Morten G. Rasch

Dynamic interplay between the collagen scaffold and tumor evolution

The extracellular matrix (ECM) is a key regulator of cell and tissue function. Traditionally, the ECM has been thought of primarily as a physical scaffold that binds cells and tissues together. However, the ECM also elicits biochemical and biophysical signaling. Controlled proteolysis and remodeling of the ECM network regulate tissue tension, generate pathways for migration, and release ECM protein fragments to direct normal developmental processes such as branching morphogenesis. Collagens are major components of the ECM of which basement membrane type IV and interstitial matrix type I are the most prevalent. Here we discuss how abnormal expression, proteolysis and structure of these collagens influence cellular functions to elicit multiple effects on tumors, including proliferation, initiation, invasion, metastasis, and therapy response.

Imaging Tumor-Stroma Interactions during Chemotherapy Reveals Contributions of the Microenvironment to Resistance

Little is known about the dynamics of cancer cell death in response to therapy in the tumor microenvironment. Intravital microscopy of chemotherapy-treated mouse mammary carcinomas allowed us to follow drug distribution, cell death, and tumor-stroma interactions. We observed associations between vascular leakage and response to doxorubicin, including improved response in matrix metalloproteinase-9 null mice that had increased vascular leakage. Furthermore, we observed CCR2-dependent infiltration of myeloid cells after treatment and that Ccr2 null host mice responded better to treatment with doxorubicin or cisplatin. These data show that the microenvironment contributes critically to drug response via regulation of vascular permeability and innate immune cell infiltration. Thus, live imaging can be used to gain insights into drug responses inxa0situ.

Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity

Protein glycosylation often changes during cancer development, resulting in the expression of cancer-associated carbohydrate antigens. In particular mucins such as MUC1 are subject to these changes. We previously identified an immunodominant Tn-MUC1 (GalNAc-α-MUC1) cancer-specific epitope not covered by immunological tolerance in MUC1 humanized mice and man. The objective of this study was to determine if mouse antibodies to this Tn-MUC1 epitope induce antibody-dependent cellular cytotoxicity (ADCC) pivotal for their potential use in cancer immunotherapy. Binding affinity of mAb 5E5 directed to Tn-MUC1 was investigated using BiaCore. The availability of Tn-MUC1 on the surface of breast cancer cells was evaluated by immunohistochemistry, confocal microscopy, and flow cytometry, followed by in vitro assessment of antibody-dependent cellular cytotoxicity by mAb 5E5. Biacore analysis demonstrated high affinity binding (KDu2009=u20091.7xa0nM) of mAb 5E5 to its target, Tn-MUC1. Immunolabelling with mAb 5E5 revealed surface expression of the Tn-MUC1 epitope in breast cancer tissue and cell lines, and mAb 5E5 induced ADCC in two human breast cancer cell lines, MCF7 and T47D. Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity suggesting that antibodies targeting glycopeptide epitopes on mucins are strong candidates for cancer-specific immunotherapies.

A New Assay for Measurement of the Liberated Domain I of the Urokinase Receptor in Plasma Improves the Prediction of Survival in Colorectal Cancer

BACKGROUNDnThe liberated domain I of the urokinase plasminogen activator receptor [uPAR(I)] is a significant prognostic marker in lung and ovarian cancer, although the uPAR(I) concentration is below the limit of quantification (LOQ) in a substantial proportion of patient samples (Lung Cancer 2005;48:349-55; Clin Cancer Res 2008;14:5785-93; APMIS 2009;117:755-61). This study was undertaken to design an immunoassay with improved functional sensitivity for measuring uPAR(I) and to evaluate the prognostic value of uPAR(I) for colorectal cancer (CRC) patients.nnnMETHODSnSurface plasmon resonance analysis identified 2 monoclonal antibodies, R3 and R20, that simultaneously bind to the liberated uPAR(I) but not to intact uPAR. We used R3 for capture and Eu-labeled R20 for detection in designing a 2-site sandwich time-resolved fluorescence immunoassay (TR-FIA 4) for measuring liberated uPAR(I). TR-FIA 4 was validated for use with citrated plasma. The prognostic value of the uPAR(I) concentration was evaluated in 298 CRC patients. The Cox proportional hazards model was used for the uni- and multivariate survival analyses.nnnRESULTSnThe LOQ was 0.65 pmol/L. Liberated uPAR(I) was measurable in all patient samples with TR-FIA 4. In the multivariate analysis that included sex, age, tumor stage, tumor localization, and adjuvant treatment, the uPAR(I) concentration measured with TR-FIA 4 (hazard ratio, 1.72; 95% CI, 1.15-2.57; P = 0.009), as well as the concentration of intact soluble uPAR plus the cleaved uPAR fragment containing domains II and III, tumor stage, and age were independent predictors of prognosis.nnnCONCLUSIONSnTR-FIA 4 has a functional sensitivity improved 4-fold over that of the previous uPAR(I) assay. The uPAR(I) concentration measured with TR-FIA 4 is an independent predictor of prognosis in CRC patients.

Antibody-mediated Targeting of the Urokinase-type Plasminogen Activator Proteolytic Function Neutralizes Fibrinolysis in Vivo

Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling processes. Most of our understanding of the role of uPA in vivo is derived from studies using genetargeted uPA-deficient mice. To enable in vivo studies on the specific interference with uPA functionality in mouse models, we have now developed murine monoclonal antibodies (mAbs) directed against murine uPA by immunization of uPA-deficient mice with the recombinant protein. Guided by enzyme-linked immunosorbent assay, Western blotting, surface plasmon resonance, and enzyme kinetic analyses, we have selected two highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In enzyme activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only was directed against the first of these reactions. We additionally provide evidence that mU1, but not mU3, successfully targets uPA-dependent processes in vivo. Hence, systemic administration of mU1 (i) rescued mice treated with a uPA-activable anthrax protoxin and (ii) impaired uPA-mediated hepatic fibrinolysis in tissue-type plasminogen activator (tPA)-deficient mice, resulting in a phenotype mimicking that of uPA;tPA double deficient mice. Importantly, this is the first report demonstrating specific antagonist-directed targeting of mouse uPA at the enzyme activity level in a normal physiological process in vivo.

Murine monoclonal antibodies against murine uPA receptor produced in gene-deficient mice: inhibitory effects on receptor-mediated uPA activity in vitro and in vivo

Binding of urokinase plasminogen activator (uPA) to its cellular receptor, uPAR, potentiates plasminogen activation and localizes it to the cell surface. Focal plasminogen activation is involved in both normal and pathological tissue remodeling processes including cancer invasion. The interaction between uPA and uPAR therefore represents a potential target for anti-invasive cancer therapy. Inhibitors of the human uPA-uPAR interaction have no effect in the murine system. To enable in-vivo studies in murine cancer models we have now generated murine monoclonal antibodies (mAbs) against murine uPAR (muPAR) by immunizing uPAR-deficient mice with recombinant muPAR and screened for antibodies, which inhibit the muPA-muPAR interaction. Two of the twelve mAbs obtained, mR1 and mR2, interfered with the interaction between muPAR and the amino-terminal fragment of muPA (mATF) when analyzed by surface plasmon resonance. The epitope for mR1 is located on domain I of muPAR, while that of mR2 is on domains (II-III). In cell binding experiments using radiolabelled mATF, the maximal inhibition obtained with mR1 was 85% while that obtained with mR2 was 50%. The IC(50) value for mR1 was 0.67 nM compared to 0.14 nM for mATF. In an assay based on modified anthrax toxins, requiring cell-bound muPA activity for its cytotoxity, an approximately 50% rescue of the cells could be obtained by addition of mR1. Importantly, in-vivo efficacy of mR1 was demonstrated by the ability of mR1 to rescue mice treated with a lethal dose of uPA-activatable anthrax toxins.

Presence of Insulin-Like Growth Factor Binding Proteins Correlates With Tumor-Promoting Effects of Matrix Metalloproteinase 9 in Breast Cancer

The stroma of breast cancer can promote the disease’s progression, but whether its composition and functions are shared among different subtypes is poorly explored. We compared stromal components of a luminal [mouse mammary tumor virus (MMTV)–Neu] and a triple-negative/basal-like [C3(1)–Simian virus 40 large T antigen (Tag)] genetically engineered breast cancer mouse model. The types of cytokines and their expression levels were very different in the two models, as was the extent of innate immune cell infiltration; however, both models showed infiltration of innate immune cells that expressed matrix metalloproteinase 9 (MMP9), an extracellular protease linked to the progression of many types of cancer. By intercrossing with Mmp9 null mice, we found that the absence of MMP9 delayed tumor onset in the C3(1)-Tag model but had no effect on tumor onset in the MMTV-Neu model. We discovered that protein levels of insulin-like growth factor binding protein-1 (IGFBP-1), an MMP9 substrate, were increased in C3(1)-Tag;Mmp9−/− compared to C3(1)-Tag;Mmp9+/+ tumors. In contrast, IGFBP-1 protein expression was low in MMTV-Neu tumors regardless of Mmp9 status. IGFBP-1 binds and antagonizes IGFs, preventing them from activating their receptors to promote cell proliferation and survival. Tumors from C3(1)-Tag;Mmp9−/− mice had reduced IGF-1 receptor phosphorylation, consistent with slower tumor onset. Finally, gene expression analysis of human breast tumors showed that high expression of IGFBP mRNA was strongly correlated with good prognosis but not when MMP9 mRNA was also highly expressed. In conclusion, MMP9 has different effects on breast cancer progression depending on whether IGFBPs are expressed.

Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells.

Matrix metalloproteinase-9 (MMP-9) is a 92-kDa soluble pro-enzyme implicated in pathological events including cancer invasion. It is therefore an attractive target for therapeutic intervention studies in mouse models. Development of inhibitors requires sufficient amounts of correctly folded murine MMP-9. Constructs encoding zymogens of full-length murine MMP-9 and a version lacking the O-glycosylated linker region and hemopexin domains were therefore generated and expressed in stably transfected Drosophila S2 insect cells. After 7 days of induction the expression levels of the full-length and truncated versions were 5 mg/l and 2 mg/l, respectively. The products were >95% pure after gelatin Sepharose chromatography and possessed proteolytic activity when analyzed by gelatin zymography. Using the purified full-length murine MMP-9 we raised polyclonal antibodies by immunizations of rabbits. These antibodies specifically identified pro-MMP-9 in incisional skin wound extracts from mice when used for Western blotting. Immunohistochemical analysis of paraffin embedded skin wounds from mice showed that MMP-9 protein was localized at the leading-edge keratinocytes in front of the migrating epidermal layer. No immunoreactivity was observed when the antibody was probed against skin wound material from MMP-9 deficient mice. In conclusion, we have generated and purified two proteolytically active recombinant murine MMP-9 protein constructs, which are critical reagents for future cancer drug discovery studies.

Discrimination of different forms of the murine urokinase plasminogen activator receptor on the cell surface using monoclonal antibodies

The urokinase plasminogen activator receptor (uPAR) is a versatile three-domain GPI-anchored protein, which binds urokinase plasminogen activator (uPA) and thereby focalises plasminogen activation on the cell surface. Generation of a proteolytic potential is essential in both normal physiological and pathological extracellular tissue remodelling processes. uPA can also cleave uPAR, resulting in liberation of the amino-terminal domain I, which encompasses binding sites for both uPA and the adhesion molecule, vitronectin. In order to localise the different uPAR forms on the plasma membrane of murine monocyte macrophage-like P388D.1 cells, we have now generated and characterised two high-affinity murine mAbs, mR3 and mR4, raised against murine uPAR. mR3 was found to recognise an epitope located in domain I of uPAR. Surface plasmon resonance analyses and cell binding studies revealed that this mAb was able to bind preformed complexes of murine pro-uPA and murine uPAR. In contrast, mR4 recognises domains II-III in uPAR and does not bind preformed pro-uPA-uPAR complexes in similar analyses. Immunofluorescence microscopy of P388D.1 cells revealed that mR3 stained the cells equally well in the presence or absence of saturation with the amino-terminal fragment of uPA, ATF. However, the signal intensity obtained using another uPAR domain I specific mAb, mR1, was significantly reduced upon ATF saturation. Furthermore, when adding ATF, mR4 selectively stained the cleaved receptor. Applying these newly generated mAbs, we additionally demonstrated that cleaved and intact uPAR was evenly distributed on the surface of these cells.

Abstract 2465: Matrix metalloproteinase 9 promotes breast cancer through regulation of insulin-like growth factor-binding proteins

The matrix metalloproteinase (MMP) family is a large family of extracellularly acting proteases. MMP9 is expressed by tumor infiltrating myeloid cells and promotes tumor progression in animal models of cancer. Yet, the identities of the in vivo MMP9 substrates are not well established. Furthermore, clinical trials with broad-spectrum MMP inhibitors have shown no therapeutic benefit. We compared the effects of deleting Mmp9 between the luminal mouse mammary tumor virus (MMTV)-Neu model of breast cancer, driven by expression of rodent HER2/ErbB2, and the basal-like C3(1) SV40 large T antigen (Tag) model, driven through inactivation of p53 and Rb by Tag. Interestingly, absence of MMP9 delayed tumor-onset in C3(1)-Tag mice, but had no effect in the MMTV-Neu mice. In line with these data, we found that low MMP9 expression indeed predicts good prognosis for breast cancer patients with mutant p53, but does not influence prognosis for patients with wild type p53. The insulin-like growth factor-binding protein-1 (IGFBP-1) is an established in vitro MMP9 substrate. IGFBP-1 sequesters IGF in the extracellular matrix, leading to reduced IGF receptor activity and thereby cancer cell proliferation. Proteolysis of IGFBP by MMP9 releases IGF from the matrix. Interestingly, the protein levels of IGFBP-1 were significantly increased in the C3(1)-Tag;Mmp9 −/− compared to C3(1)-Tag;Mmp9 +/+ tumors. In contrast, IGFBP-1 protein levels were low in the MMTV-Neu model regardless of MMP9 status. Human breast tumors did not express IGFBP-1 but instead the other family members, IGFBP-2, -3, and -4, which all are substrates for MMP9 in vitro. We found that low levels of MMP9 were a good prognostic factor only for patients with high mRNA expression levels of the IGFBPs, and not for patients with low levels of the IGFBPs. Our findings from mouse models and human patients suggest that MMP9 promotes tumor progression in breast cancer via proteolysis of IGFBPs. Furthermore, a lack of stratification of patients based on both MMP9 and IGFBPs levels might explain why MMP inhibitors showed no clinical benefit against breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2465. doi:1538-7445.AM2012-2465