IDC - American Chemical Society

Article pubs.acs.org/jpr

Host Response to Human Breast Invasive Ductal Carcinoma (IDC) as Observed by Changes in the Stromal Proteome Lavakumar A. Reddy,† Leann Mikesh,† Christopher Moskulak,‡ Jennifer Harvey,§ Nicholas Sherman,† Paola Zigrino,∥ Cornelia Mauch,∥ and Jay W. Fox*,† †

Department of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Jordan Hall, Box 441, Charlottesville, Virginia 22908, United States ‡ Department of Pathology, University of Virginia School of Medicine, P.O. Box 800904, Charlottesville, Virginia 22908, United States § Department of Radiology, University of Virginia School of Medicine, P.O. Box 800170, Charlottesville, Virginia 22908, United States ∥ Department of Dermatology, University of Cologne, Kerpener Str. 62, Cologne D-50937, Germany ABSTRACT: Following initial transformation, tumorigenesis, growth, invasion, and metastasis involves a complex interaction between the transformed tissue and the host, particularly in the microenvironment adjacent to the developing tumor. The tumor microenvironment itself is a unique outcome of the host reacting to the tumor and perhaps the tumor reacting to the host and in turn the tumor altering the host’s response to give rise to an environment that ultimately promotes tumor progression. The tumor-adjacent stromal, sometimes referred to as “reactive stromal” or the desmoplastic stroma, has received some investigative studies, but it is incomplete, and likely different tumors promote a varied response and hence different reactive stroma. In this study, we have investigated the proteomics of the host response, both in vitro and in vivo, to breast epithelial cancer, in the former using tissue culture and in the latter laser microdissection of stromal tissue both adjacent and distal to breast invasive ductal cancer (IDC). From proteomic analysis of in vitro tissue culture studies, we observed that the stroma produced is related to the invasiveness of the stimulating breast cancer cell lines but different from that observed from the stromal proteome of archival tissue. In vivo we have identified several potential markers of a reactive stroma. Furthermore, we observed that the proteome of tumor-adjacent stroma differs from that of tumor-distal stroma. The proteomic description of human breast IDC stroma may serve to enhance our understanding of the role of stroma in the progression of cancer and may suggest potential mechanisms of therapeutic interdiction. KEYWORDS: extracellular matrix, breast cancer, stroma, collagen XII, periostin, metastasis

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INTRODUCTION Worldwide, breast cancer is the second most commonly diagnosed cancer with the incidence of breast cancer increasing by 20% and mortality by 14% since 2008. It is the most common cause of cancer death in women.1 Traditionally, research directed at breast carcinogenesis has focused on mutational events in the ductal epithelium or alternations in the activity of relevant tumor suppressor genes. More recently, there has been a growing appreciation for the role the stroma plays in normal mammary development as well as carcinogenesis, tumor growth, and metastasis.2−4 The extracellular matrix (ECM) in breast is a dynamic structure changing in both the components present in the matrix as well as their relative amounts and by doing so offers a dynamic biological functionality important in normal breast development.5,6 However, if the dynamics of ECM is dysregulated, pathological conditions may occur including promotion of tumorigenesis and invasion.7 Breast mammary ducts are composed of myo-epithelial cells that produce basement membrane, a specialized ECM on which adheres mammary epithelium, thereby forming the ductal © 2014 American Chemical Society

lumen. These ducts reside within the breast stroma that is composed of fibroblasts and leukocytes along with ECM proteins including collagens, fibronectin, and a variety of proteoglycans.8 Stromal changes represented by alterations in ECM and cellular constituents are known to occur during breast development and lactation as well as in breast cancer.9 Breast stroma associated with tumors differs from normal breast stroma by virtue of its increased stiffness due to a quantitative and qualitative alteration of ECM constituents.10 These changes in the tumor-associated stroma give rise to an altered or aberrant context for signaling in the ductal epithelium11 that, in turn, could promote tumor growth and metastasis. A variety of experimental approaches have been utilized to characterize the stroma of normal as well as breast tumorassociated stroma including ultrastructural imaging, immunohistochemistry, and proteomic analyses. Both ultrastructural Special Issue: Proteomics of Human Diseases: Pathogenesis, Diagnosis, Prognosis, and Treatment Received: June 20, 2014 Published: September 22, 2014 4739

dx.doi.org/10.1021/pr500620x | J. Proteome Res. 2014, 13, 4739−4751

Journal of Proteome Research

Article

containing 10% conditioned media from one of the breast cancer cell lines. Culturing continued for 6 weeks, during which the fibroblasts secreted ECM sufficient in amount for proteomic analysis. Media was changed every other day. To harvest the fibroblast matrix, the culture flasks were first incubated with PBS containing 0.5% (v/v) Triton X-100 and 20 mM NH4OH extraction buffer to lyse the cells,15 followed by two further rinses with the buffer. The remaining cell-free matrix was solubilized in Laemmli buffer for subsequent SDSPAGE analysis. Twenty μg of matrix protein in Laemmli buffer was applied to each lane and electrophoresed on 10% SDSPAGE gels (BioRad, Hercules, CA).16 Multiple gels were run with two gels stained with Coomassie Blue for visualization of the samples and subsequent mass spectrometric analysis.

and light microscopic approaches provide a visual representation of stromal ECM composition and more importantly its organization/architecture; however, it only offers a nominal representation of the stroma ECM composition. Using a combination of histology, electron microscopy and nonlinear optical imaging, Provenzano and colleagues12 investigated epithelial-stromal interactions in mammary tumors, tumor explants, and normal mammary glands and were able to identify tumor-associated collagen structures. In these structures, there was increased collagenous stroma density near the tumor, taut collagen fibers around the tumor, as well as collagen fibers reorganized into fibers aligned in the direction of cell invasion. Similarly, increased stromal collagen density in a mouse mammary model of tumorigenesis was observed using multiphoton laser-scanning microscopy. This increase in collagen was associated with a three-fold greater risk of tumorigenesis and a more invasive phenotype producing an increase in metastasis to the lungs.13 In addition to stromal collagens, other noncollagenous stromal proteins have been demonstrated to play critical roles in normal breast development as well as tumorigenesis and invasion.5 In this investigation, we compared the in vitro stromal proteome of fibroblasts stimulated by conditioned media from human breast cancer cell lines with differing invasive/metastatic phenotypes. We also compared the in vivo stromal proteome of normal human breast tissue to that of the stromal regions adjacent and distal to invasive ductal carcinoma (IDC) in situ using laser microdissection and mass spectrometry. The results of these proteomic analyses highlight the similarities and differences observed between in vitro and in vivo experimentation. More interestingly, the in vivo studies suggest that certain ECM proteins may function as markers for tumorigenesis in mammary tissue and that there is a spatial difference in stromal composition that is dependent on proximity to the tumor. The differences in the regional stromal ECM may also provide insight into the role stoma plays in carcinogenesis, invasion, and metastasis.

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3. GeLC−MS Prep for In Vitro Fibroblast Extracellular Matrix

Individual lanes of the gels were each cut into 10 individual slices and treated with our standard sample preparation protocol.17 Briefly, each slice was cubed and transferred to a siliconized tube, followed by washing and destaining in 200 μL of 50% methanol for 4 h. The samples were then dehydrated with acetonitrile, followed by rehydration and reduction in 30 μL of 10 mM dithiolthreitol in 0.1 M ammonium bicarbonate at room temperature for 30 m, followed by alkylation with 30 μL of 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 30 m. Following removal of the solution, the samples were dehydrated again with treatment of 100 μL of acetonitrile, followed by 100 μL of ammonium bicarbonate. After another dehydration step with acetonitrile, the samples were dried by vacuum centrifugation. The gel pieces were then rehydrated in 20 ng/μL trypsin (Promega reductively alkylated, MS grade) in 50 mM ammonium bicarbonate on ice for 10 min. The digestion was allowed to go overnight at 37 °C with the peptides extracted twice from the gel pieces with 50 μL of 50% acetonitrile/5% formic acid. The extracts were then reduced to 15 μL for MS analysis. 4. Laser Microdissection of Formalin Fixed Paraffin Embedded Human Breast Tissue

EXPERIMENTAL SECTION

Formalin-fixed paraffin-embedded (FFPE) tissue samples were retrieved from the clinical archives of the University of Virginia Health System Biorepository and Tissue Research Facility according to Institutional Review Board (IRB) protocols. Normal breast samples from reduction mammoplasty procedures of women with no known cancer and IDC were obtained from mastectomy or lumpectomy procedures. For the tissue samples containing cancer, “adjacent stroma” was defined as stroma (nonepithelial tissue) adjacent to and extending from neoplastic epithelium for a distance no greater than 2 mm. “Distal stroma” was defined as nonepithelial tissue no closer than 10 mm to neoplastic epithelium. After pathological review of hemoxylin- and enosin-stained 8 μm FFPE breast tissue sections, a 2 mm2 area of stroma was laser-microdissected using a Leica ASLMD instrument (Leica Microsystems, Buffalo Grove, IL). Typically, this area yielded ∼43 μg of protein, as estimated by absorbance at 280 nm using a NanoVue Plus spectrophotometer (GE Healthcare Biosciences, Pittsburgh, PA). The laser-captured tissue was processed for mass spectrometry using an Expression Pathology Liquid Tissue MS Protein Prep Kit according to the manufacturer’s recommendations (Expression Pathology, Rockville, MD) to generate peptides from the samples. Samples were stored at −20 °C until processed for mass spectrometric analysis.

1. Cell Lines

Four cell lines from the American Type Culture Collection (ATCC, Rockville, MD) were utilized in these studies: HS68, a human fibroblast line and three human breast lines: MCF10A, MCF7, and MDA-MB-231. The breast cell lines are distinctive in their in vitro and in vivo invasiveness with the MDA-MB-231 line being invasive/malignant, MCF7 being noninvasive/ benign, and MCF10A representing normal breast epithelium.14 The breast cell lines were grown to ∼70−80% confluence at 37 °C, 7% CO2 in DMEM media (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY). The media was then replaced with serum-free DMEM, and allowed to condition for 48 h, at which point it was removed and centrifuged to clear any particular matter. HS68 fibroblasts were cultured under the same conditions as the breast cancer cell lines until ready for use. 2. In Vitro Stimulated Fibroblast Matrix Production

Production of stimulated fibroblast-secreted ECM was performed by generally following a standardized protocol from the literature.15 In brief, fibroblasts were cultured in 25 cm2 plates (Corning, NY) until 70% confluent and then rinsed with serum-free DMEM media, followed by culture in DMEM 4740



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5. Mass Spectrometry Sample Analysis

Aldrich, St. Louis, MO) was used at a dilution of 1:100. Polyclonal antibodies against human fibronectin (AB2413) and thrombospondin (AB1823) were purchased from Abcam (Cambridge MA) and used at a dilution of 1:200 and 1:40, respectively. Polyclonal antibody against human periostin was from Sigma-Aldrich, St. Louis, MO. Antibodies were incubated with sections at ambient temperature for 30 min, followed by visualization with Envision Dual Link (Dako North America) with 3,3′-diaminobenzidene tetrahydrochloride. Immunohistochemical sections were scored for relative amounts of staining with a range of no staining to heavy staining.

LC−MS/MS was performed on a Thermo Electron LTQFT with ECD mass spectrometer (in vitro fibroblast ECM) or a Thermo Electron Orbitrap XL with ETD mass spectrometer (laser microdissection FFPE tissue) with a Protana nanospray ion source connected to a laboratory-packed 8 cm × 75 μm id Phenomenex Jupiter 10 μm C18 reversed-phase capillary column.17 The LC was an Agilent 1100 microquaternary pump using two channels to form the gradient. 50% (ECM) or 1 μg equivalent (FFPE) of extract was injected onto the column and then eluted with an acetonitrile/0.1 M acetic acid gradient 2−80% at a flow rate of 0.5 μL/min over 90 min. The ion source was set at 2.5 kV. The instrument was set to acquire a full-scan mass spectrum (MS − FT/Orbitrap 60K resolution) to analyze peptide molecular masses, followed by 20 product ion spectra (MS/MS - ion trap) to identify amino acid sequences over the course of the elution. The following instrument settings were used for the LTQFT (Orbitrap XL in parentheses if different) − cap temp 210 C (265 C), FT AGC 1E6 (9E5), 1 μm scan, FT max ion time 1000 ms (500 ms), IT AGC 1E4 (8E3), IT max ion time 100 ms, ms/ms trigger 1000 counts, isolation width 3, normalized collision energy 35, activation time 30 ms (10 ms), repeat count 1, repeat duration 30 s, exclusion list size 50 (200), and exclusion duration 120 s (60 s).

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RESULTS

1. Proteomics of Breast Cell Line-Stimulated Fibroblast Extracellular Matrix

The average amount of protein extracted from the plates following removal of the fibroblasts was 80 μg. A representative SDS-PAGE of the secreted ECM for the HS68 fibroblasts grown in the presence of conditioned media from the three breast cancer cell lines is shown in Figure 1. The Coomassie

6. Mass Spectrometry Data Analysis

The data generated from each gel slice or FFPE sample was analyzed by a database search with the Sequest search algorithm (Proteome Discoverer 1.4.1) against the Uniprot Human Proteome database (downloaded 07/2014, 88 942 entries). All search data were loaded into Scaffold V 4_3_4 (www. proteomesoftware.com) with the following filters: 2 or greater unique peptides, Peptide Prophet score > 60%, Protein Prophet score > 95%, Xcorr > 1.8 (+1), 2.2 (+2), 2.5 (+3), and 3.5 (+4 or greater). The p values were determined using the two-tailed Fisher exact test. All data from each of the 10 slices of an individual gel lane (sample) were combined into a single data set and relative quantitation performed using normalized spectral counts (called Quantitative Values in Scaffold; www. proteomesoftware.com). For the laser microdissected tissue, each sample was treated as a unique sample in the Scaffold analysis, and the same Quantitative Values were used as above. Complete mass spectrometry data can be assessed with a Scaffold viewer at http://proteus.achs.virginia.edu:8080/ lablink/Welcome.do.

Figure 1. SDS-PAGE of HS68 fibroblast secreted proteomes following stimulation by breast epithelial cell lines. Lane HS-68 represents unstimulated fibroblasts; lane MCF-7 represents HS-68 cells stimulated with conditioned media from MCF-7 cells; lane MCD10A represents HS-68 cells stimulated with conditioned media from MCD-10A cells; and MDAMB231 represents HS-68 cells stimulated with conditioned media from MDA-MB-231 cells.

7. Bioinformatics

To evaluate the similarities or differences between protein abundance determined by mass spectrometry for the stroma from normal, IDC-adjacent, and IDC-distal stroma, we used the dchip software for hierarchical cluster analysis.18 8. Immunohistochemical Analysis of FFPE Breast Tissue

Four-micron sections of FFPE human breast tissue were cut and positioned on charged glass slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA). The slides were then deparaffinized, followed by a standard antigen retrieval protocol carried out on a PT Link automated slide processing system as recommended by the manufacturer (Dako North America, Carpinteria, CA). Endogenous peroxidases were blocked using the Peroxidase and Alkaline Phosphatase Blocking Reagent (Dako North America). Polyclonal antibody against collagen XII (Sigma-

blue stained lanes all appear superficially similar to that of the control, unstimulated HS68 fibroblast ECM. Table 1 contains the 50 most abundant proteins from the culture plate extractions from the 1701 proteins identified with high probability (ProteinProphet > 95%, minimum 2 peptides as per Scaffold algorithm). Most of the proteins seen in this presentation are cellular, which may be interpreted as being due to the long incubation times to generate sufficient matrix and the subsequent accumulation of cellular debris in the ECM. 4741



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Table 1. 50 Most Abundant Proteins in Breast-Cell-Conditioned Media-Stimulated Fibroblast Matrix quantitative value 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

identified protein

accession number

mw (kDa)

HS68

MCF10A

MCF7

MDA-MB-231

actin, cytoplasmic 2 vimentin tubulin beta chain prelamin-A/C annexin A2 filamin-A keratin, type II cytoskeletal 1 pyruvate kinase PKM OS=Homo sapiens GN=PKM PE=1 SV=4 Keratin, type II cytoskeletal 2 epidermal tubulin alpha-4A chain alpha-actinin-4 14-3-3 protein zeta/delta glyceraldehyde-3-phosphate dehydrogenase neuroblast differentiation-associated protein AHNAK annexin A5 elongation factor 1-alpha 1 keratin, type I cytoskeletal 10 aminopeptidase N superoxide dismutase [Mn], mitochondrial myosin-9 L-lactate dehydrogenase B chain isoform 2 of heat shock protein HSP 90-alpha isoform 2 of Transketolase keratin, type I cytoskeletal 9 serpin H1 transgelin-2 78 kDa glucose-regulated protein vinculin ubiquitin-40S ribosomal protein S27a annexin A6 peptidyl-prolyl cis−trans isomerase A retinal dehydrogenase 1 isoform 5 of caldesmon polymerase I and transcript release factor annexin A1 peptidyl-prolyl cis−trans isomerase B protein disulfide-isomerase A3 neprilysin alpha-enolase tropomyosin alpha-4 chain heat shock cognate 71 kDa protein synaptic vesicle membrane protein VAT-1 homologue peroxiredoxin-1 phosphoglycerate kinase-1 protein disulfide-isomerase galectin-1 dihydropyrimidinase-related protein 2 histone H3 (fragment) EH domain-containing proten 2 ubiquitin carboxyl-termina hydrolase isozye L1

P63261 P08670 P07437 Q5TCI8 P07355 P21333 P04264 P14618 P35908 P68366 O43707 P63104 P04406 Q09666 P08758 P68104 P13645 P15144 P04179 P35579 P07195 P07900-2 P29401-2 P35527 P50454 P37802 P11021 P18206 P62979 P08133 P62937 P00352 Q05682-5 Q6NZI2 P04083 P23284 P30101 P08473 P06733 P67936 P11142 Q99536 Q06830 P00558 P07237 P09382 Q16555 K7EK07 Q9NZN4 P09936

42 54 50 56 39 281 66 58 65 50 105 28 36 629 36 50 59 110 25 227 37 98 69 62 46 22 72 124 18 76 18 55 61 43 39 24 57 86 47 29 71 42 22 45 57 15 62 15 61 25

2379 1765 1298 1108 778 1191 750 784 799 644 534 424 562 681 399 385 550 419 268 570 294 474 272 270 276 250 412 406 110 243 222 282 144 258 282 184 248 264 219 291 329 226 264 164 202 202 369 64 199 143

2214 1987 1270 1165 1070 618 835 598 897 637 466 454 453 290 493 400 561 472 573 229 350 397 248 410 325 502 392 342 186 222 255 231 184 304 306 282 308 311 319 358 319 289 274 171 240 206 279 103 219 187

2309 1718 1341 1219 863 412 1101 538 942 580 639 537 416 143 439 359 710 329 223 287 491 379 291 517 287 461 392 297 128 204 298 256 340 272 287 233 229 135 305 438 302 236 280 192 221 213 257 89 170 209

2569 1934 1183 1139 903 574 611 572 644 664 482 449 502 250 507 404 392 413 480 388 300 347 277 302 278 338 405 226 152 183 254 215 199 300 270 229 281 253 269 318 299 266 240 167 264 242 294 108 201 191

unstimulated fibroblast matrix, compared with the stimulated fibroblast matrices. Lumican and thrombospondin-1 were observed to be at greater abundance in the breast-cell-linecondition media-stimulated fibroblast matrices compared with the unstimulated fibroblast matrix with thrombospondin-1, demonstrating the most abundance in the fibroblast matrix stimulated with the conditioned media from the metastatic breast cancer cell line MDA-MB-231.

Table 2 shows a listing of the ECM proteins identified in those samples and their relative abundance. In general, under these experimental conditions, there was not a significant correlation of the expression of canonical ECM proteins by fibroblasts stimulated with media from breast cell lines having different invasive phenotypes. However, at relatively low abundances, 12 ECM proteins were identified. Of those 12, collagen alpha3(VI) chain was in greater abundance (>3 fold) in the control, 4742



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Table 2. Extracellular Matrix Proteins Identified in Breast-Cell-Conditioned Media-Stimulated Fibroblast Matrix quantitative value 1 2 3 4 5 6 7 8 9 10 11 12

identified protein

accession number

mw (kDa)

HS68

MCF10A

MCF7

MDA-MB-2

isoform 2 of collagen alpha-3(VI) chain collagen alpha-1(I) chain collagen alpha-2(I) chain lumican isoform 5 of fibronectin thrombospondin-1 tenascin fibulin-1 collagen alpha-1(VI) chain collagen alpha-1(XII) chain collagen alpha-2(VI) chain isoform B of decorin

P12111-2 P02452 P08123 P51884 P02751-5 P07996 F5H7V9 P23142 P12109 Q99715 P12110 P07585-2

321 139 129 38 243 129 201 77 109 333 109 27

158 30 22 7 21 3 5 3 4 3 2 1

57 22 16 23 7 2 4 4 2 1 1 4

34 104 45 18 23 6 0 3 0 1 0 0

67 34 27 20 16 13 1 3 3 2 3 2

2. Proteomics of Breast Stromal Tissue

An example of laser microdissection of breast stroma is seen in Figure 2. In Figure 3 is shown the relative protein amount

Figure 3. Effect of breast tumor on stromal density. Relative amount of total protein per mm2 measured in cancer distal, cancer adjacent, and normal stroma of laser capture microdissection stroma.

the 25 ECM proteins identified with high confidence from the laser microdissection experiments. These were grouped as the following: similar abundance across all three groups; variable abundance across all three groups; higher abundance in cancer adjacent stroma; higher abundance in cancer distal stroma; and lower abundance in cancer stroma. A review of this data suggested candidates for potential markers of tumor adjacent reactive stroma. As seen in Table 4, seven ECM proteins were identified to be more abundance in IDC adjacent stroma compared with normal breast stroma or stroma distal to IDC. These included: collagen alpha-1(XII) chain, fibronectin, periostin, thromobospondin-1, thromobospondin-2, tenascin, and isoform B of collagen alpha-1(XI) chain. Five ECM proteins, mimecan, collagen alpha-1(I) chain, collagen alpha1(I) chain, isoform 2 of collagen alpha-1(XIV) chain, and isoform B of decorin, were observed to be of lower abundance in the IDC stroma, particularly stroma adjacent to IDC, compared with normal stroma.

Figure 2. Laser microdissection of breast stroma. Before and after microdissection of tissue from normal breast stroma (A and B, respectively) region, cancer distal region (C and D, respectively), and cancer adjacent area (E and F, respectively). Magnification is 200×.

associated with different areas of laser microdissected tissue from different breast samples. In general, it appears that the protein density, as estimated by protein amount/area of sample in the region adjacent to the tumor, was higher compared with that of normal breast stroma and the stroma distal to the tumor, which is in accordance with reports of increased stroma near tumors. The 50 most abundant proteins identified in breast stroma from a total of 234 identified proteins are shown in Table 3. Unlike in the in vitro generated proteome, many of the proteins identified from these tissues are known ECM proteins along with some cellular components present. In Table 4 are shown

3. Hierarchical Cluster Analysis of Stromal Proteomes from Normal Breast Stroma and Stroma Adjacent and Distal to IDC

In Figure 4 is shown the hierarchical cluster analysis of the proteomic data generated from laser microdissection of three samples each of normal breast stroma, stroma adjacent to IDC, and stroma distal to IDC. From this analysis, it is observed that 4743


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

serum albumin collagen alpha-3(VI) chain collagen alpha-1(I) chain collagen alpha-2(I) chain vimentin actin, cytoplasmic 1 collagen alpha-1(XII) chain mimecan hemoglobin collagenalpha-1(VI) chain lumican collagen alpha-2(VI) chain apolipoprotein A-I Ig kappa chain C region isoform 2 of Collagen alpha-1(XIV) chain hemoglobin subunit alpha Ugl-Y3 serotransferrin complement C3 isoform 5 of Periostin isoform B of Decorin Ig gamma-1 chain C region fibrillin-1 keratin, type II cytoskeletal 8 isoform 6 of prelamin-A/C myosin-9 Centrosomal protein of 104 kDa alpha-1-antitrypsin biglycan tubulin alpha-8 chain (fragment) Ig alpha-1 chain C region collagen alpha-1(III) chain prolargin annexin A2 HCG1983504, isoform CRA_d histone H4 neuroblast differentiation-associated protein AHNAK nollagen alpha-1(V) chain tenascin alpha-2-macroglobulin keratin, type I cytoskeletal 19 histone H2B

identified proteins P02768 P12111 P02452 P08123 P08670 P60709 D6RGG3 P20774 P68871 P12109 P51884 P12110 P02647 P01834 Q05707-2 P69905 F8W7G7 P02787 P01024 Q150630-5 P07585-2 P01857 P35555 P05787 P02545-6 P35579 O60308 P01009 P21810 C9J2C0 P01876 P02461 P51888 P07355 G3V2N6 P62805 Q09666 P20908 P24821 P01023 P08727 B4DR52

accession number 69 344 139 129 54 42 333 34 16 109 38 109 31 12 192 15 243 77 187 90 27 36 312 54 69 227 104 47 42 52 38 139 44 39 18 11 629 184 241 163 44 18

mw (kDa) 307 149 39 45 55 42 0 43 11 30 31 17 10 33 37 4 0 18 2 9 23 8 3 38 20 6 6 4 6 15 20 6 2 17 10 27 0 0 0 0 15 11

B11-normal 272 105 132 115 42 14 0 43 44 26 30 16 31 34 49 22 0 17 28 6 17 10 1 2 10 4 11 7 5 7 2 12 4 12 2 9 5 5 0 15 0 5

B12-normal

Table 3. 50 Most Abundant Proteins Identified from Laser Microdissection of Breast Stromaa

467 118 119 84 22 19 0 38 15 33 38 11 28 30 3 8 0 20 7 4 28 5 0 11 5 4 7 12 12 5 7 19 1 5 0 11 0 3 0 0 1 3

B13-normal 171 165 202 136 46 22 0 50 9 39 39 24 26 33 28 2 0 7 11 0 28 4 2 0 18 2 13 2 9 7 6 20 7 18 4 11 4 11 0 0 0 6

137 71 47 33 55 48 88 6 18 14 16 11 15 28 0 6 64 11 6 47 6 21 3 26 9 33 5 6 10 11 3 4 3 7 11 8 0 2 4 2 10 6

B08-CA 118 81 54 42 54 52 57 3 19 20 12 20 21 16 2 5 36 11 11 26 5 6 1 44 12 26 4 6 8 21 1 5 1 12 16 14 4 2 1 6 19 5

B09-CA

quantitative value B14-normal 228 132 80 45 39 28 51 13 11 29 30 23 19 32 13 6 17 17 19 58 14 7 4 16 7 9 7 5 15 11 9 12 4 7 7 11 0 1 4 2 6 8

B10-CA 269 121 80 52 43 19 1 18 60 24 26 25 28 53 27 57 0 22 12 0 6 44 2 0 11 1 9 12 16 7 16 5 10 8 7 6 0 0 0 5 0 4

B08-CD 310 124 82 55 39 16 1 20 69 33 28 22 33 30 26 82 0 24 14 5 16 21 3 0 9 0 9 6 15 5 6 8 14 14 3 9 0 0 0 9 0 6

B09-CD

337 118 85 55 53 12 0 23 53 32 31 24 32 48 21 29 0 33 20 2 12 20 5 1 11 4 11 2 11 9 11 7 7 16 6 7 0 1 0 7 0 4

B10-CD

Journal of Proteome Research Article

4744


B10-CD

2 0 6 0 12 4 1 0 0 0 9 0 3 3 2 0

the three sample types cluster on separate arms with the normal breast stromal and the stroma distal to IDC are more closely related to each other than the stroma adjacent to IDC. Thus, from this perspective as well the stromal proteomes for tissue regions can distinguish these tissue regions from one another.

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B09-CA

2 10 12 16 4 13 1 2 3 25 6 24 5 9 3 4 0 0 11 0 4 6 6 0

B08-CA

3 1 10 1 6 5 2 0

2 0 10 1 9 4 2 0

To extend and verify increased abundance of selected proteins identified by the proteomic analyses, immunohistochemical analyses were performed on 22 different samples of breast tissue: 10 normal breast tissue samples, 9 samples from stroma adjacent to IDC, and 3 samples from stroma distal (>10 mm) from IDC. All of the candidate markers (fibronectin, thrombospondin, collagen XII, and periostin) were highly stained in the stroma adjacent to the IDC (Table 5). Fibronectin and collagen XII were observed to be only modestly stained in both the normal breast stroma and stroma distal to IDC. Thrombospondin and periostin staining were absent in normal breast stroma and stroma distal to IDC. Representative images of immunohistochemical staining for these proteins are shown in Figure 5.

DISCUSSION

0 0 17 0 1 3 5 0

2 0 10 0 6 2 4 0

0 0 9 0 4 1 1 0

The alteration of stromal composition and hence structure adjacent to neoplasia has long been recognized to be a critical aspect of the host’s response to the disease.5 Numerous investigations of the desmoplastic response or “reactive stroma” of tumors have been conducted with the aim of characterizing this aspect of the tumor microenvironment as well as for seeking mechanistic insights into what, if any, role this altered stroma may play in tumor growth, invasion, and metastasis.19 Many of these studies utilized immunohistochemical approaches that are quite specific and sensitive; however, they require a preknowledge of the protein(s) one is seeking. Proteomics, to a degree, allows for an unbiased characterization of a sample, and laser microdissection of tissue further allows for spatial discrimination as to what histological regions are to be interrogated. Previously our laboratory demonstrated cross-talk between melanoma and stromal fibroblasts in vitro and in vivo using gene expression analysis.20 From these studies, we noted a correlation of the aggressiveness of the melanoma cell lines with their ability to stimulate fibroblast gene expression, and many of the transcripts observed to be upregulated by the highly invasive melanoma lines were for ECM proteins. In this investigation, we explored the ability of conditioned media from breast cancer cell lines with different invasive phenotype to stimulate the production of ECM by fibroblasts as observed by proteomic analysis of secreted matrix. Some modest differences were observed in the ECM proteomes of the fibroblasts stimulated with conditioned media (Tables 1 and 2). While no one protein or set of proteins was observed to be correlated to stimulation by a specific cell-line-conditioned media, it did appear that some increase in thrombospondin-1 abundance in the matrix was secreted by the fibroblasts stimulated with breast-cancer-cell-conditioned media (MCF7 and MDA-MB231). Also, there seemed to an increase in abundance of lumican in all fibroblast matrices in the presence of conditioned media from the cell lines. Collagen VI was observed to have a lower abundance in all of the stimulated fibroblast matrices. Overall, from the proteomic analysis of the laser-microdissected stroma, 234 proteins were identified with high confidence. These proteins were a combination of typical

CA = stroma adjacent to tumor; CD = stroma distal to tumor.

52 129 14 281 70 29 15 532 P02790 P07996 P0C0S5 P21333 P02671-2 P67936 Q5TEC6 Q15149 hemopexin thrombospondin-1 histone H2A.Z filamin-A isoform 2 of fibrinogen alpha chain tropomyosin alpha-4 chain histone H3 plectin

B14-normal

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4. Immunohistochemistry of Breast Tissue

43 44 45 46 47 48 49 50

Table 3. continued

identified proteins

accession number

mw (kDa)

B11-normal

B12-normal

B13-normal

quantitative value

B10-CA

B08-CD

B09-CD


4745


a

4746

344 109 109 312 42 139 184 70 24 142 333 243 90 241 129 130 182 66 56 52 34 139 129 192 27

P02461 P20908 P02671-2 Q07507 P02458 D6RGG3 P02751 Q15063-5 P24821 P07996 P35442 P12107-2 P15502-4 P02675 C9JC84 P20774 P02452 P08123 Q05707-2 P07585-2

mw (kDa)

P12111 P12109 P12110 P35555 P21810

accession number

CA = stroma adjacent to cancer; CD = stroma distal to cancer.

collagen alpha-3 (VI) chain collagen alpha-1(VI) chain collagen alpha-2(VI) chain fibrillin-1 biglycan Variable Abundance Across Regions collagen alpha-1(III) chain collagen alpha-1(V) chain isoform 2 of fibrinogen alpha chain dermatopontin collagen alpha-1(II) chain Higher Abundance in Cancer Adjacent Stroma collagen alpha-1(XII) chain fibronectin isoform 5 of periostin tenascin thrombospondin-1 thrombospondin-2 isoform B of collagen alpha-1(XI) chain Higher Abundance in Cancer Distal Stroma isoform 4 of elastin fibrinogen beta chain fibrinogen gamma chain Lower Abundance in Cancer Stroma mimecan collagen alpha-1(I) chain collagen alpha-2(I) chain isoform 2 of collagen alpha-1(XIV) chain isoform B of decorin

identified proteins

B12-normal

B13-normal

43 39 45 37 23

2 0 0

0 0 9 0 0 0 0

6 0 1 2 0

43 132 115 49 17

2 0 0

0 0 6 0 0 0 0

12 5 6 5 4

38 119 84 3 28

1 0 0

0 0 4 0 0 0 0

19 3 4 8 3

Similar Abundance Across Regions 149 105 118 30 26 33 30 26 33 3 1 0 6 5 12

B11-normal

Table 4. Breast Stromal Extracellular Matrix Proteins Grouped by Abundance in Specific Regions

50 202 136 28 28

4 0 0

0 0 0 0 0 0 0

20 11 4 11 7

165 39 39 2 9

B14-normal

6 47 33 0 6

3 1 1

88 64 47 4 25 8 3

4 2 5 1 1

71 14 14 3 10

B08-CA

quantitative value

3 54 42 2 5

0 1 1

57 36 26 1 10 2 1

5 2 4 1 2

81 20 20 1 8

B09-CA

13 80 45 13 14

2 2 2

51 16 58 4 1 0 2

12 1 6 3 2

132 29 29 4 15

B10-CA

20 82 55 26 16

14 0 0

1 0 5 0 0 0 0

8 0 3 8 2

124 33 33 3 15

B08-CD

18 80 52 27 6

15 3 3

1 0 0 0 0 1 0

5 0 9 6 3

121 24 24 2 16

B09-CD

23 85 55 21 12

13 5 1

0 0 2 0 0 0 0

7 1 12 6 2

118 32 24 5 11

B10-CD

Journal of Proteome Research Article



Article

Figure 4. Hierarchical clustere analysis of stromal proteomes. Hierarchical cluster analysis of extracellular matrix proteins from normal breast stroma, cancer distal, and cancer adjacent stroma.

ECM proteins as well as cellular proteins present due to cellular populations in the stroma including fibroblasts and resident macrophages among others. Hierarchal analysis of the proteomes from 10 breast tissue stromal samples organized the tissues in relationship to whether they were in close proximity to IDC (Figure 4). The stromal proteome of the normal breast tissue and that of a stromal region distal to IDC were more similar to one another than the stroma juxtaposed to IDC, indicating that the primary, reactive, desmoplastic stromal response to the cancerous tissue is somewhat spatially limited. However, the proteome distal from the IDC tissue was

localized on its own arm of the dendrogram. Therefore, although similar to the normal breast stroma, it is not identical. This suggests that the effect of the presence of cancerous tissue on stromal composition and architecture is most profound in the adjacent stroma. Also, the impact of the presence of tumor on the stromal proteome is further reaching and may have functional implications distal to the tumor (Table 4). Close inspection of the proteomes and immunohistochemistry from these three stromal regions indicated that several proteins were associated at high abundance associated with having a close proximity to IDC (collagen XII, fibronectin, 4747



Article

Table 5. Immunohistochemical Detection of Extracellular Matrix Proteins in Breast Stromaa

a

specimen

specimen type

fibronectin

thrombospondin

collagen XII

periostion

B8 CA B9 CA B10 CA B28-CA B29 CA B30 CA B31 CA B32 CA B30 CA B8 SD B9 SD B10 CD B11 N B12 N B13 N B14 N B22-N B23-N B24-N B25-N B26-N B27-N

cancer adjacent cancer adjacent cancer adjacent cancer adjacent cancer adjacent cancer adjacent cancer adjacent cancer adjacent cancer adjacent cancer distal cancer distal cancer distal normal breast normal breast normal breast normal breast normal breast normal breast normal breast normal breast normal breast normal breast

+++ +++ +++ ++ +++ +++ +++ ++ +++ + + + + + + + + + + + + +

+++ +++ +++ +++ ++ +++ +++ +++ +++ − − − − − − − − − − − − −

+++ +++ +++ +++ +++ ++ +++ +++ ++ + + + + + + + + + + + + −

+++ +++ ++ +++ +++ +++ ++ +++ +++ + + + − − − − − − − − − −

−, no expression; ***, strong expression; **, moderate expression; *, light expression.

likely depends on the specific tissue/microenvironment.31 Therefore, the presence of thrombospondin-1 in stromal regions adjacent to IDC is not surprising given the different proteome/structure of this matrix region compared with normal breast stromal matrix. Periostin is a protein that has received considerable recent experimental attention by virtue of it being associated with a variety of cancers.32−34 Periostin is expressed in a multitude of tissues and is involved with normal development and response to stress.35,36 By virtue of its multidomain structure, periostin can interact with a number of matrix proteins including tenascin-C and collagen I, thereby playing an important role in matrix organization and function.37 Experimental alteration of periostin levels in tissues can dramatically alter ECM structure, such as observed with periostin knockout mice, and manifests itself as confined tibial periostitis.27 In addition, using a mouse model for melanoma using human xenografts, Kotobuki and colleagues were able to show a dependence of periostin expression by the host for metastasis by altering the stroma microenvironment.38 In addition to the structural role of periostin in matrices, they also interact via a number of integrins, including avB3, avB5, and a6B4, giving rise to intracellular signaling.39 Activation of both the Erk and PI3-K/ Akt pathways has been demonstrated for periostin and associated with a tumorigenic function for the protein.40,41 In the breast IDC adjacent studies in this investigation, periostin abundance was significantly increased compared with the IDC distal or normal breast stroma, thereby suggesting that periostin may be playing an important role in carcinogenesis in situ and perhaps beyond for those cancers that become invasive. Two additional interesting observations are seen in this study beyond the identification of high abundance ECM proteins associated with the IDC adjacent stroma. There is an overall decrease in the abundance of mimecan in the IDC adjacent stroma compared with the IDC distal stroma and in particular with the normal breast stroma. Mimecan is a member of the

periostin, thrombospondin-1, and tenascin) (Tables 4 and 5). Collagen XII is a fibril-associated collagen with interrupted triple helices (FACIT).21,22 As the term FACIT implies, collagen XII interacts with collagen I fibrils utilizing its collagenous region. The noncollagenous regions also can interact with other matrix proteins, notably tenascin, and has been show to play a role in the stabilization of matrix structure in bones and muscle.23,24 Collectively, collagen XII has been shown to play a role in the organization of collagen fibril into bundles.24,25 Previously, using the MCF10CA1a highly malignant breast cancer cell and tissue lysates, Yen and colleagues identified collagen alpha-1(XII) chain as being expressed at significantly greater levels compared with nonmalignant cell lines or normal tissue lysates.26 In a different experimental setting, Karagiannis and colleagues have shown collagen XII to be present in the desmoplastic invasive front of colorectal cancer metastasis.27,28 From our investigation, we see an enhanced abundance of collagen XII in the stroma adjacent to IDC in conjunction with an enhanced abundance of its binding partner tenascin, suggesting a functional role for collagen XII and tenascin in this microenvironment as well. Tumor cell growth and particularly invasion is in part defined by ECM composition, and collagen fibril reorganization in the stroma is critical to provide an interstitial pathway for breast cancer cell dissemination.29 One could speculate that increased collagen XII abundance may be important in the reorganization of the collagen bundles that appears to occur adjacent to tumors as they develop and eventually invade into the stroma. The increased abundance of fibronectin and thrombospondin-1 in the stroma adjacent to the IDC is not surprising in that both of the matrix components have been observed in stromal regions adjacent to tumors. In normal biological processes, thrombospondin-1 has been demonstrated to be involved in inflammatory processes and ECM production and organization.30 Thrombospondin-1 has been implicated as being both pro- and anti-tumorigenic/invasive, and the exact role it plays 4748



Article

Figure 5. Immunohistochemical staining results of fibronectin, thrombospondin, collagen XII, and periostin. IHC staining for fibronectin in the normal breast (A), cancer distal (B), and cancer adjacent stroma (C). IHC staining for thrombospondin in the normal breast stroma (D), cancer distal (E), and cancer adjacent stroma (F). IHC staining for collagen XII in the normal breast stroma (G), cancer distal (H), and cancer adjacent stroma (I). IHC staining for periostin in the normal breast stroma (J), cancer distal (K), and cancer adjacent stroma (L). Magnification is 200×.

readily apparent that extracellular matrices are highly complex, giving rise to a vast combination of biological, structural, and functional properties associated with normal and pathological biology. In this study, we have demonstrated that stromal proteomes depend on the histological regions at which they are assessed, a feature we have previously demonstrated for dermal tissue.47 The difference we observed with these proteomes provides indications as to which proteins may be potential markers of carcinogenesis. Consideration of these results may suggest experimental approaches to query the function of these proteins in the various aspects of carcinogenesis, tumor growth, invasion, and metastasis and potentially identify novel therapeutic focus to attenuate these processes.

small leucine repeat protein (SLRP) family and has been demonstrated to interact with collagens that play a role in collagen fibrillogenesis.42 Furthermore, SLRPs have been shown to interact with collagens and provide some degree of protection from cleavage by collagenases.43 A decrease in mimecan abundance has been reported in invasive ductal breast carcinoma, suggesting that the decreased abundance of mimecan is a marker for invasive carcinoma.44 The decreased abundance of mimecan in the IDC adjacent stroma suggests, in complement to the increased abundance of other ECM proteins in the region, the generation of a very different matrix structure and thus a functionality associated with IDC potentially involved in carcinogenesis and invasion. ECM biology and tissue stroma has long held the interests of scientists in a variety of disciplines due to the central role they play in numerous normal and pathological biological processes. With the advent of new technologies including genomics, gene expression analysis, and proteomics, matrix biology is being explored at new depths. Recently, Hynes and Naba45 have defined the “matrisome” as a list of proteins found in a specific tissue as well as complete list of all of the proteins shown to be involved in a matrix. The “core matrisome” as described is composed of approximately 300 proteins along with numerous other ECM modifying proteins (such as proteinases), ECMbinding growth factors, and ECM-associated proteins.46 It is

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AUTHOR INFORMATION

Corresponding Author

*Tel: 434-924-0050. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the University of Virginia Cancer Center and the Altria Group. We wish to acknowledge the 4749



Article

(21) Gordon, M. K.; Gerecke, D. R.; Olsen, B. R. Type XII collagen; distinct extracellular matrix component discovered by cDNA cloning. Proc. Natl. Acad. Sci. U.S.A. 1987, 6040−6044. (22) Gordon, M. K.; Castagnoia, P.; Dublet, B.; Linsemayer, T. F.; Van der Rest, M.; Mayne, R.; Olsen, B. R. Cloning of a cDNA for a new member of the class of fibril-associated collagens with interrupted triple helices. Eur. J. Biochem. 1991, 201, 333−338. (23) Koch, M.; Bohrmann, B.; Matthison, M.; Hagios, C.; Trueb, B.; Chiquet, M. Large and small splice variants of collagen XII: differential expression and ligand binding. J. Cell Biol. 1995, 267, 20087−20092. (24) Didangelos, A.; Yin, X.; Mandal, K.; Saje, A.; Smith, A.; Xu, Q.; Jahangiri, M.; Mayr, M. Extracellular matrix composition and remodeling in human abdominal aortic aneurysms: a proteomics approach. Mol Cell. Proteomics 2011, 10 (8), M111.008128. (25) Chiquet, M.; Birk, D. E.; Bonnemann, C. G.; Koch, M. Collagen XII: Protecting bone and muscle integrity by organizing collagen fibrils. Int. J. Biochem. Cell Biol. 2014, 53, 51−54. (26) Yen, T-Y; Haste, N.; Timpe, C. L.; Litsakos-Cheung, C.; Yen, R.; Macher, B. A. Using a cell line breast cancer progression system to identify biomarker candidates. J. Proteomics 2013, 96, 173−183. (27) Karagiannis, G. S.; Petraki, C.; Prassas, I.; Saraon, P.; Musrap, N.; Dimitromanolakis, A.; Diamandis, E. P. Proteomic signatures of the desmoplastic invasion front reveal collagen type XII as a marker of myofibroblastic differentiation during colorectal cancer metastasis. Oncotarget 2012, 3, 267−285. (28) Karagiannis, G. S.; Berk, A.; Dimitromanolakis, A.; Diamandis, E. P. Enrichment map profiling of the cancer invasion front suggests regulation of colorectal cancer progression by the bone morphogenetic protein antagonist, gremlin-1. Mol. Oncol. 2013, 7, 826−839. (29) Gritsenko, P. H.; IIina, O.; Friedl, P. Interstitial guidance of cancer invasion. J. Pathol. 2012, 226, 185−199. (30) Calabro, N. E.; Kristofik, N. J.; Kyriakides, T. R. Thrombospondin-2 and extracellular matrix assembly. Biochim. Biophys. Acta 2014, 1840, 2396−2402. (31) Miyata, Y.; Sakai, H. Thrombospondin-1 in urological cancer: Pathological role, clinical significance and therapeutic prospects. Int. J. Mol. Sci. 2013, 14, 12249−12272. (32) Morra, L.; Moch, H. Periostin expression and epithelialmesenchymal transition in cancer; a review and update. Virchows Arch. 2011, 459, 465−475. (33) Ruan, K.; Bao, S.; Ouyang, G. The multifaceted role of periostin in tumorigenesis. Cell. Mol. Life Sci. 2009, 66, 2219030. (34) Ontsuka, K.; Kotobuki, Y.; Shiraishi, H.; Serada, S.; Ohta, S.; Yanemura, A.; Yang, L.; Fujimoto, M.; Arima, K.; Suzuki, S.; Murota, H.; Toda, S.; Kudo, A.; Conway, S. J.; Narisawa, Y.; Katayama, I.; Izuhara, K.; Naka, T. Periostin, a matricellular protein, accelerates cutaneous wound repair by activating dermal fibroblasts. Exp. Dermatol. 2012, 21, 331−336. (35) Nuzzo, P. V.; Buzzatti, G.; Ricci, F.; Rubagotti, A.; Argellati, F.; Zinoli, L.; Boccardo, F. Periostin: A novel prognostic and therapeutic target for genitourinary cancer. Clin. Genitourn. Can. 2014, 12, 301− 311. (36) Kudo, A. Periostin in fibrillogenesis for tissue regeneration: periostin actions inside and outside the cell. Cell. Mol. Life Sci. 2011, 68, 3201−7. (37) Kii, I.; Nishiyama, T.; Li, M.; Matsumoto, K.-I.; Saito, M.; Amizuka, N.; Kudo, A. Incorporation of tenascin-C into the extracellular matrix by periostin underlies an extracellular meshwork architecture. J. Biol. Chem. 2010, 285, 2028−2039. (38) Kotobuki, Y.; Yang, L.; Serada, S.; Tanemura, A.; Yan, F.; Nomura, S.; Kudo, A.; Izuhara, K.; Murota, H.; Fujimoto, M.; Katayama, I.; Tetsuji, N. Periostin accelerates human malignant melanoma progression by modifying the melanoma microenvironment. Pigm. Cell Melanoma Res. 2014, 27, 630−639. (39) Gillan, L.; Matei, D.; Fishman, D. A.; Gerbin, C. S.; Karlan, B. Y.; Chang, D. D. Periostin secreted by epithelial ovarian carcinoma is a ligand for alpha(V)beta(3) and alpha(V)beta 5 integrins and promotes cell motility. Cancer Res. 2002, 62, 5358−5364.

excellent technical assistance of the University of Virginia School of Medicine Mass Spectrometry Core.

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