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OPEN Matrisome-Associated Gene Expression Patterns Correlating with TIMP2 in Cancer David Peeney1*, Yu Fan2, Trinh Nguyen 2, Daoud Meerzaman2 & William G. Stetler-Stevenson 1

Remodeling of the extracellular matrix (ECM) to facilitate invasion and metastasis is a universal hallmark of cancer progression. However, a defnitive therapeutic target remains to be identifed in this tissue compartment. As major modulators of ECM structure and function, matrix metalloproteinases (MMPs) are highly expressed in cancer and have been shown to support tumor progression. MMP enzymatic activity is inhibited by the tissue inhibitor of metalloproteinase (TIMP1–4) family of proteins, suggesting that TIMPs may possess anti-tumor activity. TIMP2 is a promiscuous MMP inhibitor that is ubiquitously expressed in normal tissues. In this study, we address inconsistencies in the literature regarding the role of TIMP2 in tumor progression by analyzing co-expressed genes in tumor vs. normal tissue. Utilizing data from The Cancer Genome Atlas and Genotype-Tissue expression studies, focusing on breast and lung carcinomas, we analyzed the correlation between TIMP2 expression and the transcriptome to identify a list of genes whose expression is highly correlated with TIMP2 in tumor tissues. Bioinformatic analysis of the identifed gene list highlights a core of matrix and matrix- associated genes that are of interest as potential modulators of TIMP2 function, thus ECM structure, identifying potential tumor microenvironment biomarkers and/or therapeutic targets for further study.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that are the major mediators of extracellular matrix (ECM) breakdown and turnover. Humans express 23 MMPs and these play a critical role in tissue development, homeostasis and disease progression. Te expression and activity of metzincin proteinases (MMPs and ADAMs), are routinely dysregulated in a number of pathologic conditions, including cancer. Chronic infammation, oxidative stress as well as infltration/proliferation of hematopoietic and stromal cells within the tumor environment (TME) are coincident with enhanced expression and activity of MMPs and ADAMs. Tis enhanced expression is associated with, in addition to amplifed ECM proteolysis, increased bioavailability of growth factors and cytokines, inhibition of apoptosis, regulation of tumor vasculature and cellular diferentia- tion1,2. Te enzymatic activity of metzincin proteinases in the TME is primarily regulated by a family of 4 endog- enous proteins called tissue inhibitors of metalloproteinases (TIMPs). Since their original discovery almost three decades ago, several additional biological functions have been ascribed to TIMP family members such as regu- lation of receptor tyrosine kinase-dependent cell growth and cell migration3. TIMP2 is abundantly expressed in normal tissues and demonstrates broad spectrum inhibition of MMPs, as well as forming a high afnity complex with pro-MMP2 independent of the MMP-inhibitory domain4. TIMP2 has also been described as interacting with several membrane receptors such as integrin α3β15, membrane-bound MMP146, insulin-like growth factor 1 receptor (IGFR1)7 and low density lipoprotein receptor-related protein 1/2 (LRP1/2)8,9. In addition, TIMP2 and these putative receptors have been shown to exhibit multiple interactions with components of the ECM. Tese fndings have led us, and others, to posit that TIMPs are multifunctional proteins and that their specifc functions are modulated by the composition of the ECM10,11. Tus, TIMP expression in normal tissues, with low levels of metzincin protease expression, may have distinct biologic activities from those of the same TIMP when expressed under pathologic conditions that are frequently associated with high levels of metalloproteinase activity. Recent studies in our lab have shown that TIMP2 is a promising candidate for biological cancer ther- apy12,13. Our working hypothesis is that TIMP2 acts as a homeostatic mediator at the interface between cellular components and ECM in normal tissues. In the evolving TME with both tumor and stromal cells (fbroblast,

1Extracellular Matrix Pathology Section, Laboratory of Pathology, National Cancer Institute, National Institute of Health, Bethesda, Maryland, USA. 2Computational Genomics and Bioinformatics Group, Center for Biomedical Informatics & Information Technology, National Cancer Institute, National Institute of Health, Rockville, Maryland, USA. *email: [email protected]

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Resource Resident Institute Methods/Data Used Te Cancer Genome Atlas (TCGA) TCGA Research Network RNA sequencing data from over 11000 patients Genotype-Tissue Expression (GTEx) GTEx Consortium RNA sequencing data from non-tumor post-mortem tissue DEseq normalization and diferential expression of BioXpress George Washington University Medical Center matched tumor vs. normal samples cBioPortal Memorial Sloan Kettering Cancer Center RNA sequencing mRNA co-expression from TCGA Recount2 Johns Hopkins University Processed RNA sequencing data from TCGA and GTEx

Table 1. Publicly available databases and resources used in this study.

infammatory and endothelial cells) that express increasing levels of active MMPs, frst the MMP-independent functions and eventually the MMP-inhibitory activity of TIMP2 is overwhelmed. Tis results in extensive matrix remodeling with concomitant changes in ECM composition and tumor promoting functions. However, the role of TIMP2 in tumorigenesis has been subject to debate with the publication of conficting reports correlating TIMP2 with either enhanced or poor prognoses. Recent advances in genetic profling of tumors led to generation of Te Cancer Genome Atlas (TCGA), a com- prehensive map of the tumor genome and transcriptome, and the Genotype-Tissue Expression (GTEx) project for the study of tissue-specifc gene expression. Tis extensive trove of data has allowed researchers around the world to interrogate gene expression patterns in normal tissues and diseased states. It is already appreciated that an imbalance between proteases and their endogenous inhibitors contributes to ECM disruption associated with tissue pathology. However, there have been few reports specifcally addressing the profle of genes co-expressed with TIMP family members in diferent cancers and normal tissues using large patient datasets. To enrich our understanding of putative TIMP functions in the TME through identifcation of target genes for future studies, we examined co-expression patterns specifc for each member of the TIMP protease inhibitor family utilizing TCGA and GTEx datasets. Tis led us to identify previously unrecognized similarities between the patterns of genes that are co-expressed with both TIMP2 and TIMP3. Further interrogation of the TIMP2 co-expressed genes identifed highly correlated core matrisome partners that are of interest as modulators for TIMP2 function, specifcally in the TME, and are potentially useful either as targets or biomarkers of TME-directed cancer therapies. Results TIMP expression is altered in cancer tissues. To start we frst highlight changes of TIMP levels in tum- ors of diferent organs utilizing several publicly available databases and resources (Table 1). Log2-fold changes in expression of each TIMP were compared between 14 cancer types and corresponding normal tissue from the same organs, all obtained through the BioXpress database, Fig. 1A–D14. Tis analysis demonstrated the variability in TIMP expression levels across various cancer tissues when normalized to controls. As previously reported, TIMP1 expression is frequently up-regulated in cancers (Fig. 1A) and its increased expression has been associ- ated with poor prognoses in numerous studies11. In contrast, TIMP3/4 expression levels are signifcantly lower in several types of cancer. Tese changes are most pronounced for TIMP3 levels in bladder, lung squamous cell and esophageal carcinomas, as well as bladder, breast, head & neck and prostate for TIMP4, Fig. 1C,D. TIMP2 displays less dynamic, although signifcant changes in expression at similar cancer sites (bladder, breast, lung squamous cell carcinoma (SCC) and prostate) when compared to normal tissues. Unlike the growing consensus of enhanced TIMP1 expression correlating with poor cancer prognosis, the clinical signifcance of the absolute levels of TIMP 2–4 expression in general are not as clearly delineated with studies reporting variable prognostic value, as previously reviewed15. In order to more clearly understand the signifcance of TIMP2 expression levels in breast and lung carcino- mas, we assessed the levels of TIMP2 in relation to metzincin targets (MMPs and ADAM12) by simple division of the TIMP2 RPKM (Reads Per Kilobase of transcript, per Million mapped reads) by that of MMPs and ADAM12 (sum of the MMP family and ADAM12 RPKM). Tis analysis highlights a signifcant reduction in the ratio of TIMP2 versus metalloprotease RPKM in these cancer types when compared with normal breast and lung tissues, Fig. 1E. In accordance with our working hypothesis, the results suggest that the expression of these metzincin protease targets overwhelms the already low levels of TIMP2 in these pathologic tissues, which would result in a reduction of non-protease associated TIMP2 activity. We fnd that, in lung and breast carcinomas versus normal tissue, the balance between TIMP2 and these known molecular targets is clearly shifed in favor of metallopro- teinase expression in tumor tissues (Fig. 1E).

TIMP2 displays a unique gene co-expression profle in carcinomas. In order to initially examine possible relationships between individual TIMP family members and the whole transcriptome based on expres- sion data, we used cBioPortal to harvest Pearson’s correlation scores from 32 TCGA RNA-Seq studies. We then performed principle component analysis (PCA) on correlation scores to gauge the similarity or diferences for each TIMP co-expression profle amongst all cancer types within TGCA study repertoire, Fig. 2A. In the TIMP2 and TIMP4 co-expression profles we observed distinct clustering for the carcinomas. Whereas the TIMP1 and TIMP3 co-expressed gene profles from cancers of various origins were more interspersed with less apparent grouping of any specifc cancer type (carcinoma, sarcoma, hematologic). However, distinct outliers were observed in the co-expression pattern irrespective of the TIMP (e.g. kidney chromophobe cancer (KC) in the TIMP1 and TIMP2 co-expression profles). It is well documented that TIMP4 gene expression is generally quantitatively lower and more tissue restricted than TIMP215 (Supplementary Tables I and II), thus focusing our study further on TIMP2 and coinciding with our interest in identifcation of potential modifers of TIMP2 function.

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A TIMP1 B TIMP2 E 2 * 2 TIMP2 / (MMPs + ADAM12) * * 1 * 1 Breast * * +1.5 FC +1.5 FC 2.0 **** 0 0 * - 1.5 FC * - 1.5 FC -1 -1 Log2 FC ** Log2 FC 1.5 * -2 -2 1.0 -3 -3 t k C t e C Liver Colon Liver Colon Breas Adeno Breas Adeno Normalized RPKM Kidney Thyroid Bladder Kidney Thyroid 0.5 Bladder ProstateStomach ProstateStomach Lung SC Lung SC Esophageal Esophageal Head & Nec Lung Head & NeckLung Chromophobe Papillary Renal Chromophob Papillary Renal 0.0 Normal Tumor CD TIMP3 TIMP4 Lung 2 2 1.5

1 1 +1.5 FC +1.5 FC ****

0 0 1.0 * * *- 1.5 FC - 1.5 FC -1 * -1 * * Log2 FC * Log2 FC * * -2 * * -2 * 0.5 *

* Normalized RPKM -3 -3 t e C l t e C l Liver Liver Breas Colon Adeno Breas Colon Adeno Bladder Kidney Thyroid Kidney Thyroid ProstateStomach Bladder ProstateStomach 0.0 Lung SC Lung SC Esophageal Esophageal NormalTumor Head & NeckLung Head & NeckLung Chromophob Papillary Rena Chromophob Papillary Rena

Figure 1. TIMP1–4 display variable levels of diferential expression in tumor vs. normal tissue. (A–D) Diferential expression for TIMPs 1–4 in tumor vs. normal tissue were harvested through BioXpress and presented as log2 FC (fold change) following DEseq normalization and analysis of TCGA data, * indicating signifcance. (E) TIMP2 RPKM transcript counts were obtained through the Recount2 platform and normalized using division of TIMP2 RPKM by the sum of MMP and ADAM12 RPKM to show that the TIMP2:metalloproteinase balance is ofen disrupted tumors. ****p < 0.000, two-tailed t-test.

Following comparison of the pan-cancer correlating genes for each individual TIMP co-expression profle, Fig. 2B, we noted that TIMP2 and TIMP3 were highly similar, sharing 50% of their top 200 correlating genes. Whereas TIMP1/4 were much more distinct from the other family members (~75% unique identity) in their individual co-expression patterns. Te observation that, independent of cancer subtype, TIMP2 and TIMP3 exhibit a close similarity in the pattern of co-expressed genes (50% shared identity) implies a potential and pre- viously unappreciated functional parallelism may exist between these co-expressed gene sets that may extend beyond targeting of protease activity. What identifes TIMP2 as the more interesting candidate over TIMP3 is that TIMP2 generally displays the largest number of highly correlating genes (Pearson’s correlation >0.7) across the TIMP family in carcinomas, Fig. 2C. Specifc numbers of highly correlating genes for each study are supplied in Supplementary Table III. A pan-cancer hierarchical clustering analysis of Pearson’s scores (containing only genes that display at least −/+0.4 correlation for any one cancer sub-type) for TIMP2 correlating genes highlights a study cluster of 12 carcinomas that display highly similar co-expression profles for TIMP2, Fig. 2D. Tis suggests that if any of these co-expressed genes modulate TIMP2 function, then it is a common occurrence in carcinomas. To examine this potential efect in more detail we further focused our study to the co-expression profles for TIMP2 in normal and carcinomatous lung and breast tissues.

TIMP2 and protease targets exhibit a distinct expression pattern in tumors. Our earlier analysis of TIMP2 co-expression profles identifed genes that correlate with TIMP2 in cancers, highlighting a need to understand the TIMP2 co-expressed genes in normal tissues. To do this, we frst identifed a list of 334 genes of interest (GOI) consisting of 300 genes that consistently display a positive correlation with TIMP2 across all cancers types, plus 34 genes we identifed as TIMP2 interacting partners, such as the metzincin proteases (MMPs and ADAM12), as well as putative TIMP2 binding partners LRP1/2, extracellular Src kinase, integrin α3β1, and IGFR1 (listed in Supplementary Table IV). We then harvested normalized expression data for the GOI cohort from lung and breast TCGA (tumor and normal tissue) and GTEx (post-mortem normal tissue) studies using Recount2, Fig. 3. We focused specifcally on lung and breast carcinomas for several reasons. First, these are com- mon cancers that are within our earlier identifed study cluster (Fig. 2D) with signifcant impact on the global

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AC TIMP1 TIMP2 Most highly correlating genes (>0.7) 0.4 KC TGCC 15 0.6 SCM UM 0.0

0.3 10

SA PC2 (13.7%)

-0.4 (22 Totlal) PC2 (12.9%) 0.0 C Type 5 -0.8 -0.3 KC -0.2 0.0 0.2 0.4 -0.2 0.0 0.2 0.4 PC1 (18.1%) PC1 (17.0%) # of Studies 0 TIMP3 TIMP4 0.6 0.25 M TIMP1 TIMP2 TIMP3 TIMP4

0.00 0.3

-0.25 0.0 D Color Key PC2 (8.7%) PC2 (11.1%)

-0.50 -0.3 C T TGCC

-0.2 0.0 0.2 -0.25 0.00 0.25 0.50 PC1 (14.9%) PC1 (13.9%) Pearson Correlation

B Top 200 pan-cancer correlating genes TIMP2 TIMP3

TIMP1 TIMP4

Study Cluster (12 carcinomas)

Figure 2. TIMP2 displays a unique co-expression profle in carcinomas. (A) TIMP1–4 Pearson’s correlation with the whole transcriptome across the TCGA dataset (32 studies) were collected through cBioportal and principle component analysis was performed on the full data. (B) Venn diagram depicting the similarity/dissimilarity between TIMP1–4 in their co-expression profles. (C) TIMP2 displays, in general, the most highly correlating (>0.7) genes in carcinomas in comparison to other members of the TIMP family. (D) Hierarchical clustering performed on Pearson’s correlating genes (>0.4 correlation) to highlight a study cluster of 12 carcinomas that display similar TIMP2 co-expression patterns within the TCGA dataset. Abbreviations; C: Cholangiocarcinoma, KC: Kidney Chromophobe, M: Mesothelioma, SCM: Skin Cutaneous Melanoma, T: Tymoma, TGCC: Testicular Germ Cell Cancer, UM: Uveal Melanoma.

patient population16 and prior studies have suggested a correlation between TIMP2 expression levels and prog- nostic outcome in these cancer populations17–19. In addition, earlier work in our laboratory suggests that TIMP2 delivers signifcant therapeutic benefts in murine lung and breast cancer models12,20,21. PCA of the normalized gene counts for the 334 GOI harvested from Recount2 clearly distinguish breast and lung carcinoma from their normal tissue counterparts, Fig. 3A. However, upon closer inspection these data reveal no clear demarcation between the major subtypes of breast (ductal vs. lobular) or lung cancer (adenocarcinoma vs. squamous cell carcinoma). Previous studies clearly discriminate between gene expression profles of these carcinoma subtypes22,23. Te failure of TIMP2 co-expressed gene patterns to discriminate lobular vs. ductal breast cancers, or squamous vs. adenocarcinomas in the lung may suggest that the 334 GOI profle refects the host response in the tumor microenvironment (TME) rather than tumor autonomous gene expression patterns. Although this explanation awaits future refned analysis of specifc tumor compartments (malignant vs. TME),

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Breast ABC Tumor Color Key Normal and Histogram 00 0 Ductal Cluster Dendrogram

Cluster Dendrogram 00 04 Lobular (Euclidean, complete linkage) Count Normal 02

0.04 −2 −1 012

200 Row Z−Score

TCGA−AR−A2LK−01ATCGA−BH−A0HI−01ATCGA−BH−A0DS−01ATCGA−AR−A1ATCGA−D8−A1J9−01ATCGA−BH−A1EV−01ATCGA−A2−A04V−01AV−01A TCGA−BH−A5J0−01ATCGA−GM−A2D9−01ATCGA−3C−AALI−01ATCGA−A8−A08A−01ATCGA−D8−A1XD−01ATCGA−A8−A0A9−01ATCGA−A2−A0YC−01ATCGA−A8−A08G−01A TCGA−E2−A10A−01ATCGA−A8−A090−01TCGA−E9−A6HE−01ATCGA−BH−A0B4−01TCGA−A8−A07F−01ATCGA−E2−A15K−01ATCGA−OL−A66J−01ATCGA−EW−A1OZ−01AAA TCGA−E9−A2JS−01ATCGA−LL−A7T0−01ATCGA−E2−A14O−01ATCGA−LL−A50Y−01ATCGA−ATCGA−AN−A0FZ−01ATCGA−AR−A0TQ−01ATCGA−A2−A0T3−01AC−A5XU−01A TCGA−BH−A0BV−01ATCGA−E9−A1RC−01ATCGA−E9−A247−01TCGA−B6−A0WW−01TCGA−BH−A0DT−01ATCGA−C8−A26V−01ATCGA−AN−A0XR−01ATCGA−AN−A0XP−01AAA TCGA−AR−A1AS−01ATCGA−B6−A0WS−01ATCGA−D8−A73X−01ATCGA−A8−A0A1−01ATCGA−AN−A0FD−01ATCGA−D8−A73U−01ATCGA−A7−A26I−01ATCGA−A2−A04R−01A TCGA−E2−A1LB−01ATCGA−AR−A0TW−01ATCGA−BH−A18R−01ATCGA−A8−A092−01TCGA−E9−A1RA−01ATCGA−ATCGA−EW−A1P5−01Q−A0Y5−01AQ−A04L−01BAA 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TCGA−E2−A15A−01ATCGA−D8−A1JF−01ATCGA−EW−A6SD−01TCGA−C8−A12Z−01ATCGA−BH−A0H6−01ATCGA−C8−A12P−01ATCGA−A8−A084−01AA TCGA−BH−A1ES−01ATCGA−EW−A1P6−01TCGA−E2−A15C−01TCGA−AN−A046−01ATCGA−E2−A1B0−01ATCGA−BH−A203−01ATCGA−ATCGA−A8−A09I−01AO−A12D−01AAA TCGA−AR−A24R−01ATCGA−A8−A082−01TCGA−BH−A0BT−01ATCGA−A2−A0YH−01ATCGA−ATCGA−A1−A0SJ−01ATCGA−E2−A10B−01AO−A03L−01AQ−A1H3−01AA TCGA−AN−A049−01ATCGA−A8−A096−01TCGA−AN−A04A−01ATCGA−E9−A22B−01ATCGA−EW−A1PH−01TCGA−A1−A0SD−01ATCGA−LL−A5YL−01ATCGA−C8−A135−01AAA TCGA−C8−A12T−01ATCGA−B6−A0RI−01ATCGA−D8−A1XS−01ATCGA−D8−A1JD−01ATCGA−AN−A0XW−01ATCGA−E9−A24A−01ATCGA−AR−A24L−01ATCGA−E2−A1IG−01A TCGA−BH−A18U−01ATCGA−AN−A0XS−01ATCGA−E9−A1NA−01ATCGA−D8−A1JE−01ATCGA−AN−A0FY−01ATCGA−D8−A1Y1−01TCGA−A8−A06P−01ATCGA−E9−A1N5−01A TCGA−5L−AATCGA−AR−A254−01ATCGA−A2−A0SV−01TCGA−E2−A9TCGA−BH−A1EY−01ATCGA−B6−A0RH−01ATCGA−E9−A1R5−01TCGA−C8−A1HF−01AT1−01ARU−01AA TCGA−BH−A0ATCGA−D8−A1XY−01ATCGA−E2−A10F−01ATCGA−A2−A04N−01TCGA−A2−A0YD−01ATCGA−E9−A1RG−01ATCGA−A2−A0T0−01ATCGA−A8−A06R−01U−01AA TCGA−BH−A0ATCGA−A1−A0SN−01ATCGA−E2−A15L−01TCGA−E9−A1NH−01ATCGA−B6−A0RP−01ATCGA−A7−A6VX−01TCGA−A7−A26H−01TCGA−AO−A12C−01AY−01AAA TCGA−BH−A0W5−01ATCGA−A8−A086−01TCGA−ATCGA−A8−A07J−01ATCGA−A8−A08Z−01ATCGA−A2−A1FZ−01AC−A3HN−01C−A6IV−01AC−A3BB−01AAA TCGA−BH−A0HA−01ATCGA−GM−A5PX−01ATCGA−E2−A1BC−01ATCGA−B6−A40C−01TCGA−A8−A08O−01ATCGA−ATCGA−OL−A5D6−01TCGA−BH−A0EB−01AC−A3W7−01AA TCGA−BH−A1FB−01ATCGA−AR−A255−01ATCGA−ATCGA−A2−A25C−01TCGA−E2−A10E−01ATCGA−E2−A14V−01ATCGA−BH−A0GZ−01ATCGA−A1−A0SG−01AC−A23C−01AA TCGA−EW−A1IX−01ATCGA−A8−A0AD−01ATCGA−BH−A0C3−01ATCGA−D8−A27G−01ATCGA−EW−A6SC−01TCGA−BH−A0B5−01TCGA−D8−A27T−01ATCGA−A2−A0CP−01AAA TCGA−A1−A0SE−01TCGA−A2−A0CV−01ATCGA−BH−A28O−01ATCGA−A1−A0SH−01ATCGA−BH−A0EI−01ATCGA−A1−A0SM−01TCGA−C8−A12N−01ATCGA−AO−A0J5−01AAA TCGA−ATCGA−S3−AA14−01TCGA−A2−A0YL−01ATCGA−BH−A18M−01ATCGA−MS−A51U−01ATCGA−AR−A5QN−01ATCGA−A7−A4SC−01ATCGA−AN−A0FS−01AC−A2FE−01AA TCGA−BH−A28Q−01ATCGA−D8−A1Y0−01TCGA−EW−A424−01ATCGA−GM−A2TCGA−A2−A1G0−01ATCGA−E9−A1NE−01ATCGA−AO−A0JC−A6IX−01ADAA−01−01AA TCGA−BH−A1FH−01ATCGA−D8−A1JU−01ATCGA−D8−A1XU−01ATCGA−B6−A0RN−01ATCGA−D8−A1XO−01ATCGA−AR−A1AL−01TCGA−A7−A26E−01ATCGA−A8−A09V−01AA TCGA−BH−A0BM−01ATCGA−AR−A1AK−01ATCGA−B6−A0TCGA−E9−A5FK−01ATCGA−ATCGA−D8−A1JB−01ATCGA−AR−A1AX−01ATCGA−A2−A0YK−01C−A2FB−01RV−01AA TCGA−A2−A1FX−01ATCGA−AR−A252−01ATCGA−ATCGA−E2−A15I−01ATCGA−C8−A26W−01ATCGA−A2−A259−01TCGA−E2−A15P−01ATCGA−E2−A14Q−01AO−A0J9−01AA TCGA−E9−A227−01TCGA−E9−A1R0−01TCGA−OK−A5Q2−01ATCGA−BH−A0EA−01ATCGA−AR−A1AM−01ATCGA−E9−A1QZ−01ATCGA−GM−A4E0−01TCGA−EW−A2FS−01AAAA TCGA−A7−A4SA−01TCGA−ATCGA−D8−A3Z6−01ATCGA−E9−A1N9−01TCGA−A2−A3XS−01TCGA−BH−A42V−01ATCGA−LD−A7W5−01ATCGA−BH−A2L8−01AO−A03V−01AA TCGA−AN−A0XL−01TCGA−ATCGA−A7−A3J1−01ATCGA−A2−A0SY−01TCGA−OL−A66N−01TCGA−LD−A66U−01ATCGA−OL−A66L−01TCGA−A7−A425−01C−A2FO−01AAA TCGA−E9−A22H−01TCGA−AR−A24K−01ATCGA−A2−A25E−01ATCGA−A7−A0CD−01ATCGA−E2−A1IE−01ATCGA−A8−A09M−01ATCGA−ATCGA−A1−A0SF−01AC−A5XS−01AA TCGA−D8−A1XG−01ATCGA−BH−A0E7−01TCGA−A8−A07Z−01ATCGA−BH−A0B8−01TCGA−A8−A06Y−01ATCGA−B6−A0X0−01ATCGA−A2−A0CT−01ATCGA−A8−A08C−01A 100 TCGA−E2−A15R−01TCGA−EW−A1OY−01AA TCGA−A8−A07R−01TCGA−EW−A1J3−01ATCGA−BH−A1EW−01ATCGA−BH−A1FL−01ATCGA−GM−A3NW−01ATCGA−C8−A1HI−01ATCGA−ATCGA−D8−A1X8−01O−A1KS−01AA TCGA−ATCGA−S3−A6ZG−01ATCGA−A8−A09N−01TCGA−A8−A07P−01ATCGA−A8−A0AB−01TCGA−BH−A1FG−01ATCGA−BH−A1F5−01AO−A12G−01AO−A1KP−01AA TCGA−E9−A295−01TCGA−D8−A1X5−01TCGA−A2−A0EM−01TCGA−E9−A1RD−01ATCGA−A8−A0A2−01ATCGA−C8−A26Z−01ATCGA−A8−A09B−01ATCGA−B6−A0WV−01AAAA TCGA−E2−A2P5−01ATCGA−E2−A1BD−01ATCGA−E2−A1B4−01ATCGA−EW−A1P3−01TCGA−ATCGA−E2−A15E−01ATCGA−E9−A1R2−01TCGA−OL−A5O−A12A−01ADA−01AAA TCGA−PE−A5DD−01ATCGA−V7−A7HQ−01ATCGA−ATCGA−E2−A105−01TCGA−Z7−A8R6−01ATCGA−C8−A130−01ATCGA−JL−A3YX−01ATCGA−S3−A6ZH−01AO−A0J7−01AA TCGA−ATCGA−C8−A12Y−01ATCGA−A2−A0CS−01ATCGA−E2−A1L8−01TCGA−LL−A9Q3−01ATCGA−OL−A66O−01ATCGA−E2−A576−01TCGA−BH−A0HU−01AO−A126−01AA TCGA−AR−A2LO−01ATCGA−A2−A4TCGA−EW−A1PC−01TCGA−B6−A0I8−01ATCGA−A8−A093−01TCGA−ATCGA−BH−A42T−01AC−A3W6−01ARYRW−01A−01AAB 0.00 TCGA−AR−A1ATCGA−AR−A24T−01ATCGA−BH−A0DE−01ATCGA−ATCGA−B6−A0IP−01ATCGA−AR−A5QP−01ATCGA−A7−A26G−01ATCGA−E2−A1AZ−01AO−A03U−01BU−01A TCGA−E9−A1RI−01ATCGA−D8−A1JH−01ATCGA−D8−A27L−01ATCGA−BH−A18G−01ATCGA−AR−A24N−01ATCGA−A8−A07B−01ATCGA−A8−A09T−01ATCGA−A7−A0CH−01A TCGA−EW−A1J2−01ATCGA−ATCGA−A8−A07G−01ATCGA−E2−A1IO−01ATCGA−D8−A1JP−01ATCGA−BH−A0BF−01ATCGA−A7−A5ZW−01ATCGA−BH−A18I−01AC−A2B8−01A MMP7 TCGA−AR−A24O−01ATCGA−BH−A1FE−01ATCGA−BH−A208−01ATCGA−AR−A24M−01ATCGA−AR−A24P−01ATCGA−D8−A1XM−01ATCGA−BH−A0DTCGA−E2−A1IJ−01AV−01A TCGA−WT−AB44−01ATCGA−3C−AALK−01TCGA−E2−A570−01TCGA−A8−A07E−01ATCGA−AR−A2LM−01ATCGA−B6−A0X7−01ATCGA−OL−A6VQ−01ATCGA−A2−A0EW−01AAA TCGA−5L−AATCGA−GM−A3XN−01TCGA−E2−A1IF−01ATCGA−AN−A0AS−01ATCGA−BH−A0W7−01ATCGA−BH−A18K−01ATCGA−A2−A0EV−01TCGA−D8−A146−01AT0−01AAA TCGA−BH−A0BP−01ATCGA−AN−A0XO−01ATCGA−BH−A0B7−01TCGA−A2−A0T7−01ATCGA−D8−A1XB−01ATCGA−4H−AAAK−01TCGA−BH−A0DI−01ATCGA−A2−A0EO−01AA TCGA−AR−A2LQ−01ATCGA−EW−A1P1−01TCGA−E2−A1IH−01ATCGA−XX−A89A−01ATCGA−XX−A899−01TCGA−E2−A1B1−01ATCGA−A2−A0CX−01ATCGA−C8−A273−01AAA TCGA−ATCGA−D8−A27I−01ATCGA−EW−A1PG−01ATCGA−A8−A08H−01TCGA−B6−A0WX−01ATCGA−LL−A440−01TCGA−BH−A0B0−01C−A2FF−01C−A3YI−01AAA TCGA−ATCGA−A7−A426−01TCGA−A2−A25A−01ATCGA−A2−A3KC−01ATCGA−A7−A5ZX−01ATCGA−HN−A2OB−01ATCGA−A2−A0CO−01ATCGA−E9−A3X8−01AC−A3QP−01AA TCGA−BH−AB28−01TCGA−AR−A2LR−01ATCGA−BH−A0BO−01ATCGA−ATCGA−E2−A3DX−01ATCGA−C8−A8HR−01ATCGA−BH−A18M−11ATCGA−B6−A0IH−01AC−A8OS−01AA TCGA−D8−A4Z1−01ATCGA−BH−A0BA−01ATCGA−LD−A7W6−01ATCGA−A2−A0CK−01ATCGA−AR−A2LN−01ATCGA−W8−A86G−01ATCGA−OL−A5RX−01ATCGA−OL−A5RV−01A TCGA−ATCGA−D8−A145−01ATCGA−A7−A0CG−01ATCGA−D8−A27V−01ATCGA−BH−A0HP−01ATCGA−BH−A0E9−01TCGA−BH−A1ET−01ATCGA−AR−A24W−01AO−A0JJ−01AB MMP7 TCGA−ATCGA−E9−A1N5−11TCGA−BH−A0BV−11ATCGA−A2−A0ES−01C−A2FF−11A TCGA−AR−A1ATCGA−BH−A0BG−01ATCGA−A2−A0D2−01TCGA−A2−A04Q−01ATCGA−AR−A256−01ATCGA−E9−A3QA−01ATCGA−E9−A1ND−01ATCGA−AO−A129−01AQ−01AA TCGA−AR−A1ATCGA−BH−A18V−01ATCGA−BH−A0RX−01ATCGA−A2−A0YM−01TCGA−D8−A27F−01ATCGA−BH−A0DL−01ATCGA−E2−A573−01TCGA−C8−A12K−01AY−01AAA 50 TCGA−C8−A27B−01ATCGA−A8−A07O−01ATCGA−AR−A0U4−01ATCGA−A2−A0D0−01TCGA−S3−AA15−01TCGA−E2−A1LI−01ATCGA−AN−A0AT−01A PC2 (27.19% ) TCGA−BH−A0WTCGA−GM−A2DK−01ATCGA−LL−A5YM−01TCGA−AR−A0TY−01ATCGA−GM−A2DF−01AA−01AA TCGA−ATCGA−E2−A1LL−01TCGA−A2−A0EY−01TCGA−E9−A5FL−01ATCGA−B6−A0IK−01ATCGA−BH−A5IZ−01ATCGA−AN−A0G0−01ATCGA−C8−A134−01AC−A8OQ−01AAA TCGA−BH−A0H3−01ATCGA−BH−A0DP−01ATCGA−A2−A0T4−01ATCGA−A2−A25F−01ATCGA−A2−A0SW−01ATCGA−AR−A0TT−01ATCGA−A8−A0A7−01ATCGA−B6−A0RG−01A TCGA−EW−A3E8−01TCGA−A2−A4S2−01ATCGA−ATCGA−GM−A2DI−01ATCGA−BH−A18F−01ATCGA−E2−A1IL−01ATCGA−BH−A0C7−01BTCGA−A2−A0EX−01Q−A7U7−01AAB TCGA−BH−A8FZ−01ATCGA−A2−A0CL−01TCGA−A2−A0CR−01ATCGA−A7−A13H−01TCGA−BH−A1EU−01ATCGA−BH−A0B6−01TCGA−A2−A0YI−01ATCGA−GI−A2C8−01AA TCGA−A8−A09D−01TCGA−A2−A04W−01ATCGA−BH−A0DQ−01ATCGA−AN−A0FV−01ATCGA−BH−A18J−01ATCGA−D8−A1JJ−01TCGA−AR−A2LJ−01ATCGA−LD−A74U−01AAA MMP9 TCGA−ATCGA−B6−A0WY−01AC−A2QI−01A TCGA−AN−A041−01ATCGA−A7−A0TCGA−B6−A400−01TCGA−BH−A0BC−01ATCGA−GM−A2DD−01ATCGA−BH−A0HQ−01ATCGA−BH−A0BJ−01ATCGA−AR−A5QM−01ADA−01AA TCGA−B6−A408−01TCGA−A7−A56D−01TCGA−LL−A6FQ−01ATCGA−B6−A401−01TCGA−E2−A150−01TCGA−A7−A3IY−01ATCGA−A2−A0SX−01TCGA−LL−A6FR−01AAA MMP1 1 TCGA−3C−AALJ−01ATCGA−BH−A201−01ATCGA−EW−A1P7−01TCGA−LL−A73Y−01ATCGA−ATCGA−A2−A0CZ−01AC−A23G−01AA TCGA−A7−A4SF−01ATCGA−D8−A1X7−01TCGA−D8−A1XJ−01ATCGA−A8−A06X−01ATCGA−BH−A0HX−01ATCGA−AR−A1ATCGA−BH−A0DX−01ATCGA−A8−A08R−01O−01AA MMP1 TCGA−A2−A4S3−01ATCGA−C8−A26X−01ATCGA−BH−A209−01ATCGA−C8−A1HL−01ATCGA−E2−A109−01A TCGA−ATCGA−A8−A07W−01ATCGA−C8−A12W−01ATCGA−D8−A1X9−01TCGA−BH−A0E2−01TCGA−AR−A0U3−01ATCGA−A2−A0YG−01ATCGA−B6−A0I5−01AO−A1KQ−01AA TCGA−D8−A141−01ATCGA−A8−A097−01TCGA−AR−A250−01ATCGA−BH−A1F2−01ATCGA−AR−A24Q−01ATCGA−A8−A08J−01ATCGA−ATCGA−AN−A0FF−01AO−A03N−01BA TCGA−E9−A1NI−01ATCGA−AR−A24V−01ATCGA−E2−A153−01TCGA−E9−A1NF−01ATCGA−E9−A1NG−01ATCGA−AN−A0XN−01ATCGA−AC−A5EH−01C−A23E−01AAA TCGA−A7−A4SE−01TCGA−A2−A0EP−01TCGA−AR−A1AN−01ATCGA−BH−A1FN−01ATCGA−C8−A12O−01ATCGA−B6−A40B−01ATCGA−A1−A0SI−01ATCGA−E9−A226−01AA TCGA−A2−A4RX−01ATCGA−A7−A6VV−01TCGA−PL−A8LZ−01ATCGA−D8−A147−01ATCGA−OL−A5S0−01ATCGA−D8−A1JL−01ATCGA−E9−A1NC−01ATCGA−OL−A5RY−01AA MMP3 TCGA−A7−A4SD−01ATCGA−ATCGA−AN−A0AL−01TCGA−A7−A6VW−01ATCGA−E9−A244−01O−A0J4−01AAA MMP9 TCGA−A2−A0YE−01TCGA−LL−A73Z−01AA TCGA−A7−A26F−01ATCGA−A2−A0T1−01ATCGA−BH−A0BL−01TCGA−C8−A8HQ−01ATCGA−A2−A1G1−01ATCGA−D8−A27M−01ATCGA−E2−A1LH−01TCGA−D8−A1XQ−01AA AM12 TCGA−A7−A2KD−01ATCGA−BH−A0BQ−01ATCGA−ATCGA−E2−A14Z−01ATCGA−BH−A0DK−01ATCGA−AN−A0FX−01ATCGA−BH−A0W4−01ATCGA−EW−A1PD−01TCGA−AR−A5QQ−01O−A1KT−01AA MMP1 TCGA−BH−A0HF−01ATCGA−C8−A12Q−01ATCGA−BH−A0GY−01ATCGA−B6−A0I6−01ATCGA−BH−A0DZ−01A MMP11 TCGA−E2−A1LA−01ATCGA−D8−A13Z−01ATCGA−B6−A0IM−01ATCGA−ATCGA−D8−A1JC−01AO−A0J8−01A TCGA−E9−A22A−01ATCGA−ATCGA−C8−A138−01ATCGA−LL−A740−01TCGA−C8−A132−01ATCGA−A8−A07I−01ATCGA−AR−A24S−01AC−A3EH−01O−A0JM−01AAA MMP1 3 TCGA−E9−A1RF−01ATCGA−EW−A1IY−01ATCGA−EW−A1IW−01ATCGA−BH−A0BD−01ATCGA−D8−A27N−01ATCGA−D8−A27H−01ATCGA−BH−A0DG−01A MMP1 2 TCGA−C8−A8HP−01ATCGA−D8−A1JG−01BTCGA−B6−A0RS−01ATCGA−E2−A159−01TCGA−BH−A0B3−01AA 0 TCGA−C8−A12L−01ATCGA−BH−A18P−01ATCGA−EW−A1PE−01ATCGA−BH−A0ATCGA−AR−A24U−01AW−01A AD TCGA−AN−A0AM−01A MMP1 0 TCGA−E9−A1RB−01ATCGA−A7−A6VY−01TCGA−LL−A5YP−01TCGA−EW−A1P4−01TCGA−A8−A094−01TCGA−A8−A08P−01ATCGA−EW−A1OW−01AAAA TCGA−BH−A0BZ−01ATCGA−A8−A076−01TCGA−AN−A03X−01ATCGA−BH−A0C1−01BTCGA−E2−A1IN−01ATCGA−E9−A229−01TCGA−AN−A0FN−01ATCGA−BH−A0AZ−01AA MMP3 TCGA−BH−A1EO−01ATCGA−GM−A2DL−01ATCGA−E9−A1R3−01TCGA−AO−A0JE−01AA MMP1 5 TCGA−BH−A1FD−01ATCGA−BH−A1FM−01ATCGA−A2−A0T6−01A AM12 TCGA−D8−A1JK−01ATCGA−E2−A1LE−01ATCGA−A8−A08F−01ATCGA−BH−A1EX−01A TCGA−BH−A0BR−01ATCGA−E9−A1N6−01TCGA−E9−A1RH−01ATCGA−A8−A075−01TCGA−D8−A1J8−01ATCGA−C8−A278−01ATCGA−ATCGA−C8−A131−01AC−A23H−01AAA TCGA−ATCGA−E9−A22E−01ATCGA−A8−A09R−01TCGA−E2−A1L9−01TCGA−D8−A1Y2−01TCGA−BH−A0ATCGA−A2−A3XY−01TCGA−BH−A1FU−01AC−A3W5−01AV−01AAA MMP1 9 MMP2 4 TCGA−JL−A3YW−01ATCGA−BH−A6R9−01ATCGA−EW−A2FR−01A MMP2 8 TCGA−A7−A0CJ−01ATCGA−EW−A1J1−01ATCGA−BH−A0C0−01ATCGA−D8−A1TCGA−E2−A152−01TCGA−D8−A1XT−01AJA−01AA TCGA−A2−A04X−01ATCGA−BH−A0DH−01ATCGA−E2−A56Z−01ATCGA−EW−A1P0−01TCGA−BH−A18N−01ATCGA−D8−A1XR−01ATCGA−A7−A3IZ−01ATCGA−E2−A15J−01AA TCGA−A2−A3XW−01ATCGA−A8−A079−01TCGA−LL−A7SZ−01ATCGA−D8−A1XL−01TCGA−ATCGA−AR−A0U2−01ATCGA−C8−A1HN−01ATCGA−UL−AAZ6−01AQ−A04H−01BAA MMP13 TCGA−AN−A0AK−01ATCGA−BH−A0HL−01A MMP1 4 TCGA−E2−A14S−01ATCGA−BH−A0HW−01ATCGA−ATCGA−EW−A6SA−01ATCGA−B6−A0IB−01AO−A12H−01A MMP1 7 MMP2 5 TCGA−AN−A04C−01ATCGA−AR−A0TV−01ATCGA−E9−A5UP−01ATCGA−C8−A1HO−01ATCGA−OL−A5RZ−01ATCGA−C8−A12X−01A MMP12 TCGA−A2−A1G4−01ATCGA−D8−A1JT−01ATCGA−A1−A0SO−01A TCGA−ATCGA−A8−A081−01TCGA−C8−A26Y−01ATCGA−A2−A0YF−01ATCGA−E2−A156−01TCGA−A8−A06Z−01ATCGA−AN−A04D−01ATCGA−C8−A1HM−01AC−A8OR−01AA MMP2 TCGA−AR−A24H−01A MMP1 6

TIMP 2 TCGA−AO−A0JC−01A MMP10 TCGA−B6−A0IA−01ATCGA−D8−A1X6−01TCGA−OL−A6VR−01ATCGA−E9−A5UO−01ATCGA−ATCGA−S3−AA17−01TCGA−OL−A6O−A0JL−01AVO−01AA TCGA−D8−A1Y3−01TCGA−E9−A22D−01TCGA−E2−A10C−01TCGA−S3−AA0Z−01ATCGA−AR−A0TP−01ATCGA−BH−A0EE−01ATCGA−UU−A93S−01ATCGA−A2−A0CY−01AA AD MMP15 TCGA−ATCGA−D8−A140−01ATCGA−EW−A1J5−01AO−A03P−01AO−A0JG−01AC−A2BM−01A MMP23B TCGA−B6−A0IN−01ATCGA−OL−A66H−01TCGA−ATCGA−B6−A1KI−01AC−A6NO−01AA TCGA−ATCGA−E2−A1IU−01ATCGA−B6−A0IO−01ATCGA−AN−A0XT−01ATCGA−A2−A25D−01TCGA−OL−A66P−01ATCGA−OL−A5O−A03O−01AO−A0JB−01ARU−01AA MMP24 TCGA−E9−A248−01TCGA−E2−A15T−01ATCGA−GM−A2DM−01ATCGA−BH−A0HO−01ATCGA−E2−A1B5−01ATCGA−ATCGA−E9−A2JT−01ATCGA−EW−A1O−A03T−01AOV−01AA MMP19 TCGA−E9−A243−01TCGA−BH−A0DO−01BTCGA−E2−A15F−01AA MMP28 TCGA−BH−A18H−01ATCGA−A7−A0DB−01A MMP2 0 TCGA−E2−A1L6−01TCGA−GM−A2DN−01AA TCGA−BH−A0E1−01TCGA−ATCGA−A2−A0EQ−01ATCGA−B6−A0TCGA−E2−A14T−01ATCGA−LL−A5YN−01ATCGA−GM−A2DO−01ATCGA−WT−AB41−01AC−A3OD−01ART−01AA MMP8 TCGA−E2−A14R−01TCGA−BH−A0H5−01AA

MMP2 7 TCGA−A8−A08I−01A MMP14 TCGA−LL−A441−01TCGA−AR−A1ATCGA−AR−A1AR−01AW−01AA MMP25 TCGA−D8−A1XK−01A MMP17 TCGA−OL−A97C−01TCGA−HN−A2NL−01AA TIMP2 TCGA−AN−A0AJ−01ATCGA−LL−A5YTCGA−B6−A0IG−01ATCGA−C8−A275−01ATCGA−PL−A8LVO−01−01AA MMP2 TCGA−GM−A2DB−01ATCGA−AR−A0U0−01ATCGA−BH−A0E6−01TCGA−AN−A0FJ−01ATCGA−AR−A1AJ−01ATCGA−BH−A1F6−01ATCGA−E2−A15M−01ATCGA−E2−A108−01AA TCGA−A8−A07U−01TCGA−E2−A15O−01ATCGA−BH−A0HB−01ATCGA−A8−A0A6−01ATCGA−B6−A1KN−01ATCGA−A8−A08B−01ATCGA−BH−A202−01ATCGA−E2−A1B6−01AA MMP16 TCGA−PE−A5DE−01A TCGA−EW−A1IZ−01ATCGA−A2−A04U−01TCGA−A8−A06O−01ATCGA−BH−A0DD−01ATCGA−A8−A06U−01TCGA−A7−A13F−01ATCGA−GM−A2DH−01ATCGA−A8−A08T−01AA −0.04 MMP2 1 MMP2 6 TCGA−C8−A1HG−01ATCGA−E2−A1L7−01A TCGA−E9−A1R4−01TCGA−A2−A0T2−01ATCGA−A8−A091−01TCGA−B6−A0I9−01ATCGA−A2−A04T−01ATCGA−BH−A0H9−01ATCGA−D8−A27W−01ATCGA−EW−A1PA−01AAA TCGA−D8−A1XW−01ATCGA−AR−A0TS−01ATCGA−A2−A3Y0−01ATCGA−ATCGA−A2−A0CW−01ATCGA−A2−A04Y−01ATCGA−A7−A0CE−01AO−A0J2−01AO−A03M−01B TCGA−AN−A0FL−01ATCGA−B6−A1KF−01ATCGA−E2−A14P−01ATCGA−A2−A3XX−01TCGA−D8−A143−01ATCGA−A2−A0YJ−01ATCGA−E9−A228−01TCGA−AC−A62Y−01AAA MMP23B TCGA−C8−A3M8−01ATCGA−D8−A1XA−01ATCGA−A8−A07S−01ATCGA−A2−A0D1−01A TCGA−S3−AA11−01TCGA−E9−A249−01TCGA−A8−A06T−01ATCGA−OL−A5D8−01TCGA−ATCGA−AR−A2LL−01ATCGA−AN−A0FK−01AC−A3TM−01C−A3TN−01AAA MMP8 TCGA−A8−A085−01TCGA−ATCGA−PE−A5DC−01AQ−A54O−01AA MMP20 TCGA−C8−A27A−01ATCGA−BH−A1FR−01ATCGA−EW−A423−01ATCGA−A2−A3XV−01TCGA−D8−A13Y−01ATCGA−3C−AATCGA−A8−A08L−01AU−01AAA MMP27 TCGA−A2−A04P−01ATCGA−A1−A0SK−01TCGA−E9−A3HO−01ATCGA−ATCGA−BH−A6R8−01ATCGA−EW−A2FW−01ATCGA−A7−A3RF−01AC−A62V−01AA TCGA−BH−A18Q−01ATCGA−E2−A14X−01ATCGA−E2−A1IK−01ATCGA−B6−A0I2−01ATCGA−GM−A5PV−01ATCGA−B6−A0I1−01ATCGA−AN−A0AR−01ATCGA−B6−A0IQ−01A TCGA−E2−A574−01TCGA−ATCGA−EW−A1PB−01ATCGA−BH−A0BW−01ATCGA−B6−A0TCGA−GM−A3XG−01ATCGA−AR−A2LH−01AC−A6IW−01AC−A2FG−01RU−01AAA TCGA−Z7−A8R5−01ATCGA−E2−A14N−01TCGA−EW−A6SB−01ATCGA−E2−A1II−01ATCGA−AR−A0TU−01ATCGA−AR−A1AI−01ATCGA−A2−A3XU−01ATCGA−E2−A1LG−01AA MMP21 TCGA−ATCGA−A2−A0ST−01ATCGA−BH−A0BW−11ATCGA−C8−A12V−01ATCGA−EW−A3U0−01ATCGA−A2−A3XT−01ATCGA−BH−A42U−01AO−A128−01AQ−A04J−01A MMP26 TCGA−B6−A0IJ−01ATCGA−OL−A5D7−01TCGA−BH−A0B9−01TCGA−LL−A8F5−01ATCGA−B6−A402−01TCGA−B6−A3ZX−01AAA TCGA−OL−A66I−01ATCGA−B6−A0RE−01ATCGA−BH−A1F0−01ATCGA−OL−A5TCGA−EW−A1P8−01TCGA−AN−A0XU−01ATCGA−S3−AA10−01TCGA−A1−A0SP−01RW−01AAA TCGA−BH−A8G0−01ATCGA−E2−A1LK−01ATCGA−GI−A2C9−01TCGA−A2−A0CM−01ATCGA−ATCGA−BH−A18T−01ATCGA−BH−A1FC−01ATCGA−AR−A1AH−01AO−A0J6−01AA TCGA−BH−A1ET−11BTCGA−BH−A18N−11ATCGA−ATCGA−A2−A4S1−01ATCGA−E9−A1N4−01TCGA−LD−A9QF−01AC−A7VC−01C−A2QJ−01C−A2QH−01AAA TCGA−BH−A1F8−11BTCGA−E9−A1RB−11ATCGA−GI−A2C9−11TCGA−E2−A15M−11ATCGA−BH−A1EN−11ASRR1414661SRR65691TCGA−BH−A1FD−11B1 A TCGA−BH−A1FJ−11BSRR1341539SRR1380460TCGA−BH−A1FC−11ATCGA−BH−A1F6−11BTCGA−E9−A1RH−11ATCGA−ATCGA−BH−A1F0−11BC−A2FB−11A TCGA−BH−A209−11ATCGA−E9−A1R7−11TCGA−E9−A1NA−11ATCGA−BH−A0BZ−11ATCGA−BH−A0ATCGA−BH−A0BA−11ATCGA−E2−A1IG−11ATCGA−BH−A18L−11AY−11AA SRR1418729SRR1352457TCGA−BH−A0HK−11ASRR1077139SRR1439915SRR1356765SRR1429181TCGA−BH−A1EU−11A SRR1472102SRR1083959SRR1406224SRR82149SRR1357826SRR66028SRR1378903TCGA−AC−A23H−1183 A TCGA−BH−A0BJ−11ATCGA−ASRR1356746SRR1404442SRR81520SRR1336181TCGA−BH−A0C3−11ATCGA−BH−A208−11AO−A1K8 O−01A TCGA−BH−A0BC−11ATCGA−BH−A18U−11ATCGA−BH−A1EW−11BTCGA−BH−A0DQ−11ATCGA−BH−A1FU−11ATCGA−BH−A18K−11ATCGA−BH−A0DT−11ATCGA−BH−A0DG−11ATCGA−E2−A1L7−11A TCGA−BH−A0B7−11TCGA−BH−A0H5−11ASRR1318774TCGA−BH−A1FB−11ATCGA−BH−A18Q−11ATCGA−BH−A1FM−11BTCGA−BH−A1EV−11ASRR1498997 A dist(t(dmlog), method = "euclidean") TCGA−B6−A0RQ−01ATCGA−BH−A0DZ−11ATCGA−BH−A0BQ−11ATCGA−BH−A0B3−11TCGA−A2−A1G6−01ATCGA−A7−A13F−11ATCGA−BH−A1F2−11ATCGA−BH−A203−11AB TCGA−BH−A18S−11ATCGA−E2−A15K−11ATCGA−BH−A1FN−11ATCGA−BH−A1FH−11BTCGA−E9−A1N6−11TCGA−BH−A0DL−11ATCGA−A7−A0CE−11ATCGA−BH−A18V−11AA SRR1393026TCGA−BH−A1EO−11ATCGA−E9−A1NF−11ATCGA−A7−A0D9−11TCGA−A7−A5ZV−01ATCGA−BH−A0DO−11ATCGA−ATCGA−BH−A18J−11AC−A2FK−01AA TCGA−E9−A1RD−11ASRR1391511SRR1417725SRR1317282SRR1386945SRR66230SRR1389432TCGA−BH−A0DD−11A6 TCGA−BH−A1FR−11BTCGA−E2−A153−11TCGA−A7−A0CH−11ASRR1476950TCGA−E9−A1ND−11ASRR1369088SRR1430615SRR1071084 A hclust (*, "complete") TCGA−C8−A3M7−01ASRR1465693TCGA−BH−A0BT−11ATCGA−BH−A0DP−11ATCGA−BH−A0DH−11ATCGA−A1−A0SB−01TCGA−ASRR1369584C−A2FM−11AB TCGA−E2−A14U−01TCGA−E2−A15I−11ATCGA−BH−A18R−11ASRR1409397TCGA−BH−A0C0−11ATCGA−BH−A18P−11ATCGA−E9−A1N9−11TCGA−D8−A3Z5−01AA TCGA−A8−A0A4−01ATCGA−BH−A0H7−11ATCGA−ATCGA−A2−A3KD−01ATCGA−BH−A0DK−11ATCGA−BH−A0E0−11TCGA−E2−A1LB−11ATCGA−PL−A8O−A03R−01ALY −01AA TCGA−E2−A1LH−11TCGA−BH−A1FG−11BTCGA−BH−A1FE−11BTCGA−BH−A0ATCGA−BH−A0HA−11ATCGA−BH−A0E1−11TCGA−BH−A0DTCGA−AC−A62X−01AU−11V−11AA TCGA−E9−A22G−01ATCGA−E2−A14Y−01ATCGA−A8−A07C−01TCGA−E9−A1N8−01TCGA−E2−A1LS−01ATCGA−BH−A0BM−11ATCGA−E9−A1N4−11TCGA−BH−A0BS−11AA TCGA−ATCGA−A2−A3XZ−01ATCGA−B6−A409−01TCGA−B6−A0X1−01ATCGA−GM−A3XL−01O−A12F−01AO−A1KR−01AO−A124−01AO−A0JD−01AAA TCGA−E2−A15G−01ATCGA−A2−A0CU−01ATCGA−A2−A0D3−01TCGA−A2−A0ER−01ATCGA−BH−A1EN−01ATCGA−AN−A03Y−01ATCGA−C8−A1HE−01ATCGA−C8−A1HJ−01AA TCGA−A2−A0CQ−01ATCGA−GM−A2DC−01ATCGA−E2−A2P6−01ATCGA−C8−A12M−01ATCGA−S3−AA12−01TCGA−E9−A1R7−01TCGA−BH−A0W3−01ATCGA−B6−A0RM−01AA TCGA−A2−A0D4−01TCGA−ATCGA−E2−A106−01TCGA−A2−A4S0−01ATCGA−BH−A18S−01ATCGA−E9−A3Q9−01ATCGA−BH−A0HN−01AC−A2FM−01C−A4ZE−01AAAA TCGA−BH−A204−01ATCGA−BH−A0E0−01TCGA−D8−A1JI−01ATCGA−D8−A27E−01ATCGA−B6−A0X4−01ATCGA−ATCGA−E9−A1N3−01TCGA−LL−A6FP−01AO−A0JI−01AA TCGA−BH−A0BS−01ATCGA−A1−A0SQ−01ATCGA−E9−A245−01TCGA−A2−A0YT−01ATCGA−E2−A107−01TCGA−B6−A2IU−01ATCGA−AN−A0FW−01ATCGA−AO−A12B−01AA TCGA−A2−A1FW−01ATCGA−A7−A4SB−01TCGA−B6−A0IE−01ATCGA−A2−A1FV−01ATCGA−ATCGA−GM−A3NY−01ATCGA−B6−A0TCGA−A8−A09G−01AO−A12E−01ARO−01AA TCGA−C8−A12U−01ATCGA−PL−A8LX−01ATCGA−E2−A155−01TCGA−A8−A09Z−01ATCGA−LQ−A4E4−01ATCGA−BH−A0HK−01ATCGA−B6−A0X5−01ATCGA−C8−A1HK−01AA TCGA−A8−A083−01TCGA−E2−A572−01TCGA−D8−A1JS−01ATCGA−B6−A0IC−01ATCGA−5T−A9QA−01ATCGA−ATCGA−E2−A15S−01ATCGA−EW−A1C−A7VB−01AOX−01AA TCGA−B6−A1KC−01BTCGA−D8−A1XF−01ATCGA−A8−A09K−01ATCGA−ATCGA−EW−A1J6−01ATCGA−B6−A0RL−01TCGA−E9−A54Y−01AC−A3YJ−01O−A0J3−01AA TCGA−A8−A06Q−01ATCGA−C8−A133−01ATCGA−D8−A1JN−01ATCGA−BH−A8FY−01ATCGA−A2−A0ET−01ATCGA−BH−A18L−01ATCGA−D8−A1XV−01ATCGA−A7−A3J0−01A TCGA−E2−A158−01TCGA−AR−A0TR−01ATCGA−D8−A1XC−01ATCGA−E9−A54X−01ATCGA−A8−A06N−01TCGA−ATCGA−GI−A2C8−11TCGA−BH−A0H0−01AO−A125−01AAA SRR1455861SRR1433374SRR1365534SRR1357166TCGA−D8−A1JM−01ATCGA−ATCGA−LL−A442−01Q−A54N−01AC−A2BK−01AA TCGA−E2−A1LS−11ATCGA−A7−A13G−11ATCGA−BH−A0B8−11SRR1342873TCGA−E2−A1BC−11ASRR66237SRR1489589SRR14553918 A SRR1486457SRR1102949SRR1490246SRR1312822SRR1362972TCGA−BH−A0B5−11TCGA−E9−A1RC−11ATCGA−E9−A1RI−11AA SRR1400159SRR1417894SRR1076441SRR1068999SRR1339431TCGA−BH−A0H9−11ASRR1071905SRR1469279 TCGA−E9−A1NG−11ASRR1310158SRR1367024SRR1096547SRR1477273SRR1455902TCGA−A7−A0DB−11ATCGA−E2−A158−11A SRR1420238SRR1409421SRR1084674SRR1090046SRR81633SRR1070208TCGA−E9−A1RF−11ATCGA−A7−A13E−11A6 SRR1472704SRR1077920SRR1346861SRR1454009SRR1370308SRR1488196SRR1467522SRR1076219 −0.05 0.00 0.05 SRR1364871SRR66024SRR65697SRR1378948SRR1105009SRR80842SRR1075484SRR107026080 SRR1323829SRR1489803SRR1475408SRR1359262SRR1339236SRR1459561SRR1401017SRR1366790 SRR1457705SRR1410765SRR1481128SRR1481315SRR81386TCGA−BH−A204−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−BH−A0AZ−11ASRR1498416SRR1091791SRR1388281SRR658319 SRR1409620SRR1342758SRR1472506SRR1078258SRR1086772SRR1454158SRR1455807SRR1472415 SRR1456010SRR1477671SRR1335212SRR1488586SRR1388399SRR1459866SRR1437073SRR1368050 AM12 MMP 7 MMP 1 MMP 3 MMP 2 MMP 9 TIMP2 MMP13 MMP15 MMP24 MMP19 MMP11 MMP14 AD AM12 TIMP2 MMP2 MMP3 MMP9 MMP1 MMP7 MMP15 MMP24 MMP19 MMP11 MMP14 MMP13 Lung AD

Adenocarcinoma Tumor Cluster Dendrogram Color Key SSC Normal Cluster Dendrogram and Histogram Normal (Euclidean, complete linkage) 200 0

0.04 Count 1000 200 0

−2 02 Row Z−Score 150 TCGA−MP−A4SV−01A−11R−A24X−07 TCGA−55−1596−01A−01R−0946−07TCGA−49−4494−01A−01R−1206−07TCGA−22−4601−01A−01R−1443−07TCGA−60−2726−01A−01R−0851−07TCGA−63−A5ML−01A−31R−A27Q−07TCGA−68−8250−01A−11R−2296−07TCGA−NK−A5D1−01A−11R−A26W−07TCGA−L4−A4E5−01A−11R−A24X−07TCGA−49−4505−01A−01R−1206−07 TCGA−58−A46K−01A−11R−A24H−0TCGA−94−7943−01A−11R−2187−07TCGA−55−6986−01A−11R−1949−07TCGA−NC−A5HQ−01A−11R−A26W−07TCGA−77−7142−01A−11R−2045−07TCGA−68−A59J−01A−21R−A26W−07TCGA−85−8277−01A−11R−2296−07TCGA−56−7221−01A−11R−2045−07TCGA−85−8580−01A−31R−2403−077 TCGA−80−5608−01A−31R−1949−07TCGA−46−3768−01A−01R−0980−07TCGA−93−8067−01A−11R−2287−07TCGA−62−A471−01A−12R−A24H−07TCGA−63−A5MI−01A−12R−A27Q−07TCGA−44−7660−01A−11R−2066−07TCGA−39−5035−01A−01R−1443−07TCGA−52−7809−01A−21R−2125−07TCGA−77−A5GA−01A−11R−A27Q−07 TCGA−66−2786−01A−01R−0851−07TCGA−55−8203−01A−11R−2241−07TCGA−22−1017−01A−01R−0692−07TCGA−73−4659−01A−01R−1206−07TCGA−60−2698−01A−01R−0851−07TCGA−43−A56V−01A−11R−A26W−07TCGA−60−2697−01A−11R−2125−07TCGA−69−7760−01A−11R−2170−07TCGA−75−6207−01A−11R−1755−07 TCGA−56−A49D−01A−11R−A24H−07TCGA−56−7822−01A−11R−2125−07TCGA−58−8388−01A−11R−2326−07TCGA−55−6987−01A−11R−1949−07TCGA−55−7724−01A−11R−2170−07TCGA−52−7812−01A−11R−2125−07TCGA−75−5125−01A−01R−1755−07TCGA−99−8032−01A−11R−2241−07TCGA−97−7552−01A−11R−2039−07 TCGA−58−8386−01A−11R−2296−07TCGA−78−7146−01A−11R−2039−07TCGA−85−8351−01A−11R−2296−07TCGA−22−1002−01A−01R−0692−07TCGA−22−1016−01A−01R−0692−07TCGA−43−A56U−01A−11R−A26W−07TCGA−22−1005−01A−01R−0692−07TCGA−55−7727−01A−11R−2170−07TCGA−58−A46J−01A−11R−A24H−07 TCGA−86−8076−01A−31R−2241−07TCGA−67−3771−01A−01R−0946−07TCGA−55−7903−01A−11R−2170−07TCGA−86−A4JF−01A−11R−A24X−07TCGA−50−5935−01A−11R−1755−07TCGA−78−7143−01A−11R−2039−07TCGA−86−7954−01A−11R−2187−07TCGA−56−7222−01A−11R−2045−07TCGA−60−2696−01A−01R−0851−07 TCGA−50−5051−01A−21R−1858−07TCGA−78−7152−01A−11R−2039−07TCGA−MP−A4TH−01A−31R−A262−07TCGA−75−6212−01A−11R−1755−07TCGA−95−A4VP−01A−21R−A262−07TCGA−MP−A4T7−01A−11R−A24X−0TCGA−85−A510−01A−11R−A26W−07TCGA−95−7947−01A−11R−2187−07TCGA−MP−A4T6−01A−32R−A262−07 TCGA−85−7710−11A−01R−2125−07TCGA−44−2657−11A−01R−1758−07TCGA−97−A4M2−01A−12R−A24X−07TCGA−44−5645−01A−01R−1628−07TCGA−38−4632−11A−01R−1755−07TCGA−55−6979−11A−01R−1949−07TCGA−53−7626−01A−12R−2066−07TCGA−99−AA5R−01A−11R−A39D−0TCGA−56−8623−01A−11R−A28V−077 0.00 TCGA−69−A59K−01A−11R−A262−07TCGA−86−7701−01A−11R−2170−07TCGA−37−5819−01A−01R−1635−07TCGA−50−7109−01A−11R−2039−07TCGA−NC−A5HJ−01A−11R−A26W−07TCGA−44−6144−11A−01R−1755−07TCGA−97−7546−01A−11R−2039−07TCGA−L4−A4E6−01A−11R−A24H−0TCGA−91−8497−01A−11R−2403−077 Sample.Type TCGA−44−6778−01A−11R−1858−07TCGA−62−8398−01A−11R−2326−07TCGA−05−4432−01A−01R−1206−07TCGA−96−7545−01A−21R−2045−07TCGA−73−4662−01A−01R−1206−07TCGA−39−5034−01A−01R−1443−07TCGA−91−6828−01A−11R−1858−07TCGA−86−8669−01A−11R−2403−07TCGA−05−4426−01A−01R−1206−07 TCGA−MN−A4N5−01A−11R−A24X−0TCGA−91−7771−01A−11R−2170−07TCGA−77−7139−01A−11R−2045−07TCGA−78−8640−01A−11R−2403−07TCGA−NJ−A4YQ−01A−11R−A262−0TCGA−55−8508−01A−11R−2403−07TCGA−55−7995−01A−11R−2187−07TCGA−55−8205−01A−11R−2241−07TCGA−44−A479−01A−31R−A24H−0777 TCGA−64−5779−01A−01R−1628−07TCGA−44−7671−01A−11R−2066−07TCGA−56−8624−01A−11R−2403−07TCGA−85−8072−01A−31R−2247−07TCGA−96−8170−01A−11R−2296−07TCGA−86−7953−01A−11R−2187−07TCGA−62−A46R−01A−11R−A24H−07TCGA−44−2657−01A−01R−1107−07TCGA−55−6543−01A−11R−1755−07 TCGA−55−7573−01A−11R−2039−07TCGA−77−8008−11A−01R−2187−07SRR148069TCGA−51−4081−11A−01R−1758−07TCGA−98−A53C−01A−11R−A262−07TCGA−55−8087−01A−11R−2241−07TCGA−05−4424−01A−22R−1858−07TCGA−50−8457−01A−11R−2326−07TCGA−J2−A47 AG−01A−11R−A24H−07 TCGA−33−6737−01A−11R−1820−07TCGA−05−4433−01A−22R−1858−07SRR601311SRR812493SRR144182SRR149833SRR141913SRR135723SRR1446546713 TCGA−49−AARR−01A−11R−A41B−0SRR608542TCGA−91−6830−01A−11R−1949−07TCGA−22−5472−01A−01R−1635−07TCGA−62−8399−01A−21R−2326−07SRR140412TCGA−34−7107−11A−01R−1949−07SRR627437SRR6274593 7 TCGA−50−5936−11A−01R−1628−07TCGA−75−6203−01A−11R−1755−07TCGA−91−6831−11A−02R−1858−07TCGA−44−6778−11A−01R−1858−07SRR662257TCGA−43−6771−11A−01R−1820−07SRR216687SRR134693SRR146863437 Adenocarcinoma TCGA−49−AAR0−01A−21R−A39D−0TCGA−78−7537−01A−11R−2066−07TCGA−62−8395−01A−11R−2326−07TCGA−90−6837−11A−01R−1949−07TCGA−22−5472−11A−11R−1635−07TCGA−44−6148−01A−11R−1755−07TCGA−55−6985−11A−01R−1949−07TCGA−49−6761−11A−01R−1949−07TCGA−43−3394−11A−01R−1758−077 TCGA−95−A4VK−01A−11R−A262−07TCGA−99−8025−01A−11R−2241−07TCGA−44−2661−11A−01R−1758−07TCGA−05−4405−01A−21R−1858−07TCGA−55−7574−01A−11R−2039−07TCGA−55−8302−01A−11R−2326−07TCGA−55−7907−01A−11R−2170−07TCGA−55−8092−01A−11R−2241−07TCGA−97−A4M5−01A−11R−A24X−07 TCGA−77−A5GF−01A−21R−A27Q−0TCGA−66−2781−01A−01R−0851−07TCGA−50−6594−01A−11R−1755−07TCGA−69−7763−01A−11R−2170−07TCGA−J2−A4AD−01A−11R−A24H−0TCGA−44−8120−01A−11R−2241−07TCGA−97−8179−01A−11R−2287−07TCGA−55−8097−01A−11R−2241−07TCGA−NC−A5HT−01A−11R−A26W−0777 TCGA−85−7843−01A−11R−2125−07TCGA−34−2596−01A−01R−0851−07TCGA−66−2783−01A−01R−1201−07TCGA−98−A538−01A−11R−A262−07TCGA−77−8150−01A−11R−2247−07TCGA−46−6025−01A−11R−1820−07TCGA−96−A4JK−01A−11R−A24Z−07TCGA−34−5231−01A−21R−1820−07TCGA−63−A5M9−01A−11R−A26W−07 TCGA−73−4670−01A−01R−1206−07TCGA−55−A490−01A−11R−A466−07TCGA−05−4417−01A−22R−1858−07TCGA−91−6831−01A−11R−1858−07TCGA−22−5474−01A−01R−1635−07TCGA−86−8585−01A−11R−2403−07TCGA−22−4607−01A−01R−1201−07TCGA−63−6202−01A−11R−1820−07TCGA−94−7557−01A−11R−2125−07 100 TCGA−50−5044−01A−21R−1858−07TCGA−92−8063−01A−11R−2247−07TCGA−75−6214−01A−41R−1949−07TCGA−55−A48Y−01A−11R−A24H−0TCGA−77−8146−01A−11R−2247−07TCGA−44−6145−01A−11R−1755−07TCGA−50−5072−01A−21R−1858−07TCGA−85−8584−01A−11R−2403−07TCGA−05−4427−01A−21R−1858−077 TCGA−49−4487−01A−21R−1858−07TCGA−90−7964−01A−21R−2187−07TCGA−34−5927−01A−11R−1820−07TCGA−56−1622−01A−01R−0692−07TCGA−44−8117−01A−11R−2241−07TCGA−55−8507−01A−11R−2403−07TCGA−95−7944−01A−11R−2187−07TCGA−50−5930−01A−11R−1755−07TCGA−55−8089−01A−11R−2241−07 SSC TCGA−64−5774−01A−01R−1628−07TCGA−56−8082−01A−11R−2247−07TCGA−33−A4WN−01A−11R−A262−07TCGA−05−4420−01A−01R−1206−07TCGA−NC−A5HK−01A−11R−A26W−0TCGA−56−8083−01A−11R−2247−07TCGA−51−6867−01A−11R−2045−07TCGA−90−7769−01A−11R−2125−07TCGA−77−8153−01A−11R−2403−07 7 TCGA−86−8673−01A−11R−2403−07TCGA−73−4668−01A−01R−1206−07TCGA−55−6985−01A−11R−1949−07TCGA−78−7542−01A−21R−2066−07TCGA−55−7913−01B−11R−2241−07TCGA−78−7159−01A−11R−2039−07TCGA−55−8085−01A−11R−2241−07TCGA−56−7823−01B−11R−2247−07TCGA−05−4397−01A−01R−1206−07 TCGA−MP−A4SW−01A−21R−A24X−07TCGA−73−4675−01A−01R−1206−07TCGA−55−8090−01A−11R−2241−07TCGA−86−8278−01A−11R−2287−07TCGA−73−7499−01A−11R−2187−07TCGA−85−A5B5−01A−21R−A26W−07TCGA−37−A5EN−01A−21R−A26W−07TCGA−85−7696−01A−11R−2125−07TCGA−49−AAQV−01A−11R−A39D−07 SRR139172TCGA−MP−A4T8−01A−11R−A24X−0TCGA−MP−A4TD−01A−32R−A262−07TCGA−44−6777−01A−11R−1858−07TCGA−91−6835−01A−11R−1858−07TCGA−95−7567−01A−11R−2066−07TCGA−78−7145−01A−11R−2039−07TCGA−55−1595−01A−01R−0946−07TCGA−34−7107−01A−11R−1949−074 7 TCGA−L9−A8F4−01A−11R−A39D−0TCGA−56−7223−01A−11R−2045−07TCGA−33−AASI−01A−22R−A405−07TCGA−L9−A444−01A−21R−A24H−07TCGA−NJ−A55R−01A−11R−A262−07TCGA−LA−A7SW−01A−11R−A405−07TCGA−50−6591−01A−11R−1755−07TCGA−53−7624−01A−11R−2066−07TCGA−85−6560−01A−11R−1820−077 Height TCGA−NJ−A4YF−01A−12R−A262−07TCGA−55−8615−01A−11R−2403−07 TCGA−33−AAS8−01A−11R−A405−07TCGA−33−AASJ−01A−11R−A405−07TCGA−NC−A5HI−01A−11R−A26W−07TCGA−77−6843−01A−11R−1949−07TCGA−60−2711−01A−01R−0851−07TCGA−56−8304−01A−11R−2326−07TCGA−58−8392−01A−11R−2326−07TCGA−33−6738−01A−11R−1949−07TCGA−43−5668−01A−01R−1635−07 ZNormal TCGA−LA−A446−01A−21R−A24Z−07TCGA−58−8391−01A−11R−2326−07TCGA−77−8143−01A−11R−2247−07TCGA−NK−A5CR−01A−11R−A26W−07TCGA−51−4080−01A−01R−1100−07TCGA−39−5016−01A−01R−1443−07TCGA−44−5643−01A−01R−1628−07TCGA−22−5477−01A−01R−1635−07TCGA−J1−A4AH−01A−31R−A24H−07 TCGA−44−3917−01A−01R−A278−07TCGA−33−4532−01A−01R−1201−07TCGA−85−8354−01A−31R−2326−07TCGA−33−4583−01A−01R−1443−07TCGA−66−2793−01A−01R−1201−07TCGA−33−AASL−01A−11R−A405−07TCGA−64−1677−01A−01R−0946−07TCGA−O2−A52W−01A−11R−A26W−07TCGA−39−5036−01A−01R−1443−07 PC2 (21.17% ) TCGA−37−4132−01A−01R−1100−07 TCGA−66−2787−01A−01R−0980−07TCGA−37−4141−01A−02R−1100−07TCGA−MP−A4TF−01A−11R−A262−07TCGA−44−7669−01A−21R−2066−07TCGA−66−2785−01A−01R−0851−07TCGA−05−4418−01A−01R−1206−07TCGA−18−3416−01A−01R−0980−07TCGA−18−3419−01A−01R−0980−07TCGA−77−8144−01A−11R−2247−07 TCGA−77−7463−01A−11R−2045−07TCGA−38−4631−01A−01R−1755−07TCGA−NC−A5HO−01A−11R−A26W−07TCGA−63−A5MB−01A−11R−A26W−07TCGA−85−7710−01A−11R−2125−07TCGA−56−8307−01A−11R−2296−07TCGA−90−7766−01A−21R−2125−07TCGA−44−3918−01B−02R−A277−07TCGA−22−4594−01A−01R−1201−07 TCGA−18−3408−01A−01R−0980−07TCGA−91−6836−01A−21R−1858−07TCGA−66−2756−01A−01R−0851−07TCGA−66−2766−01A−01R−0851−07TCGA−NC−A5HH−01A−11R−A26W−07TCGA−63−7023−01A−11R−1949−07TCGA−63−5128−01A−01R−1443−07TCGA−98−A53J−01A−11R−A26W−07 TCGA−58−A46N−01A−11R−A24H−07TCGA−70−6723−01A−11R−1820−07TCGA−94−8490−01A−11R−2326−07TCGA−77−A5G6−01A−11R−A27Q−07TCGA−98−A53A−01A−11R−A262−07TCGA−NC−A5HR−01A−21R−A26W−07TCGA−77−6845−01A−11R−1949−07TCGA−33−4538−01A−01R−1201−07TCGA−66−2754−01A−01R−0980−07 TCGA−NC−A5HG−01A−11R−A26W−07TCGA−18−5595−01A−01R−1635−07TCGA−85−8071−01A−11R−2247−07TCGA−NC−A5HD−01A−11R−A26W−07TCGA−NC−A5HN−01A−11R−A26W−07TCGA−21−1075−01A−01R−0692−07TCGA−63−A5MU−01A−11R−A26W−07TCGA−60−2716−01A−01R−0851−07TCGA−O2−A5IB−01A−11R−A27Q−07 TCGA−22−5481−01A−31R−1949−07TCGA−44−4112−01B−06R−A277−07TCGA−77−6844−01A−11R−1949−07TCGA−56−A4BY−01A−11R−A24H−0TCGA−43−2578−01A−01R−0851−07TCGA−55−7570−01A−11R−2039−07TCGA−56−A4BX−01A−11R−A24H−0TCGA−39−5037−01A−01R−1443−07TCGA−73−A9RS−01A−11R−A41B−07 TCGA−77−8140−01A−11R−2247−07TCGA−34−2600−01A−01R−0851−07TCGA−22−5491−01A−01R−1635−07TCGA−39−5027−01A−21R−1820−07TCGA−39−5040−01A−21R−2125−07TCGA−44−2665−01B−06R−A277−07TCGA−56−7730−01A−11R−2125−07TCGA−60−2695−01A−01R−0851−07TCGA−43−7658−01A−11R−2125−07 TCGA−78−7155−01A−11R−2039−07TCGA−37−4130−01A−01R−1100−07TCGA−4B−A93V−01A−11R−A39D−0TCGA−55−8620−01A−11R−2403−07TCGA−78−7536−01A−11R−2066−07TCGA−05−4415−01A−22R−1858−07TCGA−55−7910−01A−11R−2170−07TCGA−78−7220−01A−11R−2039−07TCGA−94−7033−01A−11R−1949−077 TCGA−38−4628−01A−01R−1206−07TCGA−NJ−A55O−01A−11R−A262−07TCGA−49−4514−01A−21R−1858−07TCGA−97−A4M3−01A−11R−A24X−07TCGA−85−A4TCGA−85−8352−01A−31R−2326−07TCGA−90−A4ED−01A−31R−A24Z−0TCGA−98−A53I−01A−31R−A262−0TCGA−33−4587−01A−11R−2125−07PA−01A−11R−A24Z−0777 TCGA−97−8171−01A−11R−2287−07TCGA−53−7813−01A−11R−2170−07TCGA−44−2655−01A−01R−0946−07TCGA−05−4395−01A−01R−1206−07TCGA−L9−A5IP−01A−21R−A39D−0TCGA−91−6847−01A−11R−1949−07TCGA−49−6742−01A−11R−1858−07TCGA−78−7161−01A−11R−2039−07TCGA−55−A494−01A−11R−A24X−077 TCGA−78−7153−01A−11R−2039−07TCGA−78−7149−01A−11R−2039−07TCGA−05−4422−01A−01R−1206−07TCGA−35−3615−01A−01R−0946−07TCGA−62−A46P−01A−11R−A24H−0TCGA−49−AAR2−01A−11R−A39D−0TCGA−49−AARQ−01A−11R−A41B−0TCGA−67−6215−01A−11R−1755−07TCGA−78−7158−01A−11R−2039−07777 0 TCGA−NK−A5CT−01A−31R−A26W−07TCGA−63−5131−01A−01R−1443−07TCGA−77−7141−01A−11R−2045−07TCGA−21−1078−01A−01R−0692−07TCGA−NJ−A7XG−01A−12R−A39D−0TCGA−44−6146−01A−11R−1755−07TCGA−78−7167−01A−11R−2066−07TCGA−95−8494−01A−11R−2326−07TCGA−86−8281−01A−11R−2287−07 7 TCGA−05−4244−01A−01R−1107−07TCGA−05−5423−01A−01R−1628−07TCGA−22−A5C4−01A−12R−A27Q−07TCGA−44−5644−01A−21R−2039−07TCGA−86−8054−01A−11R−2241−07TCGA−22−5485−01A−01R−1635−07TCGA−33−4582−01A−01R−1443−07TCGA−22−4591−01A−01R−1201−07TCGA−37−4133−01A−01R−1100−07 TCGA−21−5787−01A−01R−1635−07TCGA−05−4384−01A−01R−1755−07TCGA−55−6970−01A−11R−1949−07TCGA−60−2706−01A−01R−0851−07TCGA−75−6211−01A−11R−1755−07TCGA−86−8056−01A−11R−2241−07TCGA−NJ−A4YI−01A−11R−A262−07TCGA−75−7031−01A−11R−1949−07TCGA−55−1594−01A−01R−0946−07 TCGA−91−6840−01A−11R−1949−07TCGA−05−4396−01A−21R−1858−07TCGA−86−8674−01A−21R−2403−07TCGA−86−8359−01A−11R−2326−07TCGA−85−8479−01A−11R−2326−07TCGA−77−8154−01A−11R−2247−07TCGA−22−0944−01A−01R−0692−07TCGA−94−8491−01A−11R−2326−07TCGA−49−4507−01A−01R−1206−07 TCGA−85−A4QR−01A−11R−A24Z−07TCGA−6A−AB49−01A−12R−A405−07TCGA−49−4506−01A−01R−1206−07TCGA−33−AASB−01A−11R−A405−07TCGA−33−A5GW−01A−11R−A27Q−07TCGA−85−A50Z−01A−21R−A262−07TCGA−80−5607−01A−31R−1949−07TCGA−69−7764−01A−11R−2170−07TCGA−97−7938−01A−11R−2170−07 MMP9 SRR607279SRR661937SRR656886SRR134916SRR145775SRR665658SRR148721TCGA−64−1678−01A−01R−0946−07TCGA−63−A5MY−01A−11R−A26W−07605 SRR133902SRR654838SRR607586SRR138275SRR149048SRR132638SRR807824SRR147316SRR6633449641 SRR601900SRR663038SRR602680SRR601925SRR146622SRR141364SRR140617SRR145198SRR6038002468 SRR131079SRR665429SRR148999SRR143467SRR601477SRR143400SRR613138SRR136765SRR132372751943 MMP7 SRR146000SRR147246SRR140095SRR819658SRR140110TCGA−43−5670−11A−01R−2125−07125 SRR663573SRR660414SRR216657SRR663623SRR614936SRR138411SRR145441SRR599154SRR81112105 MMP1 SRR656874SRR655601 SRR139803SRR149881SRR109807SRR107476SRR143971SRR143302SRR144277SRR138378SRR21651863497852 MMP 9 SRR137667SRR819318SRR604677SRR656153SRR11020769 SRR615129SRR141978SRR133812SRR137390SRR148926SRR612887SRR110280SRR147334SRR6621624350 SRR615189SRR627445SRR603750SRR142146SRR810461SRR143404SRR148842SRR146554SRR1467472893 −0.04 MMP1 0 SRR107215SRR135680SRR135960SRR614948SRR136271SRR661842SRR131497SRR13459305936 MMP 7 SRR107035SRR108672SRR1071568 TCGA−56−8082−11A−01R−2247−07SRR603620SRR144286SRR146688SRR603004SRR146002SRR657396SRR131956TCGA−44−6147−11A−01R−1858−072950 SRR657880SRR140981TCGA−43−7658−11A−01R−2125−07TCGA−55−6986−11A−01R−1949−07SRR660800TCGA−22−4609−11A−01R−2125−07SRR138530SRR133098SRR147120309 MMP 1 TCGA−92−7340−11A−01R−2045−07TCGA−91−6829−11A−01R−1858−07TCGA−55−6978−11A−01R−1949−07SRR147105TCGA−50−5933−11A−01R−1755−070 MMP1 1 MMP1 2 TCGA−44−6148−11A−01R−1858−07SRR140298SRR107001SRR144773TCGA−50−5931−11A−01R−1858−07TCGA−50−6595−11A−01R−1858−07TCGA−22−5478−11A−11R−1635−0765 TCGA−44−5645−11A−01R−1628−07TCGA−55−6970−11A−01R−1949−07TCGA−56−8201−11A−01R−2247−07TCGA−86−A4P8−01A−11R−A24X−07TCGA−90−7767−11A−01R−2125−07SRR814703SRR147964SRR146882SRR140668085 SRR809807SRR143384SRR600632SRR137042SRR600584SRR132135SRR817070TCGA−43−6647−11A−01R−1820−07SRR147554136 MMP1 3 TCGA−43−6773−11A−01R−1949−07SRR148076SRR13340764 SRR143433SRR604480SRR132180SRR134183SRR660981SRR147692SRR148941SRR821525SRR14091928376 SRR143482SRR109167SRR147801SRR144068SRR602058SRR138193SRR600417SRR662474SRR137702105246 MMP1 9 MMP2 8 SRR607863SRR1417088 SRR148841SRR808472TCGA−34−8454−11A−01R−2326−07SRR658897SRR131097SRR603188SRR133762SRR134440SRR1415870542 MMP10 SRR612755 SRR136465SRR133917SRR612791SRR147671SRR107311SRR137631SRR601445SRR148791SRR66296685960

05 SRR1321528 SRR108128SRR132738SRR109928SRR613414SRR141107SRR148103SRR131811SRR131220SRR66082436089 MMP11 SRR665466SRR604390SRR612239SRR821302SRR655625SRR136080SRR6030927 MMP12 SRR138802SRR133567SRR136548509 AM1 2 SRR141740SRR6024924 SRR137669SRR147915SRR135557SRR615695SRR145218SRR136580SRR109899SRR603858SRR1406534589 MMP3 MMP1 4 SRR146750SRR109878SRR140633SRR136593SRR601723058 MMP1 5 MMP2 4 SRR655064SRR131052SRR137409SRR135657SRR136134SRR108460SRR6155390275 MMP13 SRR138776SRR137573SRR147779SRR146043SRR131510SRR608630SRR21848698735 MMP1 7 SRR141394SRR216828SRR108476SRR66354906 SRR131474SRR137456SRR147552SRR140429SRR131252SRR135721SRR665217SRR147295SRR1309469743208 MMP28 SRR1335513 MMP19 SRR217013SRR657801SRR143732SRR141457SRR140813SRR6130665486 AD SRR144734SRR134702SRR133663SRR130850SRR131286SRR820596SRR627447SRR600784SRR144188706459 MMP8 MMP2 SRR817166SRR654814 TIMP2 AM12 SRR143667SRR14480541 MMP2 5 SRR811866SRR141493SRR6576114 SRR141622SRR657927SRR130949SRR134202SRR810483SRR136683SRR133542SRR61274360372 MMP 3 SRR144282SRR660152SRR614359SRR14210120 SRR809296SRR809065SRR627427SRR627457SRR627464SRR145905SRR143243SRR218509SRR662031507 MMP23B SRR143138SRR139569SRR132588SRR133703SRR137021SRR133449598160 MMP14 SRR131255SRR603776SRR656934SRR132675SRR141546SRR607839SRR599059823 MMP15 SRR1095695 MMP24 SRR132624SRR134779SRR139137SRR141611SRR133254SRR607166SRR6155519564 MMP17 SRR148699TCGA−50−5932−11A−01R−1755−07SRR147198SRR144406SRR658445SRR141495SRR817004SRR139559SRR14539759381 TIMP 2 SRR598695SRR135946SRR144367SRR818499SRR66055597 MMP 2 SRR138908SRR132237SRR148127SRR147109SRR1419864398 MMP 8 SRR149070TCGA−22−5483−11A−11R−1820−07SRR144223SRR146934SRR133365SRR661517SRR109954SRR65884901596 SRR819186SRR663112SRR137908SRR139047SRR813043SRR137469SRR132425TCGA−22−5482−11A−01R−1635−07SRR8174889760 SRR132259SRR134556SRR659037SRR147058SRR135268SRR136875SRR600900SRR108755SRR139696530497 AD SRR138116SRR14728815 MMP25 SRR132389SRR147380SRR814244SRR142920603 SRR145844TCGA−50−5939−11A−01R−1628−07TCGA−44−2655−11A−01R−1758−07TCGA−44−3398−11B−01R−1758−07SRR661601SRR654826SRR142966SRR141429SRR140550704 MMP1 6 TCGA−56−8083−11A−01R−2247−07TCGA−44−6146−11A−01R−1858−07 TCGA−55−6975−11A−01R−1949−07TCGA−38−4627−11A−01R−1758−07TCGA−77−7138−11A−01R−2045−07TCGA−55−6980−11A−01R−1949−07TCGA−77−7338−11A−01R−2045−07TCGA−77−7142−11A−01R−2045−07TCGA−51−4079−11A−01R−1758−07TCGA−91−6849−11A−01R−1949−07TCGA−77−7337−11A−01R−2045−07 SRR132338SRR139250SRR657659SRR658385SRR143506SRR140347SRR135909TCGA−22−5471−11A−01R−1635−07TCGA−33−6737−11A−01R−1820−072689 MMP2 0 TCGA−50−5935−11A−01R−1858−07TCGA−22−5491−11A−01R−1858−07TCGA−56−8309−11A−01R−2296−07TCGA−77−7335−11A−01R−2045−07SRR658885SRR608288 MMP23B TCGA−56−7823−11A−01R−2247−07TCGA−44−2665−11A−01R−1758−07TCGA−55−6982−11A−01R−1949−07TCGA−55−7816−01A−11R−2170−07TCGA−56−7580−11A−01R−2045−07TCGA−44−6777−11A−01R−1858−07TCGA−44−2662−11A−01R−1758−07 MMP2 7 TCGA−43−6143−11A−01R−1820−07TCGA−38−4625−11A−01R−1758−07TCGA−44−6145−11A−01R−1858−07TCGA−56−7730−11A−01R−2125−07TCGA−22−5481−11A−01R−1949−07TCGA−55−6969−11A−01R−1949−07TCGA−22−5489−11A−01R−1635−07 MMP2 1 MMP2 6 TCGA−44−3396−11A−01R−1758−07TCGA−22−4593−11A−01R−1820−07TCGA−55−6981−11A−01R−1949−07TCGA−93−A4JP−01A−11R−A24X−07TCGA−51−4080−11A−01R−1758−07TCGA−43−7657−11A−01R−2125−07TCGA−56−8623−11A−01R−A28V−07 TCGA−55−6983−11A−01R−1949−07TCGA−44−2668−11A−01R−1758−07TCGA−58−8386−11A−01R−2296−07TCGA−55−6968−11A−01R−1949−07TCGA−78−7162−01A−21R−2066−07SRR662954TCGA−97−8174−01A−11R−2287−07SRR141768SRR5984840 TCGA−55−6984−11A−01R−1949−07TCGA−91−6847−11A−01R−1949−07TCGA−55−6972−11A−01R−1949−07TCGA−55−6971−11A−01R−1949−07TCGA−56−7579−11A−01R−2045−07TCGA−91−6836−11A−01R−1858−07TCGA−73−4676−11A−01R−1755−07TCGA−60−2709−11A−01R−1820−07TCGA−56−7731−11A−01R−2125−07 TCGA−56−7222−11A−01R−2045−07TCGA−33−4587−11A−01R−2125−07TCGA−38−4626−11A−01R−1758−07TCGA−91−6828−11A−01R−1858−07TCGA−49−6743−11A−01R−1858−07TCGA−49−6744−11A−01R−1858−07TCGA−50−5930−11A−01R−1755−07TCGA−49−6745−11A−01R−1858−07TCGA−44−6776−11A−01R−1858−07 TCGA−MP−A4TE−01A−22R−A466−07TCGA−62−A470−01A−11R−A24H−07TCGA−MP−A5C7−01A−11R−A262−07TCGA−44−2656−01B−06R−A277−07TCGA−49−4490−11A−01R−1858−07TCGA−62−8397−01A−11R−2326−07TCGA−49−4512−11A−01R−1858−07TCGA−49−6742−11A−01R−1858−07TCGA−39−5040−11A−01R−2125−07 MMP16 TCGA−53−A4EZ−01A−12R−A24X−07TCGA−75−5146−01A−01R−1628−07TCGA−67−3770−01A−01R−0946−07TCGA−69−8255−01A−11R−2287−07TCGA−86−7955−01A−11R−2187−07TCGA−78−7166−01A−12R−2066−07TCGA−85−8070−01A−11R−2247−07TCGA−50−5946−01A−11R−1755−07TCGA−86−7713−01A−11R−2066−07 TCGA−55−A492−01A−11R−A24H−07TCGA−64−1680−01A−02R−0946−07TCGA−44−6776−01A−11R−1858−07TCGA−95−7043−01A−11R−1949−07TCGA−86−A4D0−01A−11R−A24H−07TCGA−78−7148−01A−11R−2039−07TCGA−62−A46S−01A−11R−A24H−0TCGA−97−A4M0−01A−11R−A24X−07TCGA−91−8496−01A−11R−2403−077 TCGA−50−5932−01A−11R−1755−07TCGA−55−7283−01A−11R−2039−07TCGA−55−8094−01A−11R−2241−07TCGA−78−7633−01A−11R−2066−07TCGA−62−A46O−01A−11R−A24H−07TCGA−L9−A7SV−01A−11R−A39D−0TCGA−49−4510−01A−01R−1206−07TCGA−78−7163−01A−12R−2066−07TCGA−78−7156−01A−11R−2039−077 MMP20 TCGA−50−5066−02A−11R−2090−07 MMP27 TCGA−67−3774−01A−01R−0946−07TCGA−78−8662−01A−11R−2403−07TCGA−55−8512−01A−11R−2403−07 MMP21 TCGA−50−5944−01A−11R−1755−07TCGA−44−2659−01A−01R−0946−07TCGA−55−8616−01A−11R−2403−07TCGA−78−7539−01A−11R−2066−07TCGA−78−7147−01A−11R−2039−07TCGA−55−8514−01A−11R−2403−07TCGA−97−8552−01A−11R−2403−07TCGA−73−7498−01A−12R−2187−07 MMP26 TCGA−75−6206−01A−11R−1755−07TCGA−97−8172−01A−11R−2287−07TCGA−50−6597−01A−11R−1858−07TCGA−J2−A4AE−01A−21R−A24H−07TCGA−62−A46Y−01A−11R−A24H−0TCGA−73−4677−01A−01R−1206−07TCGA−55−8206−01A−11R−2241−07TCGA−97−A4M1−01A−11R−A24X−077 TCGA−78−7540−01A−11R−2066−07TCGA−50−5942−01A−21R−1755−07TCGA−67−4679−01B−01R−1755−07TCGA−97−8176−01A−11R−2403−07TCGA−37−4129−01A−01R−1100−07TCGA−55−6972−01A−11R−1949−07TCGA−49−4486−01A−01R−1206−07TCGA−67−6217−01A−11R−1755−07TCGA−O1−A52J−01A−11R−A262−07 TCGA−63−7021−01A−11R−1949−07TCGA−94−A5I6−01A−21R−A27Q−0TCGA−55−7725−01A−11R−2170−07TCGA−91−A4BD−01A−11R−A24H−0TCGA−71−6725−01A−11R−1858−07TCGA−78−7154−01A−11R−2039−07TCGA−67−6216−01A−11R−1755−07TCGA−86−A456−01A−11R−A24H−07TCGA−44−7659−01A−11R−2066−0777 −0.08 TCGA−22−5479−01A−31R−1949−07TCGA−43−6771−01A−11R−1820−07TCGA−22−5482−01A−01R−1635−07TCGA−43−A474−01A−11R−A24H−07TCGA−18−3414−01A−01R−0980−07TCGA−18−4086−01A−01R−1100−07TCGA−96−7544−01A−11R−2045−07TCGA−56−7580−01A−11R−2045−07TCGA−21−5786−01A−01R−1635−07 TCGA−58−8390−01A−11R−2326−07TCGA−60−2708−01A−01R−0851−07TCGA−90−7767−01A−11R−2125−07TCGA−85−7698−01A−11R−2125−07TCGA−90−A59Q−01A−11R−A26W−07TCGA−78−7150−01A−21R−2039−07TCGA−21−1081−01A−01R−0692−07TCGA−22−4593−01A−21R−1820−07TCGA−69−7765−01A−11R−2170−07 TCGA−60−2721−01A−01R−0851−07TCGA−50−5933−01A−11R−1755−07TCGA−MP−A4T4−01A−11R−A262−0TCGA−35−4123−01A−01R−1107−07TCGA−NC−A5HP−01A−11R−A26W−0TCGA−85−8287−01A−11R−2296−07TCGA−85−8582−01A−21R−2403−07TCGA−85−6798−01A−11R−1949−07TCGA−51−4081−01A−01R−1100−0777 TCGA−43−7656−01A−11R−2125−07TCGA−33−4586−01A−01R−1443−07TCGA−58−A46L−01A−11R−A24H−07TCGA−50−5931−01A−11R−1755−07TCGA−56−8504−01A−11R−2403−07TCGA−85−7697−01A−11R−2125−07TCGA−56−5898−01A−11R−1635−07TCGA−69−7979−01A−11R−2187−07TCGA−21−1082−01A−01R−0692−07 TCGA−56−8308−01A−11R−2296−07TCGA−37−3783−01A−01R−1201−07TCGA−50−5066−01A−01R−1628−07TCGA−18−3412−01A−01R−0980−07TCGA−18−3411−01A−01R−0980−07TCGA−60−2722−01A−01R−0851−07TCGA−37−3789−01A−01R−0980−07TCGA−92−8065−01A−11R−2247−07TCGA−43−6773−01A−41R−1949−07 TCGA−55−6975−01A−11R−1949−07TCGA−50−5946−02A−11R−2090−07TCGA−85−A4CN−01A−11R−A24H−07TCGA−NJ−A4YP−01A−11R−A262−07TCGA−94−A4VJ−01A−11R−A24Z−07TCGA−60−2724−01A−01R−0851−07TCGA−18−4083−01A−01R−1100−07TCGA−22−5473−01A−01R−1635−07TCGA−39−5021−01A−01R−1443−07 TCGA−85−A4JB−01A−51R−A262−07TCGA−80−5611−01A−01R−1628−07TCGA−91−A4BC−01A−11R−A24H−0TCGA−68−7755−01A−11R−2125−07TCGA−NJ−A4YG−01A−22R−A262−0TCGA−46−3769−01A−01R−0980−07TCGA−64−5781−01A−01R−1628−07TCGA−56−A5DR−01A−11R−A27Q−07TCGA−75−7027−01A−11R−1949−077 TCGA−49−6745−01A−11R−1858−07TCGA−78−8648−01A−11R−2403−07SRR135239TCGA−64−5775−01A−01R−1628−07TCGA−L9−A443−01A−12R−A24H−07TCGA−63−A5MW−01A−11R−A26W−07TCGA−52−7810−01A−11R−2125−07TCGA−77−A5G1−01A−11R−A27Q−07TCGA−49−6767−01A−11R−1858−071 TCGA−49−AAR4−01A−12R−A41B−0TCGA−55−8614−01A−11R−2403−07TCGA−55−8505−01A−11R−2403−07TCGA−50−6593−01A−11R−1755−07TCGA−38−4627−01A−01R−1206−07TCGA−MP−A4SY−01A−21R−A24X−07TCGA−MP−A4T9−01A−11R−A24X−0TCGA−86−6562−01A−11R−1755−07TCGA−55−7281−01A−11R−2039−0777 TCGA−86−8672−01A−21R−2403−07TCGA−97−7554−01A−11R−2039−07TCGA−55−8207−01A−11R−2241−07TCGA−97−A4LX−01A−11R−A24X−07TCGA−43−2576−01A−01R−A32Z−07TCGA−55−6642−01A−11R−1858−07TCGA−49−4512−01A−21R−1858−07TCGA−71−8520−01A−11R−2403−07TCGA−MN−A4N4−01A−12R−A24X−07 TCGA−44−7662−01A−11R−2066−07TCGA−J2−8192−01A−11R−2241−07TCGA−MP−A4TK−01A−11R−A24X−07TCGA−77−8128−01A−11R−2247−07TCGA−97−8547−01A−11R−2403−07TCGA−49−AARE−01A−11R−A41B−07TCGA−86−8074−01A−11R−2241−07TCGA−86−8279−01A−11R−2287−07TCGA−05−4430−01A−02R−1206−07 dist(t(dmlog), method = "euclidean") TCGA−18−3409−01A−01R−0980−07TCGA−92−7341−01A−31R−2045−07TCGA−63−7022−01A−11R−1949−07TCGA−77−8145−01A−11R−2247−07TCGA−43−8115−01A−11R−2247−07TCGA−86−8075−01A−11R−2241−07TCGA−86−8055−01A−11R−2241−07TCGA−64−1679−01A−21R−2066−07TCGA−55−A48Z−01A−12R−A24X−07 TCGA−55−6981−01A−11R−1949−07TCGA−75−7030−01A−11R−1949−07TCGA−93−A4JQ−01A−11R−A24X−07TCGA−77−8130−01A−11R−2247−07TCGA−MP−A4TC−01A−11R−A24X−07TCGA−85−6175−01A−11R−1820−07TCGA−44−7672−01A−11R−2066−07TCGA−44−6775−01A−11R−A278−07TCGA−50−8459−01A−11R−2326−07 TCGA−49−AAR9−01A−21R−A41B−0TCGA−55−6983−01A−11R−1949−07TCGA−95−A4VN−01A−11R−A262−0TCGA−55−8513−01A−11R−2403−07TCGA−93−A4JO−01A−21R−A24X−07TCGA−55−A491−01A−11R−A24H−07TCGA−62−8394−01A−11R−2326−07TCGA−86−8668−01A−11R−2403−07TCGA−49−4501−01A−01R−1206−077 TCGA−78−7535−01A−11R−2066−07TCGA−97−7547−01A−11R−2039−07TCGA−44−6147−01A−11R−1755−07TCGA−64−1681−01A−11R−2066−07TCGA−97−A4M7−01A−11R−A24X−07TCGA−55−1592−01A−01R−0946−07TCGA−05−5425−01A−02R−1628−07TCGA−MP−A4TCGA−97−7553−01A−21R−2039−07TA−01A−21R−A24X−07 TCGA−55−7911−01A−11R−2170−07TCGA−62−A472−01A−11R−A24H−07TCGA−69−7973−01A−11R−2187−07TCGA−55−7576−01A−11R−2066−07TCGA−50−8460−01A−11R−2326−07TCGA−55−A4DG−01A−11R−A24H−07TCGA−86−A4P7−01A−11R−A24X−07TCGA−NJ−A55A−01A−11R−A262−07TCGA−97−7937−01A−11R−2170−07 hclust (*, "complete") TCGA−64−5778−01A−01R−1628−07TCGA−73−4676−01A−01R−1755−07TCGA−55−7914−01A−11R−2170−07TCGA−91−6835−11A−01R−1858−07TCGA−86−8073−01A−11R−2241−07TCGA−05−4410−01A−21R−1858−07TCGA−56−7582−11A−01R−2045−07TCGA−38−4626−01A−01R−1206−07TCGA−77−8007−11A−01R−2187−07 TCGA−86−6851−01A−11R−1949−07TCGA−78−7160−01A−11R−2039−07TCGA−78−8655−01A−11R−2403−07TCGA−95−7039−01A−11R−1949−07TCGA−49−4490−01A−21R−1858−07TCGA−62−A46V−01A−11R−A24H−0TCGA−44−A4SU−01A−11R−A24X−0TCGA−MN−A4N1−01A−11R−A24X−077 −0.06−0.03 0.00 0.03 0.06 TCGA−55−6980−01A−11R−1949−07TCGA−95−7948−01A−11R−2187−07TCGA−75−7025−01A−12R−1949−07TCGA−95−8039−01A−11R−2241−07TCGA−49−AARN−01A−21R−A41B−0TCGA−97−A4M6−01A−11R−A24X−07TCGA−69−7980−01A−11R−2187−07TCGA−55−A57B−01A−12R−A39D−0TCGA−05−4402−01A−01R−1206−0777 TCGA−93−7348−01A−21R−2039−07TCGA−93−7347−01A−11R−2187−07TCGA−85−A513−01A−12R−A26W−07TCGA−55−A48X−01A−11R−A24H−0TCGA−S2−AA1A−01A−12R−A39D−07TCGA−56−8622−01A−11R−2403−07TCGA−98−A53D−01A−32R−A262−07TCGA−77−A5FZ−01A−31R−A27Q−07TCGA−44−A47G−01A−21R−A24H−077 TCGA−55−6712−01A−11R−1858−07TCGA−75−6205−01A−11R−1755−07TCGA−44−A4SS−01A−11R−A24X−07TCGA−38−A44F−01A−11R−A24H−0TCGA−75−5147−01A−01R−1628−07TCGA−93−A4JN−01A−11R−A24X−07TCGA−38−6178−01A−11R−1755−07TCGA−55−8511−01A−11R−2403−07TCGA−50−5055−01A−01R−1628−077 TCGA−22−5492−01A−01R−1635−07TCGA−21−1083−01A−01R−0692−07TCGA−77−A5GH−01A−11R−A27Q−07TCGA−63−A5MP−01A−11R−A26W−07TCGA−85−8355−01A−11R−2296−07TCGA−56−8628−01A−11R−2403−07TCGA−85−7950−01A−11R−2187−07TCGA−39−5022−01A−21R−1820−07TCGA−43−6770−01A−11R−1820−07 TCGA−34−5232−01A−21R−1820−07TCGA−O2−A52S−01A−11R−A262−0TCGA−50−6590−01A−12R−1858−07TCGA−91−8499−01A−11R−2403−07TCGA−56−7582−01A−11R−2045−07TCGA−58−A46M−01A−11R−A24H−0TCGA−66−2758−01A−02R−0851−07TCGA−43−3394−01A−01R−0980−07TCGA−66−2767−01A−01R−0851−077 TCGA−55−7994−01A−11R−2187−07TCGA−43−A475−01A−11R−A24H−07TCGA−77−6842−01A−11R−1949−07TCGA−NC−A5HL−01A−11R−A26W−07TCGA−58−8387−01A−11R−2296−07TCGA−35−5375−01A−01R−1628−07TCGA−22−4613−01A−01R−1443−07TCGA−34−8456−01A−21R−2326−07TCGA−46−6026−01A−11R−1820−07 PC1 (39.69%) TCGA−56−A4ZK−01A−11R−A262−07TCGA−22−5483−01A−01R−1820−07TCGA−O2−A52Q−01A−11R−A26W−07TCGA−66−2744−01A−01R−0980−07TCGA−77−8133−01A−12R−2247−07TCGA−66−2794−01A−01R−1201−07TCGA−L3−A524−01A−11R−A262−07TCGA−86−8358−01A−11R−2326−07TCGA−75−5122−01A−01R−1755−07 TCGA−44−8119−01A−11R−2241−07TCGA−55−5899−01A−11R−1628−07TCGA−44−A47B−01A−11R−A24H−0TCGA−55−6968−01A−11R−1949−07TCGA−55−6969−01A−11R−1949−07TCGA−44−7667−01A−31R−2066−07TCGA−05−4390−01A−02R−1755−07TCGA−60−2704−01A−11R−2045−07TCGA−91−6848−01A−11R−1949−077 TCGA−39−5031−01A−01R−1443−07TCGA−56−8309−01A−11R−2296−07TCGA−85−A512−01A−11R−A26W−07TCGA−39−5019−01A−01R−1820−07TCGA−22−5489−01A−01R−1635−07TCGA−55−A4DF−01A−11R−A24H−0TCGA−MP−A4TJ−01A−51R−A262−0TCGA−56−6546−01A−11R−1820−07TCGA−50−5939−01A−11R−1628−077 TCGA−77−8148−01A−11R−2247−07TCGA−38−4630−01A−01R−1206−07TCGA−50−5045−01A−01R−1628−07TCGA−39−5030−01A−01R−1443−07TCGA−22−4604−01A−01R−1201−07TCGA−21−1076−01A−01R−0692−07TCGA−85−8049−01A−11R−2247−07TCGA−90−A4EE−01A−11R−A24Z−07TCGA−63−A5MS−01A−11R−A26W−07 TCGA−94−A5I4−01A−11R−A26W−07TCGA−22−5480−01A−01R−1635−07TCGA−34−5240−01A−01R−1443−07TCGA−60−2710−01A−01R−0851−07TCGA−85−8052−01A−11R−2247−07TCGA−05−4382−01A−01R−1206−07TCGA−66−2800−01A−01R−1201−07TCGA−05−4403−01A−01R−1206−07TCGA−44−3398−01A−01R−1107−07 TCGA−77−8156−01A−11R−2247−07TCGA−34−5239−01A−21R−1820−07TCGA−46−3766−01A−01R−0980−07TCGA−43−6143−01A−11R−1820−07TCGA−34−5929−01A−11R−1820−07TCGA−77−A5G3−01A−31R−A27Q−07TCGA−66−2792−01A−01R−0980−07TCGA−79−5596−01A−31R−1949−07TCGA−96−A4JL−01A−11R−A24Z−07 TCGA−77−8131−01A−11R−2247−07TCGA−62−A46U−01A−11R−A24H−07TCGA−XC−AA0X−01A−32R−A405−07TCGA−55−8091−01A−11R−2241−07TCGA−56−8625−01A−11R−2403−07TCGA−63−A5MV−01A−21R−A26W−07TCGA−56−A62T−01A−11R−A405−07TCGA−77−8139−01A−11R−2247−07TCGA−56−5897−01A−11R−1635−07 TCGA−55−6982−01A−11R−1949−07TCGA−05−4398−01A−01R−1206−07TCGA−44−3919−01A−02R−1107−07TCGA−55−8621−01A−11R−2403−07TCGA−55−8301−01A−11R−2287−07TCGA−86−8280−01A−11R−2287−07TCGA−55−7227−01A−11R−2039−07TCGA−66−2755−01A−01R−0851−07TCGA−37−3792−01A−01R−0980−07 TCGA−99−7458−01A−11R−2039−07TCGA−55−8510−01A−11R−2403−07TCGA−98−8022−01A−11R−2247−07TCGA−50−5049−01A−01R−1628−07TCGA−55−6979−01A−11R−1949−07TCGA−67−3772−01A−01R−0946−07TCGA−73−4658−01A−01R−1755−07TCGA−49−6744−01A−11R−1858−07TCGA−55−8096−01A−11R−2241−07 TCGA−44−7670−01A−11R−2066−07TCGA−97−8175−01A−11R−2287−07TCGA−35−4122−01A−01R−1107−07TCGA−73−4666−01A−01R−1206−07TCGA−38−4632−01A−01R−1755−07TCGA−38−7271−01A−11R−2039−07TCGA−86−8671−01A−11R−2403−07TCGA−50−5941−01A−11R−1755−07TCGA−44−2662−01A−01R−0946−07 TCGA−62−8402−01A−11R−2326−07TCGA−05−4389−01A−01R−1206−07TCGA−49−4488−01A−01R−1755−07TCGA−98−A539−01A−31R−A262−07TCGA−78−8660−01A−11R−2403−07TCGA−05−5428−01A−01R−1628−07TCGA−34−A5IX−01A−12R−A27Q−07TCGA−O2−A52N−01A−11R−A26W−07TCGA−67−3773−01A−01R−0946−07 TCGA−66−2737−01A−01R−0980−07TCGA−85−A4JC−01A−11R−A24Z−07TCGA−68−8251−01A−11R−2296−07TCGA−46−3767−01A−01R−0980−07TCGA−85−8353−01A−21R−2296−07TCGA−22−1000−01A−01R−A32Z−07TCGA−85−8664−01A−11R−2403−07TCGA−18−3410−01A−01R−0980−07TCGA−39−5011−01A−01R−1443−07 TCGA−92−8064−01A−11R−2247−07TCGA−66−2788−01A−01R−0980−07TCGA−33−4533−01A−01R−1201−07TCGA−55−A493−01A−11R−A24H−07TCGA−85−6561−01A−11R−1820−07TCGA−55−7726−01A−11R−2170−07TCGA−66−2789−01A−01R−0980−07TCGA−66−2770−01A−01R−0851−07TCGA−85−7699−01A−11R−2125−07 TCGA−43−5670−01A−21R−2125−07TCGA−39−5029−01A−01R−1443−07TCGA−L3−A4E7−01A−11R−A24Z−07TCGA−21−5782−01A−01R−1635−07TCGA−NK−A5CX−01A−11R−A26W−07TCGA−60−2723−01A−01R−0851−07TCGA−66−2795−01A−02R−0980−07TCGA−18−4721−01A−01R−1443−07TCGA−63−A5MM−01A−11R−A26W−07 TCGA−22−4599−01A−01R−1443−07TCGA−60−2712−01A−01R−0851−07TCGA−77−7465−01A−11R−2045−07TCGA−66−2759−01A−01R−0851−07TCGA−O2−A52V−01A−31R−A262−0TCGA−21−1077−01A−01R−0692−07TCGA−22−4609−01A−21R−2125−07TCGA−43−8118−01A−11R−2403−07TCGA−22−4605−01A−21R−2125−077 TCGA−43−6647−01A−11R−1820−07TCGA−56−8503−01A−11R−2403−07TCGA−44−2668−01A−01R−0946−07TCGA−56−6545−01A−11R−1820−07TCGA−43−8116−01A−11R−2247−07TCGA−21−1070−01A−01R−0692−07TCGA−66−2768−01A−01R−0851−07TCGA−63−A5MH−01A−12R−A27Q−07TCGA−43−3920−01A−01R−0980−07 TCGA−56−A4BW−01A−11R−A24H−0TCGA−55−8204−01A−11R−2241−07TCGA−85−8350−01A−11R−2296−07TCGA−39−5028−01A−01R−1443−07TCGA−98−8023−01A−11R−2247−07TCGA−70−6722−01A−11R−1820−07TCGA−66−2780−01A−01R−0851−07TCGA−60−2707−01A−01R−0851−07TCGA−85−8666−01A−11R−2403−07 7 TCGA−NC−A5HE−01A−11R−A26W−0TCGA−55−8506−01A−11R−2403−07TCGA−05−4434−01A−01R−1206−07TCGA−66−2777−01A−01R−1201−07TCGA−50−6592−01A−11R−1755−07TCGA−22−4596−01A−01R−1201−07TCGA−18−3417−01A−01R−1443−07TCGA−34−5928−01A−11R−1820−07TCGA−77−7337−01A−21R−2045−07 7 TCGA−50−5936−01A−11R−1628−07TCGA−52−7811−01A−11R−2125−07TCGA−60−2725−01A−01R−1201−07TCGA−68−7756−01A−11R−2125−07TCGA−49−6743−01A−11R−1858−07TCGA−85−8048−01A−11R−2247−07TCGA−98−A53H−01A−12R−A262−07TCGA−56−7579−01A−11R−2045−07TCGA−22−1012−01A−01R−0692−07 TCGA−60−2709−01A−21R−1820−07TCGA−64−5815−01A−01R−1628−07TCGA−55−8208−01A−11R−2241−07TCGA−49−AAR3−01A−11R−A41B−0TCGA−66−2769−01A−02R−0851−07TCGA−77−8138−01A−11R−2247−07TCGA−34−2608−01A−02R−0851−07TCGA−85−A511−01A−21R−A262−07TCGA−69−8453−01A−12R−2326−077 TCGA−68−A59I−01A−11R−A262−0TCGA−56−8305−01A−11R−2296−07TCGA−52−7622−01A−11R−2125−07TCGA−77−7338−01A−11R−2045−07TCGA−44−3396−01A−01R−1206−07TCGA−99−8028−01A−11R−2241−07TCGA−56−8201−01A−11R−2247−07TCGA−43−2581−01A−01R−0851−07TCGA−MP−A4TI−01A−21R−A24X−077 TCGA−56−7731−01A−11R−2125−07TCGA−L9−A743−01A−43R−A39D−07TCGA−38−4629−01A−02R−1206−07TCGA−34−5241−01A−01R−1443−07TCGA−56−8629−01A−11R−2403−07TCGA−56−A4ZJ−01A−11R−A262−07TCGA−69−7978−01A−11R−2187−07TCGA−44−7661−01A−11R−2066−07TCGA−49−AARO−01A−12R−A41B−07 TCGA−66−2791−01A−01R−0980−07TCGA−22−0940−01A−01R−0692−07TCGA−18−3407−01A−01R−0980−07TCGA−97−8177−01A−11R−2287−07TCGA−95−7562−01A−11R−2241−07TCGA−05−5715−01A−01R−1628−07TCGA−22−5478−01A−01R−1635−07TCGA−55−6978−01A−11R−1949−07TCGA−66−2790−01A−01R−0980−07 TCGA−33−AASD−01A−11R−A405−07TCGA−56−A5DS−01A−11R−A27Q−0TCGA−85−A4CL−01A−41R−A26W−07TCGA−66−2742−01A−01R−0980−07TCGA−83−5908−01A−21R−2287−07TCGA−66−2782−01A−01R−0851−07TCGA−63−A5MG−01A−12R−A27Q−07TCGA−77−8009−01A−11R−2187−07TCGA−22−4595−01A−01R−1201−07 7 TCGA−55−8299−01A−11R−2287−07TCGA−05−4250−01A−01R−1107−07TCGA−75−5126−01A−01R−1755−07TCGA−60−2703−01A−11R−2045−07TCGA−55−6984−01A−11R−1949−07TCGA−97−7941−01A−11R−2187−07TCGA−55−8619−01A−11R−2403−07TCGA−69−7761−01A−11R−2170−07TCGA−56−8626−01A−11R−2403−07 TCGA−63−A5MN−01A−22R−A27Q−07TCGA−77−7335−01A−11R−2045−07TCGA−92−7340−01A−21R−2045−07TCGA−85−8288−01A−11R−2296−07TCGA−96−8169−01A−11R−2296−07TCGA−85−A50M−01A−21R−A262−07TCGA−91−6829−01A−21R−1858−07TCGA−44−6774−01A−21R−1858−07TCGA−60−2715−01A−01R−0851−07 TCGA−05−4425−01A−01R−1755−07TCGA−60−2720−01A−01R−0851−07TCGA−50−5068−01A−01R−1628−07TCGA−85−A53L−01A−21R−A26W−07TCGA−22−5471−01A−01R−1635−07TCGA−21−A5DI−01A−31R−A26W−07TCGA−05−5429−01A−01R−1628−07TCGA−34−5234−01A−01R−1635−07TCGA−33−4547−01A−01R−1201−07 TCGA−77−8007−01A−11R−2187−07TCGA−98−8020−01A−11R−2247−07TCGA−77−A5GB−01B−11R−A27Q−0TCGA−77−8136−01A−11R−2247−07TCGA−85−8276−01A−11R−2296−07TCGA−85−7844−01A−11R−2125−07TCGA−90−6837−01A−11R−1949−07TCGA−91−6849−01A−11R−1949−07TCGA−55−7728−01A−11R−2187−07 7 TCGA−77−8008−01A−21R−2187−07TCGA−43−7657−01A−31R−2125−07TCGA−63−A5MJ−01A−11R−A27Q−07TCGA−21−5783−01A−41R−2187−07TCGA−68−7757−01B−11R−2296−07TCGA−85−8481−01A−11R−2326−07TCGA−50−6595−01A−12R−1858−07TCGA−37−A5EM−01A−21R−A27Q−07TCGA−60−2713−01A−01R−0851−07 TCGA−66−2765−01A−01R−0851−07TCGA−63−7020−01A−11R−1949−07TCGA−98−A53B−01A−11R−A262−07TCGA−46−3765−01A−01R−0980−07TCGA−66−2773−01A−01R−1201−07TCGA−34−8455−01A−11R−2326−07TCGA−66−2734−01A−01R−0980−07TCGA−21−1079−01A−01R−0692−07TCGA−34−5236−01A−21R−1820−07 TCGA−NC−A5HM−01A−12R−A26W−07TCGA−98−8021−01A−11R−2247−07TCGA−86−7711−01A−11R−2066−07TCGA−63−A5MT−01A−21R−A26W−07TCGA−98−7454−01A−11R−2045−07TCGA−21−1071−01A−01R−0692−07TCGA−34−8454−01A−11R−2326−07TCGA−85−A4QQ−01A−41R−A262−07TCGA−66−2753−01A−01R−0980−07 TCGA−NC−A5HF−01A−11R−A26W−07TCGA−18−5592−01A−01R−1635−07TCGA−18−3415−01A−01R−0980−07TCGA−69−7974−01A−11R−2187−07TCGA−64−1676−01A−01R−0946−07TCGA−44−2661−01A−01R−1107−07TCGA−44−A47A−01A−21R−A24H−0TCGA−60−2714−01A−01R−0851−07TCGA−49−6761−01A−31R−1949−077 TIMP2 Rank (vs. MMP2 & MMP14) TCGA−77−A5G8−01B−11R−A27Q−07TCGA−94−8035−01A−11R−2247−07TCGA−60−2719−01A−01R−0851−07TCGA−66−2763−01A−01R−0851−07TCGA−51−4079−01A−01R−1100−07TCGA−66−2757−01A−01R−0851−07TCGA−MF−A522−01A−11R−A262−07TCGA−21−1072−01A−01R−0692−07TCGA−77−7140−01A−41R−2045−07 TCGA−66−2778−01A−02R−0851−07TCGA−21−5784−01A−01R−1635−07TCGA−66−2771−01A−01R−0980−07TCGA−66−2727−01A−01R−0980−07 D TCGA−55−7815−01A−11R−2170−07TCGA−55−6971−01A−11R−1949−07TCGA−58−8393−01A−11R−2326−07TCGA−05−5420−01A−01R−1628−07TCGA−22−1011−01A−01R−0692−07TCGA−63−A5MR−01A−31R−A27Q−07TCGA−18−3421−01A−01R−0980−07TCGA−21−1080−01A−01R−0692−07TCGA−37−4135−01A−01R−1100−07 TCGA−33−4566−01A−01R−1443−07TCGA−77−7138−01A−41R−2045−07 TCGA−39−5039−01A−01R−1443−07TCGA−55−7284−01B−11R−2241−07TCGA−50−6673−01A−11R−1949−07TCGA−44−6779−01A−11R−1858−07TCGA−05−4249−01A−01R−1107−07TCGA−J2−8194−01A−11R−2241−07TCGA−99−8033−01A−11R−2241−07TCGA−33−4589−01A−01R−1443−07TCGA−18−3406−01A−01R−0980−07 TCGA−69−8253−01A−11R−2287−07TCGA−37−A5EL−01A−11R−A26W−07TCGA−39−5024−01A−21R−1820−07TCGA−NK−A7XE−01A−12R−A405−07TCGA−77−A5G7−01B−11R−A27Q−07TCGA−44−2666−01A−01R−0946−07TCGA−L9−A50W−01A−12R−A39D−0TCGA−86−7714−01A−12R−2170−07TCGA−69−8254−01A−11R−2287−07 7 MMP 2 MMP 7 MMP 9 MMP 1 TIMP 2 MMP14 MMP24 MMP15 MMP11 MMP12 TIMP 2 MMP 1 Normal Tumor MMP 2 MMP 7 MMP 9 MMP11 MMP12 MMP24 MMP15 MMP14 75 Tissue Breast Tissue 75 Lung % (Samples) 50 50

25

% (Samples) 25

0 0 123 123 TIMP2 Rank TIMP2 Rank

Figure 3. TIMP2 and it’s known molecular targets exhibit a distinct expression pattern in tumors. (A) Principle component analysis of Recount2 harvested breast and lung TCGA (tumor and normal tissue) and GTEx (normal tissue) log2 RNA sequencing data (RPKM) depicts unique expression profles for tumor tissue with regards to the 334 genes highlighted as co-expressed with TIMP2 in tumors. (B) Cluster dendrograms for TIMP2, ADAM12 and MMP expression in pooled TCGA and GTEx data highlights gene clusters that show appreciable tissue expression for further analysis (identifed by the red dashed box). (C) Hierarchical clustering of previously highlighted genes of interest (identifed by the red dashed box indicating appreciable levels of expression in Figure 3B) suggests that the TIMP:MMP/ADAM12 balance in tumors is shifed in favor of metalloproteinase activity. (D) Expression rank analysis of TIMP2, MMP2 and MMP14 expression (highest expression = Rank 1, lowest expression = Rank 3) from individual normal and tumor tissue samples reveals that TIMP2 expression levels are generally lowest of these three genes in tumor tissue. Abbreviations; SCC: squamous cell carcinoma.

these data could serve as a starting point to reveal potential new targets in the TME for therapeutic intervention that may not be as susceptible to the development of drug resistance or tolerance due to genetic shifs in the tumor cell population. We then examined expression profles from the TIMP2 GOI using defned subsets. Te frst subset of this GOI list focused exclusively on the metzincin protease targets of TIMP2, the MMPs and ADAM12 genes, shortening these lists based on expression level using cluster dendrograms, Fig. 3B. Hierarchical clustering analysis of these shortened gene lists clearly segregates normal tissue from malignant tissue based on the expression levels of

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TIMP2 and its protease targets, Fig. 3C. Tese fndings suggest that in the TME the dominant biologic function for TIMP2 may be skewed towards metalloproteinase inhibitory activity. An interesting observation is that in the breast carcinoma data MMP7 expression was elevated in a subset of tumor-matched normal tissues (grossly normal tissue obtained at the time of tumor resection) within TCGA dataset. Tis suggests a potential feld efect for the development of cancer and thus associated risk-factors, highlighting the potential utility of MMP7 as a biomarker, as previously suggested for other diseases24. It is also of note that the separate tumor groups, identifed in Fig. 3C, do not specifcally align with gross tumor classifcations and may be more indicative of other, previ- ously un-appreciated, cancer sub-types. We consistently observed a signifcant correlation in the expression of the well-known TIMP2, MMP2 and MMP14 axis6. Whilst these data are generally in agreement with the proposed dominant function of TIMP2 as a protease inhibitor, this correlation also suggests another major function of TIMP2 may be altered; namely its well-studied, counter-intuitive role in the cell surface activation of pro-MMP2 via formation of a trimolecular complex with MMP146. We found that tumor associated TIMP2 expression levels in comparison to MMP2 and MMP14 are consistently the lowest of the three genes, whereas in normal tissues TIMP2 is predominant, Fig. 3D. Tese fndings are consistent with the reported increase in MMP2 activation in tumor tissue1, as well as supportive of our hypothesis that the MMP-independent functions of TIMP2 are dom- inant in normal tissues. Genes associated with the matrisome and mesenchymal cell lineages acquire a positive corre- lation with TIMP2 in tumors. In an efort to gauge which set of genes acquire an altered correlation with TIMP2 in tumor tissues, we used the r.test() function of the package psych in R to test the diference between two sets of independent correlations following calculation of the TIMP2:GeneX correlation scores (Pearson’s) within tumor and normal tissue25. We used Bonferroni correction to adjust the p-values, highlighting sets of 229 and 208 genes (adjusted p-value < 0.05) from breast and lung adenocarcinomas, respectively, with a signifcant overlap of 149 genes between these carcinomas (>60%), as shown in Fig. 4A. Te top 10 signifcant genes from each set are shown in Table 2 (full tables in Supplementary Tables V & VI). Tese results highlight a substantially signifcant co-expression signature in which many of the highly co-expressed genes are exclusive to lung tissue. In contrast, all of the top 10 correlating genes from breast tumor tissue were also signifcant in lung tumor tissue (Table 2). From Ingenuity Core Analysis we also identifed upstream regulators of the signifcant genes for both lung and breast carcinomas,® shown in Table 3 (& Supplementary Tables VII & VIII), highlighting potential drivers of this co-expression profle such as TGFβ and WNT3A. MetaCore Pathway Analysis generates broader pathway des- ignations than Ingenuity and emphasized a number of molecular™ pathways that are modulated by members of the signifcant gene lists (Supplementary® Tables IX & X). Tese data were used to generate heatmaps delineating alterations in Pearson’s correlation with genes and their associated pathways using the breast cancer data set as an example, Fig. 4B. Using reference databases (Ingenuity and the Matrisome Project26) and manual designations, signifcant genes from the breast dataset were assigned® to one of 6 major ontologies (core matrisome, matrix regulators, matrix associated, plasma membrane, intracellular and nuclear). Cytoscape was used to visualize changes in correlation, displayed as nodes grouped into their designated ontologies, with edges depicting defned interactions (physical and genetic) between genes, harvested from BioGRID27 (Fig. 4C). Tis analysis highlights the interconnectivity of the TIMP2 correlating genes, providing further evidence that these cancer-associated changes in gene co-expression share the same drivers. To support our fndings from the comparison of Pearson’s coefcients (r.test) shown in Fig. 4, we also per- formed linear regression on the TIMP2:GeneX relationships and compared the slopes of ftted lines using the lsmeans R package (Supplementary Tables XI & XII). Alignment of this data with the r.test results reduced the signifcant gene hits to 163 and 131 for breast and lung carcinomas, respectively (Supplementary Tables XIII & XIV). Of these gene hits we identifed 77 highly signifcant genes that display an altered relationship with TIMP2 expression in both breast and lung cancer, a selection of which are shown in Table 4. Tis list represents genes that possess the same or similar ontologies to TIMP2 and exhibit a signifcantly altered correlation with TIMP2 in tumors that includes metzincin family proteases (ADAM12, MMP11, MMP14), genes associated with increased matrix deposition (collagens I, III. IV, etc.) and also genes associated with stromal activation (FAP), metastasis and pre-metastatic niche formation (FN1, SPARC and POSTN). Whilst many of these genes may acquire a tumor-associated increase in expression irrelevant of TIMP2, it is likely that a number of these genes result in an alteration of TIMP2 activities within developing tumors that contributes to ECM dysregulation. Using a pairwise comparison of linear regression in normal and tumor tissue, we highlight multiple genes for future investigation into their relationship with TIMP2 (Fig. 5). Periostin (POSTN) is a matrix-associated protein that functions as an adaptor/modulator of extracellular interactions28, and its expression has been extensively linked to many aspects of carcinogenesis including premetastatic niche formation, invasion and proliferation29,30. Interestingly, TIMP2 and POSTN have been described as having a shared distribution during bone develop- ment31. More recently, we demonstrated that stromal POSTN distribution in metastatic breast tumors of the lung is dramatically altered following TIMP2 treatment21. MMP11 and ADAM12 are molecular targets for TIMP2 that are routinely over-expressed in cancers32,33. ADAM12 exists in a membrane-bound form (ADAM12-L) and soluble form (ADAM12-S), with ADAM12-L possessing a cytoplasmic tail that interacts with Src homology domains of intracellular proteins. Although TIMP2 has been shown to associate with ADAM12-S, the afnity of the TIMP2:ADAM12-L complex is unknown34. Te expression of ADAM12 has been shown to localize to tumor vasculature and exert regulatory control over local angiogenesis33. Finally, high expression of the α1 chain of collagen XI (COL11A1) is associated with a range of diseases, in addition to an almost uniform high expression in human tumors35. It remains to be shown how the altered expression of these targets specifcally afects TIMP2 biological activity, either directly or indirectly. We propose that future studies into TIMP2 activity in the TME may reveal potential avenues for the use of TIMP2, or similar biological agents, as a cancer biotherapeutic that may ofer viable treatment options in combination with other directed anti-tumor therapies.

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A B ECM Remodeling

Fibroblast TGFβ Stimulation

Breast Cancer Lung Cancer TIMP2 Correlates TIMP2 Correlates (229) (208) EMT

80 149 59

Pearson Correlation

C Core Matrisome Intracellular Matrix Associated

LTBP2 ASPN BGN GFPT2 GLT8D2 ST3GAL2 HHIPL1 GREM1 MXRA5 FNDC1 FBXL7 ARMH4 COL15A1 ARHGAP31 DPYSL3 PDLIM3 TAGLN LRRC17 COL12A1 EMILIN1 COL5A3 EHD2 CALU TNFAIP6 COL16A1 SGCD C1QTNF2 C1QTNF1 CRISPLD2 IGFBP7 GALNT15 HMCN1 FIBIN SRPX2 COLEC12 AFAP1 MICAL2 TGFB3 CTGF POSTN CYBRD1 ACTA2 GXYLT2 KERA CMTM3 SH3PXD2A ACTN1 COL6A1 EFEMP2 FLNA DKK3 ECM2 LGALS1 LAMA4 VIM SYDE1 INHBA CPXM1 COL6A3 COL8A1 HSP90AA1 PDLIM7 PXDN COL5A2 PCOLCE MFAP4 PODNL1 MRVI1 DACT3 CALD1 ARSI FSTL1 PRKG1 MAP1A COL10A1 KIF26B ITGBL1 OGN OLFML2B COL6A2 FBLN2 SPSB1 COL8A2 KDELC1 PLXDC1 LAMB1 SH3PXD2B COL4A2 VCAN HSPG2 FERMT2 CLEC11A CCIN C1QTNF6 NOX4 PARVA COL11A1FBLN5 PTGIS CCDC80 GPC6 Correlation COL5A1 COL4A1 FBN1 CHSY3 MYL9 NID1 PHLDB1 Change THBS2 SPARC COPZ2 CALHM5 NID2 CYTH3 + MFGE8 WISP1 FN1 MSRB3 THBS1 DACT1

COL3A1 COL1A1 LAMA2 LUM COL1A2 SRPX -

HEG1 PCDH18 CALHM2

CERCAM ATP8B2 TCF4 CD248 AXL EVC ARL4C GAS1 MMP11 RUNX1 MMP8 GLIS2 SSPN LRRC15 DCHS1 RUNX2 LOX ADAMTS2 ADAM10 PDGFRA ANTXR1 ZNF469 BICC1 ADAMTS4 ANTXR2 LRRC32 DDR2 ACVR1 KIRREL1 F13A1 P3H1 PDGFRB HTRA3 LOXL2 BMP1 TSHZ3 FILIP1L MFRP PLPP4 MMP23B VGLL3 SULF1 ADAMTS12 CLIC4 LHFPL6 EDNRA LOXL3 VSTM4 LATS2 MMP19 MITF MMP2 PRICKLE1 VCL TLN1 LOXL1 ITGA11 HEPH ZEB1 TIMP3 SULF2 ITGB1 CLMP ADAMTS7 TGFB1I1 MMP3 SNAI2 NRP2 MMP10 MMP1 CAVIN1 NTM MMP13 ITGA5 PLXDC2 SERPINH1 ITGB5 TNS1 MMP15ADAMTS5 PALLD F2R MMP16 FAP ADAM12 LRP1 MRC2 CDH11 P4HA3 SERPINF1 THY1 SCARF2 SSC5D MMP9 ANGPTL2 VAT1 ADAMTS14 FZD1 MMP14 CD93

Matrisome Regulators Nuclear Plasma Membrane

Figure 4. Genes that acquire a positive correlation with TIMP2 in breast/lung tumors are related to the matrisome and a mesenchymal phenotype. (A) Venn diagram of signifcant tumor specifc TIMP2 correlating genes between breast and lung tissue. (B) Pearson’s correlation heatmap for genes associated with ECM remodeling, TGFβ stimulation of fbroblasts and epithelial-mesenchymal transition (EMT) (adapted from pathway analysis using MetaCore). (C) Cytoscape was used to visualize genes that acquire a positive correlation with TIMP2 in tumor tissue as gene ontology networks, with nodes and their color used to depict genes and their correlation change in tumor vs. normal tissue and node size used to depict the correlation score in tumor tissue (no correlation = small node, high correlation = large node). Edges depict reported interactions between nodes (harvested from BioGRID).

Discussion Over the past few decades, our understanding and appreciation of the characteristics and hallmarks of cancer progression have greatly expanded however, in these descriptions, dysregulation of ECM turnover and func- tion are ofen overlooked. In spite of this, the ECM is considered as a critical regulator of all aspects of cell and

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Top genes that acquire a positive correlation with TIMP2 in tumors Breast Lung Cor. Cor. Cor. Cor. Cor. Cor. Gene Normal Tumor Δ Lung? Gene Normal Tumor Δ Breast? POSTN −0.09 0.78 0.87 CSF1R −0.25 0.61 0.86 ADAM12 −0.05 0.82 0.87 WISP1 −0.23 0.49 0.73 KIF26B −0.16 0.71 0.87 GGT5 −0.13 0.58 0.71 LOXL2 −0.13 0.69 0.82 MICAL2 −0.09 0.61 0.70 COL11A1 −0.16 0.65 0.81 KIF26B −0.16 0.54 0.69 PLPP4 −0.03 0.74 0.77 ZNF469 −0.16 0.51 0.67 PODNL1 −0.06 0.64 0.70 FAM198B −0.08 0.58 0.67 MXRA5 0.11 0.80 0.69 COL6A3 0.09 0.76 0.67 COL10A1 0.04 0.72 0.68 MMP9 −0.26 0.39 0.65 ADAMTS12 0.11 0.75 0.64 COL11A1 −0.03 0.62 0.65

Table 2. Signifcant genes (Bonferroni correction < 0.05) that display the largest changes in Pearson’s correlation in breast and lung tissue samples. Final column indicates whether the correlated gene is also signifcant in the parallel tissue data set.

Upstream regulators of TIMP2 correlates Gene Hits Breast Regulator Molecule Type Lung TGFB1 Growth Factor 98 89 Alpha catenin Membrane Receptor 32 31 HRAS Enzyme 51 41 TWIST1 Transcription Regulator 27 27 WNT3A Cytokine 28 30 ITGB1 Membrane Receptor 26 23 ERBB2 Kinase 53 44 TGFB2 Growth Factor 21 19

Table 3. A selection of top potential upstream regulators from the signifcant genes identifed, acquired using Ingenuity Pathway Analysis. Potential upstream signaling and transcription factors that have been shown to modulate the genes of interest identifed as signifcant following r.test() analysis. Gene hits are the number of signifcant genes that can be regulated by the identifed regulator.

tissue biology including development36, homeostasis37, cell diferentiation38,39, wound healing40 and cell motil- ity41. As major regulators of ECM proteolysis and turnover, MMPs have a key role in all of the aforementioned biological processes. In turn, as major regulators of metalloproteinase proteolysis, TIMPs are vital regulators of ECM stability, structure and composition. Aberrant MMP activity has long been implicated in carcinogenesis and metastasis42, an observation that led to the hasty implementation of failed clinical trials with MMP inhibi- tors. Since this failure, diverse functions of the metzincin family of metalloproteinases have been identifed, both protease-dependent43 and -independent44. Analogous to the metzincin proteases, diverse functions have been unveiled for the TIMP family of proteins, both metzincin inhibitory-dependent and -independent45. Of the TIMPs, TIMP2 is most widely expressed and observed in all normal tissues, with TIMP4 being the most restricted in its tissue expression15. Tis ubiquitous expression supports the notion that TIMP2 displays homeo- static functions independent of its MMP-inhibitory activity. Interestingly, TIMP2-defcient mice do not exhibit any gross morphological or phenotypic efects in health46. However, under conditions of pathological-induced stress, such as tumor development, we have shown that TIMP2-defcient mice show an unfavorable phenotype12. Furthermore, TIMP2 defciencies are associated with abnormal motor function and cognitive dysfunction47,48. In addition, we have shown that administration of TIMP2, both directed and systemic, can reduce primary tumor burden, normalize tumor-associated vasculature, reduce infltration of myeloid-derived suppressor cells and inhibit metastatic niche gene signatures at metastatic target sites12,13. Distinct from these fndings, there have been conficting reports linking TIMP2 biological activity49,50 and expression51 to poorer prognoses. Although the balance between TIMPs and MMPs has been described as a critical indicator of ECM proteol- ysis, it should be appreciated that ECM composition is regulated by highly complex mechanisms that are infu- enced by stromal cell activity and infltration, infammatory signaling and other chemical/physical cues within the tissue microenvironment52. Te balance between TIMPs and MMPs has been briefy investigated in some instances however we sought to gain an understanding of TIMP2 expression relative to the whole transcriptome in health and malignancy.

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Lung Breast Gene Slope Δ Slope Δ Ontology COL10A1 −0.34 −0.65 Core Matrisome COL11A1 −0.30 −0.48 Core Matrisome COL1A1 −7.82 −9.42 Core Matrisome COL1A2 −3.82 −6.16 Core Matrisome COL3A1 −7.32 −9.75 Core Matrisome COL4A1 −0.29 −0.41 Core Matrisome COL5A1 −0.61 −0.56 Core Matrisome COL6A3 −0.66 −0.65 Core Matrisome COL8A2 −0.10 −0.19 Core Matrisome FN1 −2.39 −3.62 Core Matrisome LAMA2 0.06 0.04 Core Matrisome POSTN −0.84 −3.24 Core Matrisome SPARC −2.82 −6.02 Core Matrisome ADAM12 −0.05 −0.16 Matrix Regulators ADAMTS12 −0.06 −0.05 Matrix Regulators FAP −0.05 −0.08 Matrix Regulators MMP11 −0.90 −1.02 Matrix Regulators MMP14 −0.87 −0.92 Matrix Regulators RUNX1 −0.04 −0.07 Nuclear RUNX2 −0.01 −0.02 Nuclear

Table 4. A selection of signifcant genes from both analyses that occur in both breast and lung datasets. Table depicting genes that display high signifcance in both r.test (Pearson’s correlation) and lstrends (linear regression slopes) analyses in both breast and lung cancer.

Tumor TumoTumorr A 6 Normal NormalNormal 10

5

8 4

6 Breast 3 POST N ADAM12

4 2 AM12 (log2 RPKM) POSTN (log2 RPKM) AD 1 2

0 0 Tumor 3456789 3456789 TIMP2TIMP (log22 RPKM) TIMP2 (log2TIMP2 RPKM) Normal

7 B 10 TumoTumorr NormalNormal 6

8 5

4 6 1 Lung 3

4 COL11A 2

L11A1 (log2 RPKM ) 1 MMP11 (log2 RPKM)

2 CO 0

−1 0

2345678 2345678 TIMP2 (log2 RPKM) TIMP2 (log2TIMP2 RPKM)

Figure 5. Pairwise comparison of linear regression slopes for TIMP2 versus GeneX. Example linear regressions for TIMP2 versus highly signifcant genes across both analyses in breast (B) and lung (C) tissue.

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Our choice to utilize Pearson’s correlation and linear regression to assess the relationship between TIMPs and GeneX refects our belief that classical methods of diferential expression are not sufcient for understanding dynamic changes in matrix biology through health and disease. Application of these methods to interrogate the balance between ECM structural components, regulators (TIMPs and MMPs) and associated genes could potentially reveal biological consequences that are not evident when using diferential expression analysis. We observed a signifcant overlap in the co-expression profle of TIMP2 and TIMP3, in addition to the more dynamic scale for down-regulation of TIMP3 expression versus TIMP2 in tumors. Tis observation that, independent of cancer subtype, TIMP2 and TIMP3 exhibit a close similarity in the pattern of co-expressed genes (50% shared identity) reveals an unexpected parallel functionality for these TIMPs and that the co-expressed gene sets may extend functional efects beyond direct protease activity. However, further consideration of these potential efects is beyond the scope of the current study and awaits future examination utilizing specifc knockdown of putative upstream/downstream gene targets. As noted earlier, previous reports have highlighted that TIMP2 inhibits tumor-associated angiogenesis and leads to a ‘normalization’ of tumor vasculature12,13. Similar to TIMP2, TIMP3 has been identifed as a critical reg- ulator of numerous microvascular endothelial cell functions, and also as a critical regulator of pericyte function required for proper management of vascular permeability through multiple MMP-independent and -dependent functions53,54. Tis highlights a level of redundancy between TIMP2 and TIMP3 activities and raises the intrigu- ing idea that combinatorial administration of TIMP2 and TIMP3 to tumors may augment previous anti-tumor fndings observed with TIMP2. With regards to carcinomas, TIMP2 displays a strong correlation with a large number of genes, many of which belong to components of the matrisome and supporting our idea that TIMP2 function may be modulated at a post-transcriptional level in tumors. Signifcantly, we observed that the 334 GOI subset of TIMP2 co-expressed genes readily distinguish breast and lung carcinomas from normal controls, however, it is somewhat surprising that this gene subset did not diferentiate breast or lung carcinoma subtypes (ductal vs. lobular; adenocarcinoma vs. squamous) that are easily diferentiated by conventional gene expression profling22,23. We posit this gene list potentially refects a host response of stromal origin, autonomous of direct tumor cell expression. Tus, this 334 GOI cohort may refect novel tumor microenvironment gene targets and/or biomarkers. Te matrisome is an ensemble of genes that make up the ECM proteome. Tis consists of structural (core) components such as collagens and glycoproteins, matrisome-regulators (including metalloproteinases and TIMPs) and a large number of matrisome-associated genes as defned by the Matrisome Project26. In addition, putative targets for TIMP2 include non-protease targets such as integrin α3β1, IGF1R and LRP1/255–58. Tese non-protease targets also exhibit extensive interactions with components of the ECM and/or with each other, collectively this extensive network of putative TIMP2 targets/partners further strengthens our hypothesis that altered ECM composition determines TIMP2 biological functions. It is well documented that TIMP2 is a broad spectrum inhibitor of MMPs59 in addition to ADAM1234, with the afnity of these inhibitory complexes ranging from sub-fM to low-nM60,61. Surprisingly, there have been few, if any, studies into the fate of these TIMP:MMP/ADAM12 complexes or whether combinations of these impart modulatory efects on TIMP2 function. To add further complexity to the system, many of TIMP2’s molecular targets can be proteolytically cleaved by metalloproteinase activity62–64. We focused our in-depth analyses on breast and lung tumors, since we have found that TIMP2 can deliver therapeutic efects in murine models of these cancers and that they formed the nucleus of our identifed study cluster. With regards to TIMP2:MMP expression, breast tumors seemed to be more consistent with an almost ubiquitous increase in MMP11 and a smaller number of patients exhibiting high MMP9 expression. Lung tumors, on the other hand, displayed a more inconsistent pattern of MMP expression, with subsets of tumors showing high levels of diferent MMPs such as MMP14, MMP1, MMP7, MM9 and MMP12. It would be of interest to assess whether any of the observed patterns of MMP expression are associated with clinical features (such as tobacco use, radon exposure, occupational hazard, etc.) or genetically distinct tumor subtypes defned by specifc driver mutations. Lung tumor tissue also displayed a unique profle of highly signifcant TIMP2 co-expressed genes (Table 2), the potential causes and consequences of which are likely related to a complex disease etiology. Trough our understanding of MMP and TIMP expression in healthy versus neoplastic tissues, we may be able to elucidate mechanisms by which metalloproteases can alter TIMP functions. Interestingly, MMP2 and TIMP2 expression remained highly correlated in both health and disease. MMP2 is secreted as a zymogen (pro-MMP2) which forms a high afnity complex with TIMP2 via its C-terminal hemopexin domain and the non-inhibitory C-terminal of TIMP24. Counterintuitively, the activation of MMP2 is largely dependent on its endogenous inhib- itor TIMP246. Tis activation is mediated via a tri-molecular complex between TIMP2:pro-MMP2 complex and membrane-tethered MMP14, a complex that allows a free MMP14 to cleave the pro-domain of MMP2 and leads to the release of active MMP2. In tumor tissue, the balance between TIMP2, MMP2 and MMP14 is tipped in favor of MMP14 and MMP2 suggesting that the levels of MMP2 activation in tumor tissue are increased. MMP14 demonstrates weak proteolytic activity against collagen IV, however MMP2 is a potent collagen IV protease that supports invasion into the collagen IV rich basement membrane65. Interestingly, it has recently been described that TIMPless (TIMP family deficient) fibroblasts acquire cancer-associated fbroblast-like features66 that, in combination with our study, suggests that there is a yet-to-be determined regulatory relationship between MMPs, TIMPs and fbroblast activity. Indeed, expression of both MMPs and TIMPs are most closely associated with cells from the stromal compartment, including both resident and infltrating cells1. On this premise one might anticipate that TIMP expression should increase in tumors, particularly those that are highly fbrotic in nature such as breast, lung and pancreatic carcinoma. As described above, our analysis found that the genes which acquire a strong correlation with TIMP2 are associated with the extracellular matrix and cells of a mesenchymal lineage and surprisingly do not diferentiate carcinoma sub- types (Fig. 3A). Te implications and downstream signifcance of which are currently under investigation. Te

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research described here is limited by the fact that expression data is used as the sole source of analysis. Regulation of extracellular protease activity is broad and complex, including but not limited to: transcriptional expression, pro-enzyme activation, localization (intracellular, matrix bound, cell surface bound), presence of binding part- ners, cleavage and post-translational modifcations. Metalloprotease researchers are currently limited in their ability to broadly assess metzincin protease regulation and activity in situ, highlighting the importance of tran- scriptomic data to identify target groups for in depth functional analyses. Tis study highlights a number of matrisome-associated genes and regulators for future investigations into their role in the regulation of TIMP2 biological functions.We propose that targeting the imbalance between MMPs and their inhibitors to restore the enhanced TIMP2 expression observed in normal tissues may prove a valuable treatment option for a number of carcinomas. Methods Diferential expression of TIMP family genes in cancer tissue. Log2 fold change expression results for TIMP1–4 were obtained for tumor tissue versus corresponding normal tissue via the BioXpress database14 for 14 cancer studies and presented as histograms for visualization purposes. Tis database uses DESeq to assess diferential expression67.

Identifcation of focused gene list. To support the identifcation of discrete gene signatures associated with TIMP2 expression in tumors, we generated a list of 334 genes that consistently display a high correlation with TIMP2 in tumors or have the capacity to regulate TIMP2 function. Pearson’s correlation for TIMP2 versus the transcriptome for each TCGA study were harvested using cBioPortal’s co-expression function68.

Harvesting of RNA sequencing data. TCGA and GTEx RangedSummarizedExperiment (RSE) gene-level objects were downloaded from the Recount2 (https://jhubiostatistics.shinyapps.io/recount/) project and con- verted to RPKM (Reads Per Kilobase of transcript per Million mapped reads) using the getRPKM() function through the recount R package. Legacy universally unique identifers (UUIDs), which are used as sample IDs in the Recount2 TCGA data, were converted to sample barcodes using the GenomicDataCommons R package and manual download of the JSON manifest fles from the selected study using the Genomic Data Commons legacy archive (https://portal.gdc.cancer.gov/legacy-archive/). TCGA clinical data was obtained from cBioPortal using the R package cgdsr using the getClinicalData() function and samples were marked with the appropriate identifcation depending on tissue of origin and cancer sub-type. Formalin-fxed parafn-embedded samples and duplicates were removed before further analysis.

Co-expression analysis. To identify genes that acquire a correlation with TIMP2 specifcally in tumors, Recount2 processed TCGA tumor and pooled TCGA/GTEx normal tissue RPKM counts were analyzed to calcu- late Pearson’s correlation for TIMP2 versus GeneX in normal and tumor tissue. We then used the r.test() function of the R package psych to perform a z test of the Fisher’s z transformed correlations, divided by the standard error of the diference25, to test the diference between two independent (tumor and normal) correlations. Additionally, linear regression using the lm() function in R was performed followed by pairwise comparison of linear regres- sion slopes using the lstrends() and pairs() function of the R package lsmeans. Acquired p-values for both analyses were corrected for multiple testing using Bonferroni Correction.

Visualization of results. R.test() signifcant genes from the breast tissue data were assigned one of six spe- cifc gene ontology identifcations (core matrisome, matrix regulators, matrix associated, plasma membrane, intracellular and nuclear) based on reference databases (Ingenuity and the Matrisome Project26) and manual defnitions. Pathway analysis of signifcant r.test() genes was performed® using MetaCore and highly signifcant pathways and gene hits were incorporated into Pearson’s heatmaps using the ggplot2 package™ in R. Linear regres- sion plots were generated using the R package plotly with 95% confdence intervals. Data availability Te authors confrm that the data supporting the fndings of this study are available within the article [and/or] its Supplementary Materials.

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