM04, Vector Laboratories); anti-vimentinantibody (1:5000, V6389, Sigma-Aldrich); anti–cytokeratin 19 antibody(1:5000, 3863-1, Epitomics); anti–c-Myc (C-19) antibody (1:100, sc-788,Santa Cruz Biotechnology); and anti–androgen receptor (N-20) antibody(1:150, sc-816, Santa Cruz Biotechnology). All biotin-labeled secondaryantibodies, including anti-rabbit antibody (1:300, BA-1000), anti-ratantibody (1:300, BA-9400), and anti-mouse antibody (1:300, BA-9200),were purchased from Vector Laboratories. All histological results werereviewed by two experienced mouse pathologists (J.R.W. and S.M.)from the Tri-Institutional Laboratory of Comparative Pathology whowere blinded to the Raman data and used published consensus reportsfor lesion grading (42).

Statistical analysisStatistical analysis was performed in Excel (Microsoft). Detailed in-formation on the sample size is described in the figure legends. All valuesin figures are presented as means ± SD unless otherwise noted in the textand figure legends. Statistical significance was calculated on the basis of theStudent’s t-test (two-tailed, unpaired), and the level of significance was set atP < 0.05.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/271/271ra7/DC1MethodsFig. S1. Characterization of SERRS nanostars.Fig. S2. Tissue distribution and biliary excretion of SERRS nanostars.Fig. S3. Potential clinical applications of SERRS nanostars.Table S1. Zeta potentials of nanostars.References (43, 44)

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Acknowledgments: We thank C. Sawyers, M. Evans, and J. Wongvipat for providing Hi-Mycmice; J. P. Morris IV for KPC mice; and V. Mittal for MMTV-PyMT mice. We also thank S. Leach,C. Iacobuzio-Donahue, J. Lewis, and A. K. Mitchell for critically reviewing the manuscript. Wethank the MSKCC Electron Microscopy and Molecular Cytology core facilities for their technicalsupport. Ultrasound imaging to monitor the tumor sizes in KPC mice in the Olive Laboratorywas performed by the Small Animal Imaging Shared Resource within the Columbia UniversityHerbert Irving Comprehensive Cancer Center (P30CA013696). S. I. Abrams and J. A. Joyceprovided the MMTV-PyMT breast cancer model–derived cell line AT-3. C. Sawyers providedthe Myc-CaP cell line established from a prostate tumor in the Hi-Myc mouse model. Funding:NIH grant R01 EB017748 (M.F.K.); NIH grant K08 CA163961 (M.F.K.). M.F.K. is a Damon Runyon-Rachleff Innovator supported (in part) by the Damon Runyon Cancer Research Foundation (grantDRR-29-14). MSKCC Center for Molecular Imaging and Nanotechnology grant (M.F.K.); MSKCCTechnology Development grant (M.F.K.); Geoffrey Beene Cancer Research Center at MSKCC

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grant award (M.F.K.) and Shared Resources Award (M.F.K.); The Dana Foundation Brain andImmuno-Imaging grant (M.F.K.); Dana Neuroscience Scholar Award (M.F.K.); Bayer HealthCarePharmaceuticals/Radiological Society of North America Research Scholar grant (M.F.K.); MSKCCBrain Tumor Center grant (M.F.K.); Society of MSKCC Research grant (M.F.K.); M.A.W. is supportedby a National Science Foundation Integrative Graduate Education and Research TraineeshipGrant (NSF, IGERT 0965983 at Hunter College); R25T Molecular Imaging for Training in OncologyProgram grant from the National Cancer Institute (NCI) (2R25-CA096945; principal investigator:H. Hricak; fellow: J.M.S.); and Specialized Program of Research Excellence in Soft Tissue Sarcomafrom the NCI (grant P50 CA140146; S.S.). Acknowledgments are also extended to the grant-funding support provided by the MSKCC NIH Core grant (P30-CA008748). Author contributions:S.H. designed and synthesized the SERRS nanostars, designed and performed the experiments,analyzed the data, and wrote the manuscript. R.H. performed animal experiments, histologicalprocessing, and immunohistochemical staining; analyzed the data; and wrote the manuscript.M.A.W. designed the SERRS nanostars and wrote the paper. H.K. performed tumor resections, har-vested tissues, processed tissues for IHC, and assisted in Raman imaging. J.M.S. performed Ramanimaging and processed tissues for IHC. M. Spaliviero performed tumor resections, harvested tissues,processed tissues for IHC, and assisted in Raman imaging. J.R.W., S.M., and K.P.O. analyzed andperformed histopathological staging of the specimens. R.O., K.L.P., S.A.S., and M. Saborowski as-

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sisted in generation of animal models. E.C.H., S.S., S.W.L., and K.P.O. provided mouse models andadvice. R.G.B. edited the manuscript and provided advice. M.F.K. supervised the study, designedthe experiments, analyzed data, coordinated all investigators, and wrote the manuscript. The man-uscript was critically evaluated and approved by all co-authors. Competing interests: S.H., M.A.W.,and M.F.K. are inventors of the pending international patents PCT/US13/57636 and PCT/US13/76475 and U.S. provisional patent 62/020,089; M.F.K. is the inventor of the pending internationalpatent PCT/US14/17508. Data and materials availability: The SERRS nanostars are available via amaterial transfer agreement.

Submitted 27 March 2014Accepted 12 December 2014Published 21 January 201510.1126/scitranslmed.3010633

Citation: S. Harmsen, R. Huang, M. A. Wall, H. Karabeber, J. M. Samii, M. Spaliviero, J. R. White,S. Monette, R. O’Connor, K. L. Pitter, S. A. Sastra, M. Saborowski, E. C. Holland, S. Singer,K. P. Olive, S. W. Lowe, R. G. Blasberg, M. F. Kircher, Surface-enhanced resonance Ramanscattering nanostars for high-precision cancer imaging. Sci. Transl. Med. 7, 271ra7 (2015).

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imagingSurface-enhanced resonance Raman scattering nanostars for high-precision cancer

Holland, Samuel Singer, Kenneth P. Olive, Scott W. Lowe, Ronald G. Blasberg and Moritz F. KircherWhite, Sébastien Monette, Rachael O'Connor, Kenneth L. Pitter, Stephen A. Sastra, Michael Saborowski, Eric C. Stefan Harmsen, Ruimin Huang, Matthew A. Wall, Hazem Karabeber, Jason M. Samii, Massimiliano Spaliviero, Julie R.

DOI: 10.1126/scitranslmed.3010633, 271ra7271ra7.7Sci Transl Med

detection.devices are further developed for the clinic, the SERRS nanostars are sure to find a place in human tumorbreast cancer, prostate cancer, and sarcoma with high precision. As endoscopic and handheld Raman imaging nanostars were used to image macro- and microscopic malignant lesions in animal models of pancreatic cancer,they shone nearly 400 times brighter than their ''nonresonant'' counterparts during Raman imaging. The SERRS new silica encapsulation method and a reporter molecule that was ''in resonance'' with the laser, which meant thatnear-infrared laser, these nanostars emit a unique photonic signature (Raman ''fingerprint''). The authors used a

75-nm star-shaped gold cores wrapped in Raman reporter molecule-containing silica. When hit by a−anostars'' new generation of cancer imaging agents, called ''surface-enhanced resonance Raman scattering (SERRS) nagents, which home in on cancerous tissues to signal the presence of disease. Harmsen and colleagues created a

Microscopic tumors may be difficult for the naked eye to see, but they are no match for nanosized imagingSeeing Nanostars

ARTICLE TOOLS http://stm.sciencemag.org/content/7/271/271ra7

MATERIALSSUPPLEMENTARY http://stm.sciencemag.org/content/suppl/2015/01/16/7.271.271ra7.DC1

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REFERENCES

http://stm.sciencemag.org/content/7/271/271ra7#BIBLThis article cites 43 articles, 16 of which you can access for free

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registered trademark of AAAS. is aScience Translational MedicineScience, 1200 New York Avenue NW, Washington, DC 20005. The title

(ISSN 1946-6242) is published by the American Association for the Advancement ofScience Translational Medicine

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