Contrast Agent Mass Spectrometry Imaging Reveals Tumor

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Contrast Agent Mass Spectrometry Imaging Reveals Tumor Heterogeneity Alessandra Tata,‡,† Jinzi Zheng,† Howard J. Ginsberg,§,⊥ David A. Jaffray,†, and Arash Zarrine-Afsar*,†,§,⊥, ∥

∥

Demian R. Ifa,‡

†

Techna Institute for the Advancement of Technology for Health, University Health Network, Toronto, Ontario M5G-1P5, Canada Department of Chemistry, York University, 4700 Keele Street, Toronto, Ontario M3J-1P3, Canada § Department of Surgery, University of Toronto, 149 College Street, Toronto, Ontario M5T-1P5, Canada ⊥ Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, 30 Bond Street, Toronto, Ontario M5B-1W8, Canada ∥ Department of Medical Biophysics, University of Toronto, 101 College Street, Suite 15-701, Toronto, Ontario M5G 1L7, Canada Downloaded by GEORGETOWN UNIV on August 26, 2015 | http://pubs.acs.org Publication Date (Web): July 15, 2015 | doi: 10.1021/acs.analchem.5b01992

‡

S Supporting Information *

ABSTRACT: Mapping intratumoral heterogeneity such as vasculature and margins is important during intraoperative applications. Desorption electrospray ionization mass spectrometry (DESI-MS) has demonstrated potential for intraoperative tumor imaging using validated MS profiles. The clinical translation of DESI-MS into a universal label-free imaging technique thus requires access to MS profiles characteristic to tumors and healthy tissues. Here, we developed contrast agent mass spectrometry imaging (CA-MSI) that utilizes a magnetic resonance imaging (MRI) contrast agent targeted to disease sites, as a label, to reveal tumor heterogeneity in the absence of known MS profiles. Human breast cancer tumors grown in mice were subjected to CA-MSI using Gadoteridol revealing tumor margins and vasculature from the localization of [Gadoteridol +K]+ and [Gadoteridol+Na]+ adducts, respectively. The localization of the [Gadoteridol+K]+ adduct as revealed through DESI-MS complements the in vivo MRI results. DESI-MS imaging is therefore possible for tumors for which no characteristic MS profiles are established. Further DESI-MS imaging of the flux of the contrast agent through mouse kidneys was performed indicating secretion of the intact label.

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applications often utilizes MS profiles characteristic of various disease states that must be available a priori to the analysis. This requirement in turn has motivated many ex vivo studies to identify and validate disease specific MS profiles with utility in intraoperative MSI. In this realm, DESI-MS imaging of known lipidic markers has been used to identify tumor regions and to also perform tumor “grading” on the basis of endogenous lipid profiles unique to each tumor class.10,12,15,16,18,19 Here, validation with conventional pathology methods are used to interpret DESI-MS images.9,15,20 For as much as tumor type identification and tumor subclass grading may fully utilize the wealth of information present in cancer MS lipid profile libraries, the act of merely mapping a tumor site within a healthy tissue does not necessarily require access to such libraries. In the work presented here, we extend the utility of intraoperative DESI-MS imaging to tumor site mapping without the need for disease MS profiles. Here we describe DESI-MS imaging of an exogenous MRI contrast agent

reast cancer is the second leading cause of death from all cancers in women.1 With better means to assess tumor boundaries becoming available, breast conserving surgery (BCS) is the most common method to locally manage this cancer. Although intraoperative pathology methods do exist to reveal breast cancer regions,2 the difficulties associated with dealing with fatty breast tissue material during pathology assessments have constituted a shift in focus toward utilizing alternative preoperative and intraoperative imaging technologies.1−3 Mass spectrometry imaging (MSI) is an emerging technology that can map the chemical content of biological tissues in a spatially resolved manner.4,5 The recent development of ambient desorption electrospray ionization (DESI) technique4,6−8 has opened up the prospect of intraoperative molecular imaging9−11 to identify disease states for effective diagnosis,9,10,12−14 and to map out the affected areas for intervention.8,12,13,15−17 Both these feats are achieved on the basis of mass spectra characteristic of the disease state in a label free manner. Although de novo mapping of disease regions within a healthy tissue on the basis of drastic changes in tissue molecular fingerprints at the disease boundary may be feasible, current implementation of DESI-MS imaging for intraoperative © 2015 American Chemical Society

Received: March 23, 2015 Accepted: July 3, 2015 Published: July 3, 2015 7683

DOI: 10.1021/acs.analchem.5b01992 Anal. Chem. 2015, 87, 7683−7689

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Downloaded by GEORGETOWN UNIV on August 26, 2015 | http://pubs.acs.org Publication Date (Web): July 15, 2015 | doi: 10.1021/acs.analchem.5b01992

Analytical Chemistry

Figure 1. DESI-MS of Gadoteridol in the mouse kidney. (A) Mass spectra of labeled and control kidney. (B) DESI-MS images of the major ionic species of Gadoteridol: [Gadoteridol+K]+ of m/z 598.1, [Gadoteridol+Na]+ of m/z 582.1, and [2Gadoteridol+Na+K]+2 of m/z 590.1. Here, we also show an H&E image of the consecutive slice. The boxed area represents the boundary between medulla and the cortex of the kidney. The region with the most intense Gadoteridol signal corresponds to the medulla. (C) Dynamic contrast-enhanced (DCE) MR images of a live mouse showing the flux of Gadoteridol through the kidney at various time points after the administration of Gadoteridol. A fast spin echo image (T2 weighted) indicating normal kidney anatomy is given for comparison. Here, the arrows point to the kidney harvested, sliced and analyzed with mass spectrometry in panels A and B. 7684


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Analytical Chemistry Gadoteridol23 within a solid tumor structure, revealing cancer regions in a mouse model of the human breast cancer tumor without utilizing published lipid MS profile of this cancer.16,19,24 Our findings further buttress the versatility of intraoperative DESI-MS as a general platform to identify cancer regions in combination with the administration of a clinically approved contrast agent such as Gadoteridol that offers MR signal enhancement for many tumor types.25 Our approach further provides a means to glean insights into how passively targeted contrast agents localize within tumor structures, a subject that is only recently being investigated with mass spectrometry.26−28Additional work is required to demonstrate the clinical utility of the contrast agent mass spectrometry imaging (CAMSI) using a multitude of tumor cases, and to investigate the possibility of utilizing other contrast agents as MS labels for rapid disease site identification.

Mass spectra were acquired as full scans, in the positive ion mode, over the mass range from m/z 500 to 900. Typical instrumental parameters used were 4.5 kV capillary voltage and 275 °C capillary temperature. A H2O−MeOH (1:1) solution was used as the spray solvent and delivered at the flow rate of 1.5 μL min−1. Methanol (MeOH) and ultra pure water (H2O), both HPLC-MS grade, were purchased from Sigma-Aldrich (Oakville, ON, Canada). The sprayer-to-surface distance was 1.0 mm, the sprayer to inlet distance was 6−8 mm, an incident spray was set at 54°, and a collection angle of 10° was used. Then, the glass slides containing two 20 μm consecutive slices were mounted on a lab-built 2D moving stage (described elsewhere7) using tape and subjected to 2-dimensional DESIMS and DESI-MS/MS analysis. The DESI-MS geometry was optimized, DESI-MS and MS/MS verification of the contrast agent was obtained using the first slice and DESI-MS imaging was performed using the second tissue slice present on the same glass slide without altering the geometry and collection parameters. To acquire DESI-MS images from the control and contrast agent containing samples, the tissues were scanned using the lab built moving stage described above in horizontal rows separated by 150 μm vertical steps until the entire sample was imaged. The lines were scanned at a constant velocity in the range of 248 to 414 m/s and the scan time was set in the range from 0.43 to 0.56 s. The MS parameters were further tuned, the DESI collection geometry was adjusted, and MS/MS verification of the contrast agent was obtained using the first slice and DESI imaging was performed using the second tissue slice present on the same glass slide without altering the collection geometry and collection parameters. For each tissue slice subjected to DESI-MS imaging, a consecutive 5 μm slice was taken for standard staining and pathology assessments. The software platform ImageCreator version 3.0 was used to convert the Xcalibur 2.0 mass spectra files (.raw) into a format compatible with BioMap (freeware, http://www.maldi-msi.org/ ), which was used to process the mass spectral data and to generate 2D spatially resolved ion images. Tentative assignments of lipids seen in the positive ion mode in kidney and tumor samples was made by comparing experimental m/z values with published ESI results.29,30 For each tissue slice subjected to DESI-MS imaging, a consecutive 5 μm slice was taken for standard staining and pathology assessments. Tissue Extraction and Analysis with ICP-MS. A 200 μm thick-slice of tumor was sectioned into three pieces, and the total tissue weight in each section was determined using an analytic balance. Gadoteridol was extracted using excess volume (4 mL for every 1 mg of tissue material in two rounds) of 10% perchloric acid, through mixing and vortexing followed by a 30 min centrifugation at 21000g taking the supernatant that was then diluted 5× with double distilled water. For ICP-MS both the standard Gadoteridol solutions and the extracted samples were taken up in excess 2% nitric acid (1000-fold dilution) and were subjected to ICP-MS quantification using NexIon 350 ICP-MS (PerkinElmer).


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EXPERIMENTAL SECTION Dynamic Contrast Enhanced Magnetic Resonance (DCE-MR) Imaging of Tumor Bearing Mice and ex Vivo Sample Preparation for Mass Spectrometry. All animal studies were conducted in accordance with institutional guidelines and approved by the Animal Ethics and Use Committee. Female Severe Combined ImmunoDeficient (SCID) mice (Harlan) were inoculated with 3 × 106 human MDA-MB-231 triple negative breast cancer cells in their lower mammary fat pad and housed for 3 weeks to allow tumor growth up to 1 cm in diameter (caliper measurements). ProHance (279.3 mg/mL of Gadoteridol, Bracco Imaging) was administered intravenously into the tail vein of tumor bearing SCID mice using a 29 to 31 gauge needle attached to either a syringe or a catheter. 10 μL/25 g of body weight of ProHance was administered to each animal. MR imaging was conducted using a 7T preclinical scanner (BioSpec 70/30USR, Bruker). The animals were induced using a 5% of isofluorane/air mixture and then transferred onto the imaging stage using a 2% isofluorane/air mixture. Throughout the imaging sessions, the animals’ breathing was monitored using a respiratory pad and a respiratory tracking system. Each MR imaging session included a precontrast T2-weighted anatomical scan, a precontrast T1map of the tumor region, a 10 min T1-weighted gadoliniumcontrast enhanced dynamic imaging scan, and a postcontrast T1-map of the tumor region. After significant washout of the first Gadoteridol injection (at 25 min postinjection), each animal received a second i.v. injection of Gadoteridol (100 μL/25g). At 5 min postinjection, the animals were sacrificed with an overdose of isoflurane and subjected to the surgical removal of kidneys and tumors. Extracted tissues were subsequently frozen on liquid N2 vapor and stored at −80 °C until they were sectioned using a cryotome (CM 1950 (Leica)) with a thickness of 20 μm. The tissue sections were thaw mounted onto glass slides for DESIMS imaging analysis. DESI-MS and DESI-MS Imaging Experiments. All MS experiments were performed using a Thermo Fisher Scientific LTQ mass spectrometer (San Jose, CA, USA). Data were acquired and processed using Xcalibur 2.0 (Thermo Fisher Scientific). Initially, a stock solution of the contrast agent ProHance (Gadoteridol, 279.3 mg/mL) from Bracco Imaging containing 500 mM Gadoteridol was used to optimize the spray solvent, and to tune the instrument parameters. To this aim, a volume of 1 μL was spotted on a glass slide, allowed to dry out for 10 min, and then analyzed by DESI-MS and DESI-MS/MS.

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RESULTS AND DISCUSSION Figure 1A shows the DESI-MS spectrum of Gadoteridol in a mouse kidney intravenously injected with Gadoteridol contrast agent and sacrificed at 5 min postinjection. In this spectrum, both the endogenous lipids characteristic to the mouse kidney29,30 (Table S1 of the Supporting Information) as well as the ions [Gadoteridol+Na]+ of m/z 582.1, [Gadoteridol+K]+ 7685


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Figure 2. DESI-MS of Gadoteridol in human breast cancer tumor grown in mice. (A) The mass spectra of labeled and control mouse tumor. (B) DESI-MS images of the major ionic species of Gadoteridol: [Gadoteridol+K]+ of m/z 598.1, [Gadoteridol+Na]+ of m/z 582.1 and [2Gadoteridol +Na+K]+2 of m/z 590.1. Here, we also show an H&E image of the consecutive slice. We show two zoomed in views indicating the boundary between the tumor and the adjacent muscle tissue (Red box) as well as a necrotic area within the tumor (Yellow box). (C) Dynamic ContrastEnhanced (DCE) MR images of a live mouse showing contrast enhancement in the tumor at various time points after the administration of 7686


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Analytical Chemistry Figure 2. continued


Gadoteridol. A fast spin echo image (T2 weighted) prior to Gadoteridol injection is also shown illustrating the anatomy of the tumor region. Arrows point to the tumor harvested and analyzed in panels A and B.

Figure 3. Localization of [Gadoteridol+Na]+ of m/z 582.1 to the periphery of the tumor where blood vessels are present. (A) An immunostained image of the tumor using anti-CD31 antibody revealing the localization of major blood vessels. (B) A zoomed in view of the boxed area in (A) further indicating blood vessels with arrows. (C) An overlay between CD-31 image and the distribution of [Gadoteridol+Na]+ ion of m/z 582.1 from DESI-MS. Analysis of the rest of the image shows similar colocalization with blood vessels throughout the entire tumor structure.

of m/z 598.1 and [2Gadoteridol+Na+K]+2 of m/z 590.1 typical of the standard Gadoteridol compound (Figure S2A of the Supporting Information) are seen. For comparison, Figure S2A of the Supporting Information shows the mass spectrum of the standard Gadoteridol compound spotted on the surface of a glass slide using DESI-MS along with the MS/MS fragmentation pattern of the major [Gadoteridol+Na]+ ion of m/z 582.1 and [Gadoteridol+K]+ ion of m/z 598.1 adducts, showing characteristic losses of water (564.1 m/z), CO2 (m/z 538.1), and 2CO2 (m/z 494.1) molecules (insets of Figure S2A of the Supporting Information). Here, the species of m/z 590.1 corresponds to [2Gadoteridol+Na+K]+2. When fragmented, this ion results in fragments of m/z 582.1 and 598.1 (data not shown). Inside the kidney (Figure S2B of the Supporting Information), the ratio between [Gadoteridol+Na]+ and [Gadoteridol+K]+ adducts is altered compared to what is seen with the standard compound under identical spray, collection, and instrument tuning conditions. However, the common fragmentation pattern seen between ex vivo tissueborne Gadoteridol and the standard compound (insets of Figure S2A,B of the Supporting Information) corroborates the presence of Gadoteridol inside mouse kidneys. The only Gadolinium (Gd) containing molecule present in the mass spectra of the mouse kidneys was the intact label. Since our approach utilized Gadoteridol as a mass spectrometry label, it was important to conduct imaging of the kidney to ensure secretion of the intact label. Figure S1 of the Supporting Information illustrates the results of the kinetic dynamic contrast enhanced magnetic resonance (DCE-MR) imaging of Gadoteridol, passively targeted to breast cancer tumors in live mice (n = 2) under anesthesia with isofluorane. In both cases, at 5 min postintravenous injection of Gadoteridol into the tail vein, the tumor area exhibited maximal contrast enhancement. The contrast agent was seen to penetrate the tumor core from the peripheries. Similarly, as seen in DCE-MR images at 5 min postinjection (Figure 2C and Figure S1 of the Supporting Information), MS/MS analysis confirms the presence of Gadoteridol in breast cancer tumors used in this study (inset

of Figure S2C of the Supporting Information). Figure 1B illustrates the spatial distribution of Gadoteridol within the mouse kidney through 2-dimensional DESI-MS imaging with 150 μm spatial resolution. In the kidney, [Gadoteridol+Na]+ of m/z 582.1, [Gadoteridol+K]+ of m/z 598.1, and [2Gadoteridol +Na+K]+2 of m/z 590.1 adducts all localize to the medulla region, as evident by H&E staining of the consecutive slice (Figure 1B). Here, we also present the DCE-MR images of contrast enhancement in a live mouse showing the flux of Gadoteridol through the same kidney that was subsequently harvested and subjected to DESI-MS imaging postinjection (Figure 1C). After a complete washout of the Gadoteridol signal from the primary injection-delivered for the purpose of kinetic MR imaging-took place, a secondary intravenous injection into the tail vein was performed and the kidneys were harvested at 5 min postinjection for MSI. For comparison, a fast spin echo image of the same mouse prior to Gadoteridol injection is presented, indicating normal anatomy (Figure 1C). Figure 2A shows the DESI-MS spectrum of Gadoteridol inside the breast cancer tumor from the mouse intravenously injected with this contrast agent and sacrificed at 5 min postinjection. Here, similar to the kidney results in Figure 1, MS signals characteristic to both Gadoteridol (Figure S2A of the Supporting Information) as well as endogenous tumor lipids were observed29 (see Table S1 of the Supporting Information for assignments). Figure 2B shows the spatial distribution pattern of Gadoteridol inside breast cancer tumor. Consistent with the results of the DCE-MR imaging (Figure S1 of the Supporting Information) that the contrast agent penetrates the tumor structure from the peripheries, DESI-MS imaging reveals the highest Gadoteridol signal at the outer edge of the tumor (Figure 2B). However, there is a striking dissimilarity between Gadoteridol localization pattern in the kidney and what is seen within the breast cancer tumor (Figure S3 of the Supporting Information). [Gadoteridol+K]+ of m/z 598.1 was seen throughout the tumor except in regions revealed by H&E to be necrotic (Figure 2B). The [Gadoteridol+Na]+ of m/z 582.1 and [2Gadoteridol+Na+K]+2 of m/z 590.1 adducts, on the 7687


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the muscle tissue at the tumor boundary, and is confined to the areas of epithelial origin (via PCK immunohistochemistry) that are cancerous, attesting to the utility of contrast agent mapping not only in revealing tumor regions but also in mapping the tumor boundary itself. The same observation is made using breast tumor samples from independent mice. Further systematic assessment of the tumor boundary mapping with DESI-MS imaging of contrast-enhanced tissues must be conducted to demonstrate clinical utility. Here, the localization of [Gadoteridol+Na]+ of m/z 582.1 to the regions of major vasculature, if shown to be a widespread signature of angiogenesis in all tumors, could be utilized during resections with MS guidance to ensure homeostasis during surgery. Figure 5 shows the result of quantitative ICP-MS characterization of the amount of Gadolinium (Gd) element in three representative regions along a 200 μm thick section of the breast cancer tumor. As can be seen here, there is a good agreement between the ICP-MS results quantifying the amount of Gd from all contributing adducts in each tumor subsection and the relative abundance of Gadoteridol therein as revealed by DESI-MS imaging. This observation further validates the relative abundances of tumor-borne Gadoteridol seen in DESIMS images of the contrast-enhanced breast cancer tumor (Figure 5). A caveat is that during ICP-MS analysis, speciation information is completely lost. As such, mapping intratumoral heterogeneity on the basis of contrast agent ionic species with ICP-MS is not possible.

other hand, were observed at the periphery of the tumor. Further, immunohistochemistry staining of the tumor using anti-CD31 antibody to reveal blood vessels suggests that [Gadoteridol+Na]+ of m/z 582.1 adduct colocalizes well with major vasculature within the tumor structure (Figure 3). From comparing DESI-MS images to H&E staining (Figure 2B), the necrotic region of the breast cancer tumor is seen to possess no Gadoteridol signal, as expected. Here, a cautious interpretation of DESI-MS imaging results, assuming no matrix effect at play in necrotic regions, may suggest that the lack of contrast enhancement in these areas during MRI is likely due to the physical absence of the contrast material itself. As such, mass spectrometry imaging may shed light on how passively targeted exogenous imaging agents are distributed in biological tissues. A caveat here is that the assignment of necrotic areas from lack of DESI-MS signal alone is not possible. The lack of [Gadoteridol +K]+ of m/z 598.1 signal can only be taken to indicate necrosis if further buttressed by H&E or MRI. Passive targeting contrast agents to tumor sites is widely used for signal enhancement in many other ubiquitously used clinical imaging modalities. Figure 4 shows an overlay between DESI-

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CONCLUSIONS

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ASSOCIATED CONTENT

Our work here describes the complementary use of two techniques of ex vivo ambient spectrometry imaging and in vivo contrast-enhanced MR imaging for accelerated identification and confirmation of tumor boundaries. On the basis of intratumoral distribution of an exogenous medical imaging contrast agent passively targeted to tumor sites, we have been able to show that intratumoral heterogeneity such as areas of vasculature and margins could be mapped using the distribution of contrast agent adducts specific to each tumor subregion. This approach complements current efforts that utilize lipid profiles to characterize tumors by mass spectrometry imaging.

Figure 4. Analysis of the localization of [Gadoteridol+K]+ of m/z 598.1 at the boundary between tumor and healthy tissue. (A) Immunostained images of breast cancer using pancytokeratin (PCK) reveal the tumor region. (B) An overlay between DESI-MS image of [Gadoteridol+K]+ of m/z 598.1 and the PCK image. (C). An overlay between DESI-MS image of [Gadoteridol+K]+ of m/z 598.1 and the H&E image of the same tumor indicating that healthy tissue at the tumor boundary in panel A is muscle. Both overlays suggest that [Gadoteridol+K]+ is excluded from the healthy tissue at the boundary. Similar results were obtained using the independent tumor imaged in Figure 2 (see Figure S4 of the Supporting Information).

S Supporting Information *

MS image of [Gadoteridol+K]+ of m/z 598.1 with H&E and pancytokeratin (PCK) immunostained images of the breast cancer tumor. As seen here, the contrast agent is excluded from

Additional information as noted in text. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01992.

Figure 5. ICP-MS analysis of the amount of Gadolinium (Gd) in breast cancer tumor. To corroborate the ion abundances seen in DESI-MS images, we sectioned the tumor into 3 areas depicted in panel A and extracted Gadoteridol for ICP-MS analysis. The obtained Gd amounts from ICP-MS analysis reflect the ion abundances seen with DESI-MS from all contributing adducts combined (panels B, C, and D). 7688


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AUTHOR INFORMATION

Corresponding Author

*A. Zarrine-Afsar. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Milan Ganguly for assistance and advice in histology and are indebted to Manuela Ventura for providing tumor cell lines. We acknowledge technical support from Linyu Fan and are thankful to Nasim Monfared for comments and a critical reading of the paper.


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Contrast Agent Mass Spectrometry Imaging Reveals Tumor

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