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Metastases are hard to detect and treat, and they cause most cancer-related deaths. The relative lack of therapies targeting metastases represents a major unmet clinical need. The extracellular matrix (ECM) forms a major component of the tumor microenvironment in both primary and metastatic tumors, and certain ECM proteins can be selectively and abundantly expressed in tumors. Nanobodies against ECM proteins that show selective abundance in metastases have the potential to be used as vehicles for delivery of imaging and therapeutic cargoes. Here, we describe a strategy to develop phage-display libraries of nanobodies against ECM proteins expressed in human metastases, using as immunogens entire ECM-enriched preparations from triple-negative breast cancer (TNBC) and colorectal carcinoma (CRC) metastases to different organs as immunogens. In parallel, LC-MS/MS-based proteomics were used to define a metastasis-associated ECM signature shared by metastases from TNBC and CRC, and this conserved set of ECM proteins was selectively elevated in other tumors. As proof of concept, selective and high-affinity nanobodies were isolated against an example protein from this signature, Tenascin-C (TNC), known to be abundant in many tumor types and to play a role in metastasis. TNC was abundantly expressed in patient metastases and widely expressed across diverse metastatic sites originating from several primary tumor types. Immuno-PET/CT showed that anti-TNC nanobodies bind TNBC tumors and metastases with excellent specificity. We propose that such generic nanobodies against tumors and metastases are promising cancer-agnostic tools for delivery of therapeutics to tumor and metastatic ECM.

Metastasis is the deadliest phase of cancer progression. Experimental models using immunodeficient mice have been used to gain insights into the mechanisms of metastasis. We report here the identification of a “metastasis aggressiveness gene expression signature” derived using human melanoma cells selected based on their metastatic potentials in a xenotransplant metastasis model. Comparison with expression data from human melanoma patients shows that this metastasis gene signature correlates with the aggressiveness of melanoma metastases in human patients. Many genes encoding secreted and membrane proteins are included in the signature, suggesting the importance of tumor-microenvironment interactions during metastasis. (Mol Cancer Res 2008;6(5):760–9)

Supplementary Figure S5. Mena isoform expression does not affect Integrin alphav, beta1 or FAK levels.

Supplementary Table S4 from Gene Expression Changes in an Animal Melanoma Model Correlate with Aggressiveness of Human Melanoma Metastases

Supplementary Figures S1-S2 from Gene Expression Changes in an Animal Melanoma Model Correlate with Aggressiveness of Human Melanoma Metastases

Supplementary Figure Legends

The prognosis for pancreatic ductal adenocarcinoma (PDAC) remains poor despite decades of effort. The abundant extracellular matrix (ECM) in PDAC comprises a major fraction of the tumor mass and plays various roles in promoting resistance to therapies. However, nonselective depletion of ECM has led to poor patient outcomes. Consistent with that observation, we previously showed that individual matrisome proteins derived from stromal cells correlate with either long or short patient survival. In marked contrast, those derived from cancer cells correlate strongly with poor survival. Here, we studied three cancer cell–derived matrisome proteins that are significantly overrepresented during PDAC progression, AGRN (agrin), SERPINB5 (serine protease inhibitor B5), and CSTB (cystatin B). Using both overexpression and knockdown experiments, we demonstrate that all three are promoters of PDAC metastasis. Furthermore, these proteins operate at different metastatic steps. AGRN promoted epithelial-to-mesenchymal transition in primary tumors, whereas SERPINB5 and CSTB enhanced late steps in the metastatic cascade by elevating invadopodia formation and in vivo extravasation. All three genes were associated with a poor prognosis in human patients and high levels of SERPINB5, secreted by cancer cells and deposited in the ECM, correlated with poor patient prognosis. This study provides strong evidence that cancer cell–derived matrisome proteins can be causal in promoting tumorigenesis and metastasis and lead to poor patient survival. Therefore, compared with the bulk matrix, mostly made by stromal cells, precise interventions targeting cancer cell–derived matrisome proteins, such as AGRN, SERPINB5, and CSTB, may represent preferred potential therapeutic targets.Significance:This study provides insights into the biological roles of cancer cell–derived matrisome proteins in PDAC and supports the notion that these proteins are protumorigenic and better therapeutic targets.

Supplementary Figures S1 to S10. Supplementary Figure S1. Quality control western blots; Supplementary Figure S2. Label-free matrisome protein abundance; Supplementary Figure S3. Label-free percentage protein abundance; Supplementary Figure S4. Proteins identified in only one TMT 10-plex; Supplementary Figure S5. Tumor-cell-only proteins; Supplementary Figure S6. Abundance of tumor-cell-derived and stroma-derived matrisome proteins in metastases to different tissues; Supplementary Figure S7. Stromal proteins elevated in metastases relative to normal tissue; Supplementary Figure S8. Alterations in SERPINB1 are associated with lower progression-free survival; Supplementary Figure S9. Additional proteins evaluated for altered metastatic tropism; Supplementary figure S10. Gene Set Enrichment Analysis of ECM proteins and predicted upstream regulators in brain metastases.

List of qPCR primers.

Study summary.

This file contains figures in the supplementary information. These data complements data in the main figures. It contains: Figure S1: Microfluidic Assay for cell migration in 3D ECM; Figure S2: Macrophages increase cancer cell invasion rate in 3D ECM; macrophages increase cancer cell migration total speed but decrease directedness on 2D substrates; Figure S3: MMP activity is crucial for cancer cell migration, and macrophages enhance cancer cell MMP expression; Figure S4: The abilities of macrophages to enhance cancer cell migration dynamics and the expression of MMPs are due primarily to the paracrine factors secreted by macrophages; Figure S5: Neutralizing antibody blocking is specific to factors released by macrophages; Figure S6: Blocking TNFalpha and TGFbeta1 secreted by macrophages significantly reduced macrophage-induced MMP expression and macrophage-enhanced cancer cell invasion rate; Figure S7: TNFalpha and TGFbeta1 secreted by macrophages mostly accounts for macrophage-enhanced cancer cell migration total speed and directedness; Figure S8: TGFbeta1 has a larger effect on cancer cell migration total speed than TNFalpha. TNFalpha and TGFbeta1 synergistically increase cancer cell migration directedness; Figure S9: Cancer cell migration total speed is correlated with cancer cell expression of MT1-MMP, while cancer cell migration directedness is correlated with cancer cell expression of MMP1; Figure S10: Inhibiting both TNFalpha and TGFbeta1 in a mice model of breast tumor effectively reduces lung metastasis; Figure S11: TNFalpha and TGFbeta1 co-treatment synergistically up-regulate cancer cell MMP1 protein production and secretion. TGFbeta1 is mainly responsible for the increase in MT1-MMP protein production; Figure S12: TNFalpha and TGFbeta1 co-treatment synergistically enhance NF-κB nuclear localization in MDA-MB-435S cells. Fig. S1 and S2 is related to Fig. 1; Fig. S3 and S4 is related to Fig. 2; Fig. S5, S6, S7 is related to Fig. 3; Fig. S8-11 is related to Fig. 4; Fig. S12 is related to Fig. 6

Tumor integrin β1 (ITGB1) contributes to primary tumor growth and metastasis, but its specific roles in extravasation have not yet been clearly elucidated. In this study, we engineered a three-dimensional microfluidic model of the human microvasculature to recapitulate the environment wherein extravasation takes place and assess the consequences of β1 depletion in cancer cells. Combined with confocal imaging, these tools allowed us to decipher the detailed morphology of transmigrating tumor cells and associated endothelial cells in vitro at high spatio-temporal resolution not easily achieved in conventional transmigration assays. Dynamic imaging revealed that β1-depleted cells lacked the ability to sustain protrusions into the subendothelial matrix in contrast with control cells. Specifically, adhesion via α3β1 and α6β1 to subendothelial laminin was a critical prerequisite for successful transmigration. β1 was required to invade past the endothelial basement membrane, whereas its attenuation in a syngeneic tumor model resulted in reduced metastatic colonization of the lung, an effect not observed upon depletion of other integrin alpha and beta subunits. Collectively, our findings in this novel model of the extravasation microenvironment revealed a critical requirement for β1 in several steps of extravasation, providing new insights into the mechanisms underlying metastasis. Cancer Res; 76(9); 2513–24. ©2016 AACR.

Sample information and results.

Wild-type YAP does not enhance metastasis in zebrafish embryos.

In vitro proliferation assay for the overexpression and knockdown sets of cells.

Supplementary Tables S1 through S5. Supplementary Table S1. Overview of nomenclature; Supplementary Table S2. Overview of tandem mass tag (TMT) assignment; Supplementary Table S3. shRNA sequences; Supplementary Table S4. sgRNA sequences; Supplementary Table S5. qPCR primers used.

IPA predictions of potential upstream regulators

This is supplemental figure legends.

Movie over 12 hours of a whole zebrafish embryo injected with YAP-AA over-expressing A375 cells

Movie of A375 cells over-expressing YAP-AA in the inter-segmental vessels (ISVs) over the course of ~3 hours.

The oncogene YAP has been shown previously to promote tumor growth and metastasis. However, how YAP influences the behavior of tumor cells traveling within the circulatory system has not been as well explored. Given that rate-limiting steps of metastasis are known to occur while tumor cells enter, travel through, or exit circulation, we sought to study how YAP influences tumor cell behavior within the circulatory system. Intravital imaging in live zebrafish embryos revealed that YAP influenced the distribution of tumor cells within the animal following intravenous injection. Control cells became lodged in the first capillary bed encountered in the tail, whereas cells overexpressing constitutively active YAP were able to travel through this capillary plexus, reenter systemic circulation, and seed in the brain. YAP controlled transit through these capillaries by promoting active migration within the vasculature. These results were corroborated in a mouse model following intravenous injection, where active YAP increased the number of circulating tumor cells over time. Our results suggest a possible mechanism whereby tumor cells can spread to organs beyond the first capillary bed downstream from the primary tumor. These results also show that a specific gene can affect the distribution of tumor cells within an animal, thereby influencing the global pattern of metastasis in that animal.Significance:These findings demonstrate that YAP endows tumor cells with the ability to move through capillaries, allowing them to return to and persist in circulation, thereby increasing their metastatic spread.See related commentary by Davidson, p. 3797

Movie of A375 control cells in the tail over the course of 12 hours following injection.

Movie showing blood flow through the Duct of Cuvier of a 2-day-old zebrafish embryo.

Original mass spectrometry data used for analysis

Movie over 12 hours of a whole zebrafish embryo injected with EV control A375 cells

Time-lapse confocal microscopy (20X) of one representative region of interest with control MDA-MB 231 extravasating (green) from in vitro microvascular networks (HUVEC; red).

File contains: Supplementary Materials and Methods, Supplementary Figure Legends and Supplementary References

Supplementary materials and methods

Supplementary Data from Agrin Loss in Barrett's Esophagus-Related Neoplasia and Its Utility as a Diagnostic and Predictive Biomarker

Purpose:Sessile serrated lesions (SSL) are precursors to colon carcinoma, and their distinction from other polyps, in particular hyperplastic polyps (HP), presents significant diagnostic challenges. We evaluated expression patterns in colonic polyps of previously identified colon carcinoma–associated extracellular matrix (ECM) proteins to identify markers distinguishing SSLs from other polyps.Experimental Design:Gene-expression analyses of ECM proteins were performed using publicly available data on preneoplastic colonic polyps. In parallel, we evaluated by IHC the expression of agrin (AGRN) in over 400 colonic polyps, including HP, SSL with and without dysplasia, traditional serrated adenomas (TSA), and tubular adenomas (TA), and compared the consistency of standard histologic diagnosis of SSLs by experienced gastrointestinal pathologists with that of AGRN IHC.Results:Differential gene expression analysis and IHC identified AGRN, serine peptidase inhibitor (SERPINE2), and TIMP metallopeptidase inhibitor 1 (TIMP1) elevated in SSLs and HPs but decreased in TAs and absent in normal colon. AGRN-positive basal laminae were noted in all TA, TSA, HP, and SSL in distinguishable patterns, whereas other polyps and normal mucosa were negative. SSL with or without dysplasia consistently showed IHC staining for AGRN in the muscularis mucosae, which was absent in HP, TSA, TA, and other polyps. In contrast, histologic evaluation showed only weak interobserver agreement (kappa value = 0.493) in distinguishing SSLs.Conclusions:Muscularis mucosae–based AGRN immunostaining is a novel biomarker to distinguish SSL from HP, TSA, and TA, with a specificity of 97.1% and sensitivity of 98.9% and can assist in diagnosis of morphologically challenging colonic polyps.

This files contains detailed methods on procedures used in this study, which complements the method described in the main paper. It also expands on the methods described in the main paper. Specifically, it contains method on Microfluidic 3D cell migration assay; 2D Cell Migration Assay; Immunofluorescent staining, confocal microscopy, and DQ-Collagen I Release Assay; ELISA; Cancer cell-macrophage co-culture for western blot analysis; Treatment of cancer cells for western blot analysis; Protein isolation and western blot analysis; Total RNA extraction and mRNA detection; In vivo metastasis assay

Sequences of the miR-30-based shRNAs targeting different human (for MDA-MB-231, A-375 MA2, SUM 159) or mouse (for 4T1) integrin subunits used in this study ; 4T1 cells stably expressing miR30-based shRNAs targeting the indicated integrin subunits were assayed at least 3 times by live-cell flow cytometry for surface expression of the indicated integrin .

Movie of A375 EV control cells in the inter-segmental vessels (ISVs) over the course of ~3 hours.

Movie over 12 hours of the head of a zebrafish embryo injected with EV control A375 cells.

The ability of a cancer cell to migrate through the dense extracellular matrix within and surrounding the solid tumor is a critical determinant of metastasis. Macrophages enhance invasion and metastasis in the tumor microenvironment, but the basis for their effects is not fully understood. Using a microfluidic 3D cell migration assay, we found that the presence of macrophages enhanced the speed and persistence of cancer cell migration through a 3D extracellular matrix in a matrix metalloproteinases (MMP)-dependent fashion. Mechanistic investigations revealed that macrophage-released TNFα and TGFβ1 mediated the observed behaviors by two distinct pathways. These factors synergistically enhanced migration persistence through a synergistic induction of NF-κB–dependent MMP1 expression in cancer cells. In contrast, macrophage-released TGFβ1 enhanced migration speed primarily by inducing MT1-MMP expression. Taken together, our results reveal new insights into how macrophages enhance cancer cell metastasis, and they identify TNFα and TGFβ1 dual blockade as an antimetastatic strategy in solid tumors. Cancer Res; 77(2); 279–90. ©2016 AACR.

Purpose:There is an unmet need for identifying novel biomarkers in Barrett's esophagus that could stratify patients with regards to neoplastic progression. We investigate the expression patterns of extracellular matrix (ECM) molecules in Barrett's esophagus and Barrett's esophagus–related neoplasia, and assess their value as biomarkers for the diagnosis of Barrett's esophagus–related neoplasia and to predict neoplastic progression.Experimental Design:Gene-expression analyses of ECM matrisome gene sets were performed using publicly available data on human Barrett's esophagus, Barrett's esophagus–related dysplasia, esophageal adenocarcinoma (ADCA) and normal esophagus. Immunohistochemical expression of basement membrane (BM) marker agrin (AGRN) and p53 was analyzed in biopsies of Barrett's esophagus–related neoplasia from 321 patients in three independent cohorts.Results:Differential gene-expression analysis revealed significant enrichment of ECM matrisome gene sets in dysplastic Barrett's esophagus and ADCA compared with controls. Loss of BM AGRN expression was observed in both Barrett's esophagus–related dysplasia and ADCA. The mean AGRN loss in Barrett's esophagus glands was significantly higher in Barrett's esophagus–related dysplasia and ADCA compared with non-dysplastic Barrett's esophagus (NDBE; P < 0.001; specificity = 82.2% and sensitivity = 96.4%). Loss of AGRN was significantly higher in NDBE samples from progressors compared with non-progressors (P < 0.001) and identified patients who progressed to advanced neoplasia with a specificity of 80.2% and sensitivity of 54.8%. Moreover, the combination of AGRN loss and abnormal p53 staining identified progression to Barrett's esophagus–related advanced neoplasia with a specificity and sensitivity of 86.5% and 58.7%.Conclusions:We highlight ECM changes during Barrett's esophagus progression to neoplasia. BM AGRN loss is a novel diagnostic biomarker that can identify patients with NDBE at increased risk of developing advanced neoplasia.

Legend for Supplementary Figures S1-S8, Supplementary Tables S1-S2, and Supplementary Movie S1.

Marker selection analysis to identify proteins elevated in each metastatic tissue

In vitro microfluidics-based extravasation assays (S1); Knockdown of β1 integrin results in a reduction in extravasation rate (S2); Extravasation in vivo (S3); Analysis of adhesion rate in an endothelial monolayer microfluidic flow assay (S4); Visualization and scoring of transendothelial migration events in the vertical monolayer device (S5); Tumor cell protrusion dynamics in 3D collagen 1/fibrin mix matrices (S6); β1 integrin is required for invasion past the basement membrane (S7); Role of alpha integrins subunits in transendothelial migration in microvascular networks (S8).

List of published results of the identified proteins.

Gene sets used to perform GSEA

Legends for the supplemental figures and videos.

Movie over 12 hours of the head of a zebrafish embryo injected with YAP-AA-over-expressing A375 cells.

Movie of A375 cells over-expressing YAP-AA in the tail over the course of 12 hours following injection.