Immune‐mediated ECM depletion improves tumour perfusion and payload delivery
2019; Springer Nature; Volume: 11; Issue: 12 Linguagem: Inglês
10.15252/emmm.201910923
ISSN1757-4684
AutoresYen Yeow, Venkata Ramana Kotamraju, Xiao Wang, Meenu Chopra, Nasibah Azme, Jiansha Wu, Tobias Schoep, Derek S. Delaney, Kirk W. Feindel, Ji Li, Kelsey M. Kennedy, Wes M. Allen, Brendan F. Kennedy, Irma Larma, David D. Sampson, Lisa M. Mahakian, Brett Z. Fite, Hua Zhang, Tomas Friman, Aman P. Mann, Farah Aziz, M. Priyanthi Kumarasinghe, Mikael Johansson, Hooi C. Ee, George C. Yeoh, Lingjun Mou, Katherine W. Ferrara, Hector Billiran, Ruth Ganß, Erkki Ruoslahti, Juliana Hamzah,
Tópico(s)Glioma Diagnosis and Treatment
ResumoArticle11 November 2019Open Access Source DataTransparent process Immune-mediated ECM depletion improves tumour perfusion and payload delivery Yen Ling Yeow Yen Ling Yeow Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Venkata Ramana Kotamraju Venkata Ramana Kotamraju Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Search for more papers by this author Xiao Wang Xiao Wang Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Meenu Chopra Meenu Chopra Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Nasibah Azme Nasibah Azme Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Jiansha Wu Jiansha Wu Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Tobias D Schoep Tobias D Schoep Telethon Kids Institute, Subiaco, WA, Australia Search for more papers by this author Derek S Delaney Derek S Delaney orcid.org/0000-0002-1185-121X Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Kirk Feindel Kirk Feindel Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Ji Li Ji Li Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Kelsey M Kennedy Kelsey M Kennedy Department of Electrical, Electronic & Computer Engineering, School of Engineering, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Wes M Allen Wes M Allen Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Department of Electrical, Electronic & Computer Engineering, School of Engineering, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Brendan F Kennedy Brendan F Kennedy Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Department of Electrical, Electronic & Computer Engineering, School of Engineering, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Irma Larma Irma Larma Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, Perth, WA, Australia Search for more papers by this author David D Sampson David D Sampson Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, Perth, WA, Australia Department of Electrical, Electronic & Computer Engineering, School of Engineering, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Lisa M Mahakian Lisa M Mahakian Department of Biomedical Engineering, University of California Davis, Davis, CA, USA Search for more papers by this author Brett Z Fite Brett Z Fite Department of Biomedical Engineering, University of California Davis, Davis, CA, USA Search for more papers by this author Hua Zhang Hua Zhang Department of Biomedical Engineering, University of California Davis, Davis, CA, USA Search for more papers by this author Tomas Friman Tomas Friman Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Search for more papers by this author Aman P Mann Aman P Mann Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Search for more papers by this author Farah A Aziz Farah A Aziz Sir Charles Gairdner Hospital, Perth, WA, Australia Search for more papers by this author M Priyanthi Kumarasinghe M Priyanthi Kumarasinghe PathWest Laboratory Medicine, QE2 Medical Centre, Perth, WA, Australia Search for more papers by this author Mikael Johansson Mikael Johansson Sir Charles Gairdner Hospital, Perth, WA, Australia Search for more papers by this author Hooi C Ee Hooi C Ee Sir Charles Gairdner Hospital, Perth, WA, Australia Search for more papers by this author George Yeoh George Yeoh Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Lingjun Mou Lingjun Mou Sir Charles Gairdner Hospital, Perth, WA, Australia Search for more papers by this author Katherine W Ferrara Katherine W Ferrara Department of Biomedical Engineering, University of California Davis, Davis, CA, USA Search for more papers by this author Hector Billiran Hector Billiran Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Search for more papers by this author Ruth Ganss Ruth Ganss Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Erkki Ruoslahti Erkki Ruoslahti Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Search for more papers by this author Juliana Hamzah Corresponding Author Juliana Hamzah [email protected] orcid.org/0000-0002-3802-5073 Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Yen Ling Yeow Yen Ling Yeow Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Venkata Ramana Kotamraju Venkata Ramana Kotamraju Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Search for more papers by this author Xiao Wang Xiao Wang Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Meenu Chopra Meenu Chopra Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Nasibah Azme Nasibah Azme Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Jiansha Wu Jiansha Wu Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Tobias D Schoep Tobias D Schoep Telethon Kids Institute, Subiaco, WA, Australia Search for more papers by this author Derek S Delaney Derek S Delaney orcid.org/0000-0002-1185-121X Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Kirk Feindel Kirk Feindel Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Ji Li Ji Li Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Kelsey M Kennedy Kelsey M Kennedy Department of Electrical, Electronic & Computer Engineering, School of Engineering, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Wes M Allen Wes M Allen Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Department of Electrical, Electronic & Computer Engineering, School of Engineering, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Brendan F Kennedy Brendan F Kennedy Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Department of Electrical, Electronic & Computer Engineering, School of Engineering, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Irma Larma Irma Larma Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, Perth, WA, Australia Search for more papers by this author David D Sampson David D Sampson Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, Perth, WA, Australia Department of Electrical, Electronic & Computer Engineering, School of Engineering, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Lisa M Mahakian Lisa M Mahakian Department of Biomedical Engineering, University of California Davis, Davis, CA, USA Search for more papers by this author Brett Z Fite Brett Z Fite Department of Biomedical Engineering, University of California Davis, Davis, CA, USA Search for more papers by this author Hua Zhang Hua Zhang Department of Biomedical Engineering, University of California Davis, Davis, CA, USA Search for more papers by this author Tomas Friman Tomas Friman Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Search for more papers by this author Aman P Mann Aman P Mann Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Search for more papers by this author Farah A Aziz Farah A Aziz Sir Charles Gairdner Hospital, Perth, WA, Australia Search for more papers by this author M Priyanthi Kumarasinghe M Priyanthi Kumarasinghe PathWest Laboratory Medicine, QE2 Medical Centre, Perth, WA, Australia Search for more papers by this author Mikael Johansson Mikael Johansson Sir Charles Gairdner Hospital, Perth, WA, Australia Search for more papers by this author Hooi C Ee Hooi C Ee Sir Charles Gairdner Hospital, Perth, WA, Australia Search for more papers by this author George Yeoh George Yeoh Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Lingjun Mou Lingjun Mou Sir Charles Gairdner Hospital, Perth, WA, Australia Search for more papers by this author Katherine W Ferrara Katherine W Ferrara Department of Biomedical Engineering, University of California Davis, Davis, CA, USA Search for more papers by this author Hector Billiran Hector Billiran Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Search for more papers by this author Ruth Ganss Ruth Ganss Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Erkki Ruoslahti Erkki Ruoslahti Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA Search for more papers by this author Juliana Hamzah Corresponding Author Juliana Hamzah [email protected] orcid.org/0000-0002-3802-5073 Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Author Information Yen Ling Yeow1, Venkata Ramana Kotamraju2, Xiao Wang1, Meenu Chopra1, Nasibah Azme1, Jiansha Wu1, Tobias D Schoep3, Derek S Delaney1, Kirk Feindel4, Ji Li1, Kelsey M Kennedy5, Wes M Allen1,5, Brendan F Kennedy1,5, Irma Larma4, David D Sampson4,5, Lisa M Mahakian6, Brett Z Fite6, Hua Zhang6, Tomas Friman2, Aman P Mann2, Farah A Aziz7, M Priyanthi Kumarasinghe8, Mikael Johansson7, Hooi C Ee7, George Yeoh1, Lingjun Mou7, Katherine W Ferrara6, Hector Billiran2,9, Ruth Ganss1, Erkki Ruoslahti2 and Juliana Hamzah *,1 1Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Perth, WA, Australia 2Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA 3Telethon Kids Institute, Subiaco, WA, Australia 4Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, Perth, WA, Australia 5Department of Electrical, Electronic & Computer Engineering, School of Engineering, The University of Western Australia, Perth, WA, Australia 6Department of Biomedical Engineering, University of California Davis, Davis, CA, USA 7Sir Charles Gairdner Hospital, Perth, WA, Australia 8PathWest Laboratory Medicine, QE2 Medical Centre, Perth, WA, Australia 9Present address: Department of Biology, Xavier University of Louisiana, New Orleans, LA, USA *Corresponding author. Tel: +61 8 6151 0732; E-mail: [email protected] EMBO Mol Med (2019)11:e10923https://doi.org/10.15252/emmm.201910923 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract High extracellular matrix (ECM) content in solid cancers impairs tumour perfusion and thus access of imaging and therapeutic agents. We have devised a new approach to degrade tumour ECM, which improves uptake of circulating compounds. We target the immune-modulating cytokine, tumour necrosis factor alpha (TNFα), to tumours using a newly discovered peptide ligand referred to as CSG. This peptide binds to laminin–nidogen complexes in the ECM of mouse and human carcinomas with little or no peptide detected in normal tissues, and it selectively delivers a recombinant TNFα-CSG fusion protein to tumour ECM in tumour-bearing mice. Intravenously injected TNFα-CSG triggered robust immune cell infiltration in mouse tumours, particularly in the ECM-rich zones. The immune cell influx was accompanied by extensive ECM degradation, reduction in tumour stiffness, dilation of tumour blood vessels, improved perfusion and greater intratumoral uptake of the contrast agents gadoteridol and iron oxide nanoparticles. Suppressed tumour growth and prolonged survival of tumour-bearing mice were observed. These effects were attainable without the usually severe toxic side effects of TNFα. Synopsis This study establishes a new approach to treat solid tumours by immune modulation and ECM depletion. The developed agent, TNFα-CSG, has dual capacity as an immunotherapeutic and ECM reducing agent which improves tumour perfusion and drug delivery. Extracellular matrix (ECM) targeting peptide, CSG, specifically recognises the ECM in multiple types of murine and human tumours. CSG targeting effectively delivers TNFα into tumours and elicits immune cell infiltration into the ECM. Immune-mediated ECM degradation by a cocktail of proteases reduces tumour stiffness and decompression of tumour blood vessels, and enhances tumour perfusion. TNFα-CSG treatment sensitizes tumours for enhanced uptake of diagnostic nanoparticles. The anti-tumour effects of TNFα-CSG are primarily mediated by T cells. Introduction A dense network of highly disorganised ECM is the central feature of desmoplastic tumours and often encountered in aggressive and treatment-resistant cancers (Pickup et al, 2014). Tumour ECM is made of overproduced scaffolds of collagen, non-collagenous glycoproteins and glycosaminoglycans (Mouw et al, 2014). Collectively, these ECM components represent an obstructive physical barrier that determines how migrating cells and circulating compounds enter and exit a tumour. In diagnostic and therapeutic settings, the ECM barrier restricts penetration, and thereby tumour uptake of various imaging agents and anti-cancer therapeutics (Hellebust & Richards-Kortum, 2012; Salmon et al, 2012; Choi et al, 2013). Destroying the fibrotic ECM barrier to increase the vulnerability of a tumour presents a compelling adjuvant strategy. For instance, ECM can be disrupted by employing locally injected or ectopically expressed enzymes (Guedan et al, 2010; Caruana et al, 2015), systemic application of modified hyaluronidase (PEGPH20) (Provenzano et al, 2012), fibrinolytic therapy with tissue plasminogen activator (Kirtane et al, 2017), treatment with inhibitors of ECM production (Olive et al, 2009; Diop-Frimpong et al, 2011; Vennin et al, 2017) or physical means, such as photothermal therapy (Marangon et al, 2017). Thus far, however, existing approaches lack the specificity to target and degrade tumour ECM and, therefore, tend to cause systemic toxicity (Kirtane et al, 2017). Moreover, some of the strategies used to degrade or reduce tumour ECM have also been implicated in promoting metastases (Ozdemir et al, 2014; Sevenich & Joyce, 2014; Schmaus & Sleeman, 2015; Rath et al, 2017). Thus, new approaches to dealing with the ECM barrier in tumours are needed. We have devised a strategy that is based on targeting of an immune-modulating cytokine into tumour ECM. Screening of phage-displayed peptide libraries in live mice has been used to identify peptides that specifically recognise tumours (Ruoslahti, 2016). The tumour-homing peptides identified in this manner can be used in selective delivery of payloads to tumours. For example, targeted delivery of cytokines into tumours can improve therapeutic outcomes (Hamzah et al, 2008; Johansson et al, 2012; Johansson-Percival et al, 2015). Here, we identified a peptide that specifically recognises tumour ECM and used it to target a cytokine to the ECM. TNFα is a pleiotropic cytokine known to promote innate and adaptive immune responses (Talmadge et al, 1987). It can stimulate multiple types of immune cells to secrete proteases that are capable of degrading ECM (Vaday et al, 2000; Melamed et al, 2006). An in vitro study shows that TNFα bound to fibronectin in ECM attracts monocytes and triggers their activation into MMP9-secreting cells (Vaday et al, 2000). We hypothesised that TNFα, when specifically targeted to tumour ECM, may have similar effects, leading to ECM degradation and enhanced penetration of compounds to tumours. Here, we demonstrate that TNFα fused to a new tumour ECM-recognising peptide specifically accumulates in desmoplastic tumours, increasing immune cell accumulation and ECM degradation. Results Identification and analysis of a tumour ECM-binding peptide We designed a phage library screening protocol aimed at identifying peptides specific for tumour ECM. The screen consisted of an initial in vitro biopanning of a library of random seven-amino acid peptides flanked by a cysteine residue on each side (general structure: CX7C) on Matrigel™. Matrigel is an ECM preparation derived from a mouse tumour that produces copious amounts of basement membrane (BM)-type ECM consisting primarily of laminin, nidogen-1 (also known as entactin) and collagen IV. There are also traces of heparan sulphate proteoglycan (perlecan), along with some growth factors. The enriched phage pool from 3 in vitro rounds was subsequently subjected to 4 rounds of in vivo screening in mice bearing MDA-MB-435 human breast cancer xenograft tumours. A 9-amino acid peptide, CSGRRSSKC (termed CSG), and its variants were present in multiple copies in the final phage pool (Appendix Fig S1A–D). CSG was selected for further study. We compared the binding of synthetic carboxyfluorescein (FAM)-labelled CSG to tumour sections. Appendix Fig S1E and F shows robust binding to sections of neuroendocrine pancreatic tumours from genetically engineered RIP1-Tag5 mice which are strongly fibrotic (Ganss & Hanahan, 1998). CREKA, a previously identified peptide that binds to fibrin deposited on the vessel walls of tumour vessels and to tumour stroma (Simberg et al, 2007), showed weaker (about 10-fold less) binding which was essentially restricted to tumour vessels, and there was no detectable binding of a peptide with no tumour-binding activity (ARA). When injected intravenously (i.v.) into tumour-bearing mice, CSG specifically accumulated in orthotopically implanted murine 4T1 breast and RIP1-Tag5 tumours (Fig 1). Excluding the kidneys where circulating peptides are excreted, normal organs showed only background fluorescence (Fig 1A). The specificity of the tumour accumulation was confirmed by histological detection and quantification of FAM-peptide homing using an antibody against fluorescein (Fig 1B and C). The accumulation of CSG in these tumours was at least 10- to 15-fold higher than in the normal tissues. CSG also accumulated in tumours in other mouse models, including the ALB-Tag hepatocellular carcinoma (HCC) (Ryschich et al, 2006), MMTV-PyMT breast carcinoma and transplanted CT26 colon carcinoma (Appendix Fig S1G). Figure 1. CSG specifically recognises mouse and human tumours, and binds to ECM A–C. Mice bearing orthotopically implanted 4T1 breast cancers and RIP1-Tag5 (RT5) tumours were i.v. injected with 0.1 μmol of FAM-CSG, and tissues were collected after 1-h circulation. (A) Photographic image of tissues from 4T1 tumour-bearing mouse under bright light and UV illuminator. (B and C) Distribution of FAM-CSG in different tissues including tumours (4T1 T and RT5 T), kidney (K), vertebrae (V), lung (LG), liver (LV), intestine (I), muscle (MU), spleen (SP), heart (H), pancreas (P), brain (B), lymph node (LN) and skin (SK), detected by immunoperoxidase staining with anti-FITC antibody. Representative staining (brown) is shown for each tissue in (B) and as mean ± SEM of percentage area per tissue section stained with anti-FITC antibody (n = 3; *P < 0.05 and **P < 0.005, tumour compared to other tissues except kidney by one-way ANOVA test with Tukey's correction) in (C). Scale bars: 100 μm. D, E. Human breast tumour (Hu BT) and normal breast tissues (Hu NB): 8-μm serial tissue sections were incubated for 30 min with 1 μM FAM-CSG or FAM-ARA, in the presence or absence of 1 mM unlabelled CSG peptide. CSG (brown) was detected as in panels (B and C). (D) Representative micrographs of corresponding tissues stained with anti-FITC antibody (brown) are shown for an individual patient sample. Scale bars: 150 μm. (E) Bar charts show mean ± SEM of percentage area per tissue section stained with anti-FITC antibody (N = 4 Hu NB and N = 7 Hu BT; ***P < 0.001 and ****P < 0.0001 by one-way ANOVA test with Tukey's correction). Source data are available online for this figure. Source Data for Figure 1 [emmm201910923-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint To determine whether CSG also recognises human tumours, we assessed FAM-CSG binding on fresh biopsies of primary human breast carcinomas from seven mastectomy patients. CSG bound selectively in all tumour specimens (Figs 1D and E, and EV1A), whereas there was negligible binding to the adjacent normal breast tissue (Fig 1D). The FAM-CSG binding was inhibited by unlabelled CSG, and the ARA control peptide showed no binding to the tumours (Figs 1D and E, and EV1A). CSG-specific tumour binding was also detected on fresh biopsies of primary human pancreatic adenocarcinoma and HCC from three pancreatectomy and hepatectomy patients, respectively (Fig EV1B). Therefore, CSG binding is conserved in human fibrotic cancers. Click here to expand this figure. Figure EV1. CSG-specific binding in human cancers A, B. 8-μm tissue sections were incubated for 30 min with 1 μM FAM-CSG or FAM-ARA. Micrographs of corresponding tissues stained with anti-FITC antibody (brown) are shown for (A) individual breast cancer patients and a representative (B, left) PDAC and (B, right) HCC patient samples. (B) Bar charts on the right show mean ± SEM of percentage area per tissue section stained with anti-FITC antibody (N = 3 PDAC and HCC patient samples; *P < 0.05 and **P < 0.005 by Student's t-test). Download figure Download PowerPoint As CSG was first isolated based on its in vitro binding to Matrigel, we used a CSG affinity matrix to isolate the CSG target molecule from a dilute solution of Matrigel. Elution of the affinity matrix with soluble CSG peptide produced several bands, which were identified by mass spectrometry as laminin subunits alpha-1 and gamma-1, and nidogen-1. These proteins were absent in eluates obtained with the CREKA control peptide but appeared upon subsequent elution of the same matrix with CSG (Fig 2A). These results indicate that the target of CSG is laminin–nidogen-1, which exists as a complex in ECM (Timpl et al, 1990). We next studied the expression of laminin and nidogen-1, as well as collagen IV, the third member of the basement membrane complex, in tumours. The three proteins co-localised in all tissues; however, their abundance was greater in tumours than in normal tissues (Figs 2B and EV2A). As indicated by co-staining for laminin and the blood vessel marker CD31 (Fig 2C), laminin in normal mouse envelops the blood vessels as part of the basement membrane complex. In contrast, in tumours the basement membrane complex showed widespread expression separate from the vessels (Fig 2C) and showed strong overlaps with fibrillar collagens (collagen I and trichrome staining; Fig EV2B and C). These results show that tumour ECM differs from normal ECM in abundance and location. Figure 2. CSG specifically binds tumour ECM with affinity to laminin–nidogen-1 complexMatrigel extract was fractionated by affinity chromatography on CSG-coupled columns. Bound proteins were eluted with 2 mM CSG or control CREKA peptide solutions and separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. A. Top: Silver staining shows multiple bands in the Matrigel extract (lane 1), laminin–nidogen-1 complex (lane 2) and purified laminin (lane 3), in comparison with 4 bands eluted with CSG peptide which did not appear in the first CSG elution (lane 4) but appeared in eluted fractions 2 (lane 5) and 3 (lane 6). These bands were no longer visible in subsequent elution (lane 7). Bottom: Silver staining shows the absence of bands when CSG-coupled column was eluted with the control CREKA peptide (lanes 1–3); subsequent elution of the column with the CSG peptide shows the 4 previously shown bands (lane 5). The 4 bands (top gel, lanes 5 and 6) were identified by mass spectrometry as laminin subunit α-1 (Lamα-1), laminin subunit γ-1 (Lamγ-1) and 2 nidogen-1 bands of 140 and 110 kDa. B. Normal pancreas from a C3H mouse (N Pan) and a RIP1-Tag5 tumour (RT5 T) were stained for the indicated ECM components. Representative micrographs are shown in the top panel. Bar graphs in the bottom panel show quantification of the area positive for each ECM protein (mean ± SEM; n = 3, *P < 0.05, **P < 0.005 and ***P < 0.001 by multiple t-tests). C. Tissues as shown in (B) were stained for laminin (lam, green) and blood vessels (CD31, red). Representative micrographs are shown. The bar graph depicts the ratio of laminin over CD31 staining (mean ± SEM; n = 5, **P < 0.005 by Student's t-test). Arrows: areas positive for laminin expression but lack CD31 staining. D. Representative micrograph of a RIP1-Tag5 tumour (T) with normal pancreas (N Pan) showing CSG binding (green; in vitro binding was performed as indicated in Appendix Fig S1E), laminin staining (lam; red) and CSG–laminin co-localisation (yellow). E. Co-staining analysis of in vitro bound CSG or CREKA (green) compared to indicated ECM markers or CD31+ tumour blood vessels (red). Representative micrographs (left) and corresponding bar graphs (right) show co-localisation of indicated markers with CSG or CREKA (mean ± SEM; n = 4, ****P < 0.001 by one-way ANOVA test with Tukey's correction). Data information: Scale bars for (B, C and E): 50 μm. Scale bars (D): 200 μm. Source data are available online for this figure. Source Data for Figure 2 [emmm201910923-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. CSG binding co-localises with high abundant tumour ECM A. Normal human breast (Hu NB) and human breast carcinoma (Hu BT) were stained for the indicated ECM components. Representative micrographs are shown (left) and corresponding bar graphs (right) quantifying the area positive for each ECM marker (mean ± SEM; n = 3, ***P < 0.001 and ***P < 0.0001 by multiple t-tests). Scale bars: 50 μm. B. Co-staining analysis of 4T1 (4T1 T) and RIP1-Tag5 (RT5 T) tumours for laminin (lam, red) and collagen I (Col-I-FITC, green) expression. Scale bars: 50 μm. C. Top panel: Similar analysis as in panel (B) for Hu BT tumour. Lower panel: Serial tissue sections of Hu BT comparing immunostaining of laminin and trichrome. Scale bars: 300 μm. D, E. Co-staining of FAM-CSG (green) and ECM markers or CD31 (red) on Hu BT (D) and 4T1 tumour (E). Representative micrographs are s
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