Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 18 de 18
Filter
Add more filters










Publication year range
1.
Cancer Biol Ther ; 25(1): 2296048, 2024 12 31.
Article in English | MEDLINE | ID: mdl-38206570

ABSTRACT

CD73 is a cell surface 5'nucleotidase (NT5E) and key node in the catabolic process generating immunosuppressive adenosine in cancer. Using a murine monoclonal antibody surrogate of Oleclumab, we investigated the effect of CD73 inhibition in concert with cytotoxic therapies (chemotherapies as well as fractionated radiotherapy) and PD-L1 blockade. Our results highlight improved survival in syngeneic tumor models of colorectal cancer (CT26 and MC38) and sarcoma (MCA205). This therapeutic outcome was in part driven by cytotoxic CD8 T-cells, as evidenced by the detrimental effect of CD8 depleting antibody treatment of MCA205 tumor bearing mice treated with anti-CD73, anti-PD-L1 and 5-Fluorouracil+Oxaliplatin (5FU+OHP). We hypothesize that the improved responses are tumor microenvironment (TME)-driven, as suggested by the lack of anti-CD73 enhanced cytopathic effects mediated by 5FU+OHP on cell lines in vitro. Pharmacodynamic analysis, using imaging mass cytometry and RNA-sequencing, revealed noteworthy changes in specific cell populations like cytotoxic T cells, B cells and NK cells in the CT26 TME. Transcriptomic analysis highlighted treatment-related modulation of gene profiles associated with an immune response, NK and T-cell activation, T cell receptor signaling and interferon (types 1 & 2) pathways. Inclusion of comparator groups representing the various components of the combination allowed deconvolution of contribution of the individual therapeutic elements; highlighting specific effects mediated by the anti-CD73 antibody with respect to immune-cell representation, chemotaxis and myeloid biology. These pre-clinical data reflect complementarity of adenosine blockade with cytotoxic therapy, and T-cell checkpoint inhibition, and provides new mechanistic insights in support of combination therapy.


Subject(s)
Antibodies, Monoclonal , Sarcoma , Animals , Mice , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Immunosuppressive Agents , Adenosine , Fluorouracil/pharmacology , Fluorouracil/therapeutic use , Tumor Microenvironment
2.
Clin Transl Immunology ; 9(9): e1177, 2020.
Article in English | MEDLINE | ID: mdl-33005415

ABSTRACT

OBJECTIVES: Loss of tumor-inherent type I interferon (IFN) signalling has been closely linked to accelerated metastatic progression via decreased immunogenicity and antitumor immunity. Previous studies in murine models of triple-negative breast cancer (TNBC) demonstrate that systemic IFN inducers are effective antimetastatic agents, via sustained antitumor CD8+ T-cell responses. Repeated systemic dosing with recombinant IFNs or IFN inducers is associated with significant toxicities; hence, the use of alternate intratumoral agents is an active area of investigation. It is critical to investigate the impact of intratumoral agents on subsequent metastatic spread to predict clinical impact. METHODS: In this study, the local and systemic impact of the intratumoral Toll-like receptor (TLR) 7/8 agonist 3M-052 alone or in combination with anti-PD1 was evaluated in metastatic TNBC models. The IFN-α receptor (IFNAR1) blocking antibody, MAR1-5A3, along with immune-deficient mice and ex vivo assays are utilised to examine the key targets of this agent that are critical for an antimetastatic response. RESULTS: Single intratumoral administration of 3M-052 reduced mammary tumor growth, induced a T-cell-inflamed tumor microenvironment (TME) and reduced metastatic spread to lung. Metastasis suppression was reliant on IFN signalling and an antitumor immune response, in contrast to primary tumor growth inhibition, which was retained in NSG and CD8+ T-cell-depleted mice. 3M-052 action was demonstrated via dendritic cell activation and production of type I IFN and other pro-inflammatory cytokines to initiate a T-cell-inflamed TME and promote tumor cell antigen presentation. CONCLUSION: This work supports neoadjuvant TLR agonist-based immunotherapeutics as realistic options for immune activation in the TME and long-term metastatic protection in TNBC.

3.
J Immunother Cancer ; 7(1): 244, 2019 09 11.
Article in English | MEDLINE | ID: mdl-31511088

ABSTRACT

BACKGROUND: Immune checkpoint blockade (ICB) promotes adaptive immunity and tumor regression in some cancer patients. However, in patients with immunologically "cold" tumors, tumor-resident innate immune cell activation may be required to prime an adaptive immune response and so exploit the full potential of ICB. Whilst Toll-like receptor (TLR) agonists have been used topically to successfully treat some superficial skin tumors, systemic TLR agonists have not been well-tolerated. METHODS: The response of human immune cells to TLR7 and 8 agonism was measured in primary human immune cell assays. MEDI9197 (3M-052) was designed as a novel lipophilic TLR7/8 agonist that is retained at the injection site, limiting systemic exposure. Retention of the TLR7/8 agonist at the site of injection was demonstrated using quantitative whole-body autoradiography, HPLC-UV, and MALDI mass spectrometry imaging. Pharmacodynamic changes on T cells from TLR7/8 agonist treated B16-OVA tumors was assessed by histology, quantitative real time PCR, and flow cytometry. Combination activity of TLR7/8 agonism with immunotherapies was assessed in vitro by human DC-T cell MLR assay, and in vivo using multiple syngeneic mouse tumor models. RESULTS: Targeting both TLR7 and 8 triggers an innate and adaptive immune response in primary human immune cells, exemplified by secretion of IFNα, IL-12 and IFNγ. In contrast, a STING or a TLR9 agonist primarily induces release of IFNα. We demonstrate that the TLR7/8 agonist, MEDI9197, is retained at the sight of injection with limited systemic exposure. This localized TLR7/8 agonism leads to Th1 polarization, enrichment and activation of natural killer (NK) and CD8+ T cells, and inhibition of tumor growth in multiple syngeneic models. The anti-tumor activity of this TLR7/8 agonist is enhanced when combined with T cell-targeted immunotherapies in pre-clinical models. CONCLUSION: Localized TLR7/8 agonism can enhance recruitment and activation of immune cells in tumors and polarize anti-tumor immunity towards a Th1 response. Moreover, we demonstrate that the anti-tumor effects of this TLR7/8 agonist can be enhanced through combination with checkpoint inhibitors and co-stimulatory agonists.


Subject(s)
Dendritic Cells/immunology , Heterocyclic Compounds, 3-Ring/pharmacology , Killer Cells, Natural/immunology , Melanoma, Experimental/drug therapy , Stearic Acids/pharmacology , Toll-Like Receptor 7/agonists , Toll-Like Receptor 8/agonists , Tumor Microenvironment/immunology , Adaptive Immunity , Adjuvants, Immunologic/pharmacology , Animals , Apoptosis , Cell Proliferation , Female , Humans , Immunotherapy , Male , Melanoma, Experimental/immunology , Melanoma, Experimental/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Ovalbumin/immunology , Rats, Sprague-Dawley , Tumor Cells, Cultured
4.
J Immunother Cancer ; 5(1): 63, 2017 08 15.
Article in English | MEDLINE | ID: mdl-28807001

ABSTRACT

BACKGROUND: T-cell checkpoint blockade and MEK inhibitor combinations are under clinical investigation. Despite progress elucidating the immuno-modulatory effects of MEK inhibitors as standalone therapies, the impact of MEK inhibition on the activity of T-cell checkpoint inhibitors remains incompletely understood. Here we sought to characterize the combined effects of MEK inhibition and anti-CTLA-4 mAb (anti-CTLA-4) therapy, examining effects on both T-cells and tumor microenvironment (TME). METHODS: In mice, the effects of MEK inhibition, via selumetinib, and anti-CTLA-4 on immune responses to keyhole limpet haemocyanin (KLH) immunization were monitored using ex vivo functional assays with splenocytes. In a KRAS-mutant CT26 mouse colorectal cancer model, the impact on the tumor microenvironment (TME) and the spleen were evaluated by flow cytometry. The TME was further examined by gene expression and immunohistochemical analyses. The combination and sequencing of selumetinib and anti-CTLA-4 were also evaluated in efficacy studies using the CT26 mouse syngeneic model. RESULTS: Anti-CTLA-4 enhanced the generation of KLH specific immunity following KLH immunization in vivo; selumetinib was found to reduce, but did not prevent, this enhancement of immune response by anti-CTLA-4 in vivo. In the CT26 mouse model, anti-CTLA-4 treatment led to higher expression levels of the immunosuppressive mediators, Cox-2 and Arg1 in the TME. Combination of anti-CTLA-4 with selumetinib negated this up-regulation of Cox-2 and Arg1, reduced the frequency of CD11+ Ly6G+ myeloid cells, and led to the accumulation of differentiating monocytes at the Ly6C+ MHC+ intermediate state in the tumor. We also report that MEK inhibition had limited impact on anti-CTLA-4-mediated increases in T-cell infiltration and T-cell activation in CT26 tumors. Finally, we show that pre-treatment, but not concurrent treatment, with selumetinib enhanced the anti-tumor activity of anti-CTLA-4 in the CT26 model. CONCLUSION: These data provide evidence that MEK inhibition can lead to changes in myeloid cells and immunosuppressive factors in the tumor, thus potentially conditioning the TME to facilitate improved response to anti-CTLA-4 treatment. In summary, the use of MEK inhibitors to alter the TME as an approach to enhance the activities of immune checkpoint inhibitors warrants further investigation in clinical trials.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Benzimidazoles/administration & dosage , Colorectal Neoplasms/drug therapy , Tumor Microenvironment/drug effects , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized , Benzimidazoles/pharmacology , Biomarkers, Tumor/genetics , Cell Line, Tumor , Cellular Reprogramming/drug effects , Colorectal Neoplasms/genetics , Drug Synergism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Mutation , Proto-Oncogene Proteins p21(ras)/genetics , Xenograft Model Antitumor Assays
5.
Clin Cancer Res ; 23(22): 6893-6903, 2017 Nov 15.
Article in English | MEDLINE | ID: mdl-28821560

ABSTRACT

Purpose: The development of new treatments and their deployment in the clinic may be assisted by imaging methods that allow an early assessment of treatment response in individual patients. The C2A domain of Synaptotagmin-I (C2Am), which binds to the phosphatidylserine (PS) exposed by apoptotic and necrotic cells, has been developed as an imaging probe for detecting cell death. Multispectral optoacoustic tomography (MSOT) is a real-time and clinically applicable imaging modality that was used here with a near infrared (NIR) fluorophore-labeled C2Am to image tumor cell death in mice treated with a TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) agonist and with 5-fluorouracil (5-FU).Experimental Design: C2Am was labeled with a NIR fluorophore and injected intravenously into mice bearing human colorectal TRAIL-sensitive Colo205 and TRAIL-resistant HT-29 xenografts that had been treated with a potent agonist of TRAILR2 and in Colo205 tumors treated with 5-FU.Results: Three-dimensional (3D) MSOT images of probe distribution showed development of tumor contrast within 3 hours of probe administration and a signal-to-background ratio in regions containing dead cells of >10 after 24 hours. A site-directed mutant of C2Am that is inactive in PS binding showed negligible binding. Tumor retention of the active probe was strongly correlated (R2 = 0.97, P value < 0.01) with a marker of apoptotic cell death measured in histologic sections obtained post mortem.Conclusions: The rapid development of relatively high levels of contrast suggests that NIR fluorophore-labeled C2Am could be a useful optoacoustic imaging probe for detecting early therapy-induced tumor cell death in the clinic. Clin Cancer Res; 23(22); 6893-903. ©2017 AACR.


Subject(s)
Cell Death , Molecular Imaging , Photoacoustic Techniques , Tomography , Animals , Biomarkers , Cell Line, Tumor , Disease Models, Animal , Female , Flow Cytometry , Fluorescent Dyes , Heterografts , Humans , Mice , Microscopy, Fluorescence , Molecular Imaging/methods , Tomography/methods
6.
Clin Cancer Res ; 23(13): 3416-3427, 2017 07 01.
Article in English | MEDLINE | ID: mdl-28069723

ABSTRACT

Purpose: To generate and characterize a murine GITR ligand fusion protein (mGITRL-FP) designed to maximize valency and the potential to agonize the GITR receptor for cancer immunotherapy.Experimental Design: The EC50 value of the mGITRL-FP was compared with an anti-GITR antibody in an in vitro agonistic cell-based reporter assay. We assessed the impact of dose, schedule, and Fc isotype on antitumor activity and T-cell modulation in the CT26 tumor model. The activity of the mGITRL-FP was compared with an agonistic murine OX40L-FP targeting OX40, in CT26 and B16F10-Luc2 tumor models. Combination of the mGITRL-FP with antibodies targeting PD-L1, PD-1, or CTLA-4 was analyzed in mice bearing CT26 tumors.Results: The mGITRL-FP had an almost 50-fold higher EC50 value compared with an anti-murine GITR antibody. Treatment of CT26 tumor-bearing mice with mGITRL-FP-mediated significant antitumor activity that was dependent on isotype, dose, and duration of exposure. The antitumor activity could be correlated with the increased proliferation of peripheral CD8+ and CD4+ T cells and a significant decrease in the frequency of intratumoral Tregs. The combination of mGITRL-FP with mOX40L-FP or checkpoint inhibitor antagonists enhanced antitumor immunity above that of monotherapy treatment.Conclusions: These results suggest that therapeutically targeting GITR represents a unique approach to cancer immunotherapy and suggests that a multimeric fusion protein may provide increased agonistic potential versus an antibody. In addition, these data provide, for the first time, early proof of concept for the potential combination of GITR targeting agents with OX40 agonists and PD-L1 antagonists. Clin Cancer Res; 23(13); 3416-27. ©2017 AACR.


Subject(s)
Glucocorticoid-Induced TNFR-Related Protein/immunology , Melanoma, Experimental/immunology , Oncogene Proteins, Fusion/administration & dosage , Tumor Necrosis Factors/immunology , Animals , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/immunology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CTLA-4 Antigen/antagonists & inhibitors , CTLA-4 Antigen/immunology , Disease Models, Animal , Glucocorticoid-Induced TNFR-Related Protein/administration & dosage , Humans , Melanoma, Experimental/genetics , Melanoma, Experimental/therapy , Membrane Glycoproteins/agonists , Membrane Glycoproteins/immunology , Mice , OX40 Ligand , Oncogene Proteins, Fusion/immunology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Tumor Necrosis Factors/agonists , Tumor Necrosis Factors/genetics
7.
Cancer Immunol Res ; 5(1): 29-41, 2017 01.
Article in English | MEDLINE | ID: mdl-27923825

ABSTRACT

Murine syngeneic tumor models are critical to novel immuno-based therapy development, but the molecular and immunologic features of these models are still not clearly defined. The translational relevance of differences between the models is not fully understood, impeding appropriate preclinical model selection for target validation, and ultimately hindering drug development. Across a panel of commonly used murine syngeneic tumor models, we showed variable responsiveness to immunotherapies. We used array comparative genomic hybridization, whole-exome sequencing, exon microarray analysis, and flow cytometry to extensively characterize these models, which revealed striking differences that may underlie these contrasting response profiles. We identified strong differential gene expression in immune-related pathways and changes in immune cell-specific genes that suggested differences in tumor immune infiltrates between models. Further investigation using flow cytometry showed differences in both the composition and magnitude of the tumor immune infiltrates, identifying models that harbor "inflamed" and "non-inflamed" tumor immune infiltrate phenotypes. We also found that immunosuppressive cell types predominated in syngeneic mouse tumor models that did not respond to immune-checkpoint blockade, whereas cytotoxic effector immune cells were enriched in responsive models. A cytotoxic cell-rich tumor immune infiltrate has been correlated with increased efficacy of immunotherapies in the clinic, and these differences could underlie the varying response profiles to immunotherapy between the syngeneic models. This characterization highlighted the importance of extensive profiling and will enable investigators to select appropriate models to interrogate the activity of immunotherapies as well as combinations with targeted therapies in vivo Cancer Immunol Res; 5(1); 29-41. ©2016 AACR.


Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Drug Discovery , Drug Evaluation, Preclinical , Animals , B7-H1 Antigen/antagonists & inhibitors , CTLA-4 Antigen/antagonists & inhibitors , Comparative Genomic Hybridization , DNA Copy Number Variations , Disease Models, Animal , Drug Synergism , Exome , Gene Expression Regulation, Neoplastic/drug effects , Genomics/methods , High-Throughput Nucleotide Sequencing , Immunomodulation/drug effects , Immunomodulation/genetics , Mice , Molecular Targeted Therapy , Mutation , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/metabolism , Signal Transduction/drug effects , Transcriptome , Tumor Microenvironment/drug effects , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology
8.
Oncotarget ; 7(14): 18508-20, 2016 Apr 05.
Article in English | MEDLINE | ID: mdl-26918344

ABSTRACT

Despite the availability of recently developed chemotherapy regimens, survival times for pancreatic cancer patients remain poor. These patients also respond poorly to immune checkpoint blockade therapies (anti-CTLA-4, anti-PD-L1, anti-PD-1), which suggests the presence of additional immunosuppressive mechanisms in the pancreatic tumour microenvironment (TME). CD40 agonist antibodies (αCD40) promote antigen presenting cell (APC) maturation and enhance macrophage tumouricidal activity, and may therefore alter the pancreatic TME to increase sensitivity to immune checkpoint blockade. Here, we test whether αCD40 transforms the TME in a mouse syngeneic orthotopic model of pancreatic cancer, to increase sensitivity to PD-L1 blockade. We found that whilst mice bearing orthotopic Pan02 tumours responded poorly to PD-L1 blockade, αCD40 improved overall survival. αCD40 transformed the TME, upregulating Th1 chemokines, increasing cytotoxic T cell infiltration and promoting formation of an immune cell-rich capsule separating the tumour from the normal pancreas. Furthermore, αCD40 drove systemic APC maturation, memory T cell expansion, and upregulated tumour and systemic PD-L1 expression. Combining αCD40 with PD-L1 blockade enhanced anti-tumour immunity and improved overall survival versus either monotherapy. These data provide further support for the potential of combining αCD40 with immune checkpoint blockade to promote anti-tumour immunity in pancreatic cancer.


Subject(s)
Antibodies, Monoclonal/pharmacology , B7-H1 Antigen/antagonists & inhibitors , CD40 Antigens/agonists , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/therapy , Tumor Microenvironment/immunology , Animals , Antibodies, Monoclonal/immunology , B7-H1 Antigen/immunology , CD40 Antigens/immunology , CTLA-4 Antigen/antagonists & inhibitors , CTLA-4 Antigen/immunology , Cell Transformation, Neoplastic , Disease Models, Animal , Female , Mice , Mice, Inbred C57BL , Pancreatic Neoplasms/pathology , Random Allocation
9.
Cancer Immunol Res ; 3(9): 1052-62, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25943534

ABSTRACT

Programmed cell-death 1 ligand 1 (PD-L1) is a member of the B7/CD28 family of proteins that control T-cell activation. Many tumors can upregulate expression of PD-L1, inhibiting antitumor T-cell responses and avoiding immune surveillance and elimination. We have identified and characterized MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1 and is uniquely engineered to prevent antibody-dependent cell-mediated cytotoxicity. In vitro assays demonstrate that MEDI4736 is a potent antagonist of PD-L1 function, blocking interaction with PD-1 and CD80 to overcome inhibition of primary human T-cell activation. In vivo MEDI4736 significantly inhibits the growth of human tumors in a novel xenograft model containing coimplanted human T cells. This activity is entirely dependent on the presence of transplanted T cells, supporting the immunological mechanism of action for MEDI4736. To further determine the utility of PD-L1 blockade, an anti-mouse PD-L1 antibody was investigated in immunocompetent mice. Here, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 colorectal cancer cells. The antitumor activity of anti-PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of HMGB1 within CT26 tumors. Taken together, our results demonstrate that inhibition of PD-L1 function can have potent antitumor activity when used as monotherapy or in combination in preclinical models, and suggest it may be a promising therapeutic approach for the treatment of cancer. MEDI4736 is currently in several clinical trials both alone and in combination with other agents, including anti-CTLA-4, anti-PD-1, and inhibitors of IDO, MEK, BRAF, and EGFR.


Subject(s)
Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacology , B7-H1 Antigen/antagonists & inhibitors , Animals , Antibodies, Monoclonal/administration & dosage , Antibody-Dependent Cell Cytotoxicity/drug effects , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , B7-1 Antigen/metabolism , B7-H1 Antigen/metabolism , Binding, Competitive , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Female , Humans , Lymphocyte Activation/drug effects , Lymphocyte Culture Test, Mixed , Melanoma/immunology , Melanoma/pathology , Melanoma/prevention & control , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Organoplatinum Compounds/administration & dosage , Oxaliplatin , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/prevention & control , Programmed Cell Death 1 Receptor/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
10.
FASEB J ; 27(2): 581-9, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23104982

ABSTRACT

Fibroblast activation protein (FAP) is a serine protease selectively expressed on tumor stromal fibroblasts in epithelial carcinomas and is important in cancer growth, adhesion, and metastases. As FAP enzymatic activity is a potent therapeutic target, we aimed to identify inhibitory antibodies. Using a competitive inhibition strategy, we used phage display techniques to identify 53 single-chain variable fragments (scFvs) after three rounds of panning against FAP. These scFvs were expressed and characterized for binding to FAP by surface plasmon resonance and flow cytometry. Functional assessment of these antibodies yielded an inhibitory scFv antibody, named E3, which could attenuate 35% of FAP cleavage of the fluorescent substrate Ala-Pro-7-amido-4-trifluoromethylcoumarin compared with nonfunctional scFv control. Furthermore, a mutant E3 scFv was identified by yeast affinity maturation. It had higher affinity (4-fold) and enhanced inhibitory effect on FAP enzyme activity (3-fold) than E3. The application of both inhibitory anti-FAP scFvs significantly affected the formation of 3-dimensional FAP-positive cell matrix, as demonstrated by reducing the fibronectin fiber orientation from 41.18% (negative antibody control) to 34.06% (E3) and 36.15% (mutant E3), respectively. Thus, we have identified and affinity-maturated the first scFv antibody capable of inhibiting FAP function. This scFv antibody has the potential to disrupt the role of FAP in tumor invasion and metastasis.


Subject(s)
Gelatinases/antagonists & inhibitors , Gelatinases/immunology , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/immunology , Serine Endopeptidases/immunology , Single-Chain Antibodies/immunology , Animals , Antibody Affinity , Endopeptidases , Flow Cytometry , Gelatinases/genetics , Gelatinases/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Molecular Targeted Therapy , Mutagenesis, Site-Directed , Mutant Proteins/genetics , Mutant Proteins/immunology , Neoplasms/drug therapy , Neoplasms/enzymology , Peptide Library , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Single-Chain Antibodies/genetics , Single-Chain Antibodies/metabolism , Surface Plasmon Resonance
11.
Biol Chem ; 393(12): 1405-16, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23667900

ABSTRACT

The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer.To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyper plasia(MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L,reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression.


Subject(s)
Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Breast/pathology , Cathepsin B/metabolism , Cell Transformation, Neoplastic/metabolism , Epithelial Cells/pathology , Apoptosis/drug effects , Breast/cytology , Breast/drug effects , Breast/enzymology , Breast Neoplasms/drug therapy , Cathepsin B/antagonists & inhibitors , Cathepsin L/antagonists & inhibitors , Cathepsin L/metabolism , Cell Culture Techniques , Cell Line, Tumor , Cell Survival/drug effects , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/pathology , Cysteine Proteinase Inhibitors/pharmacology , Dipeptides/pharmacology , Disease Progression , Epithelial Cells/drug effects , Epithelial Cells/enzymology , Female , Humans , Leucine/analogs & derivatives , Leucine/pharmacology , Models, Biological
12.
BMC Cancer ; 11: 245, 2011 Jun 13.
Article in English | MEDLINE | ID: mdl-21668992

ABSTRACT

BACKGROUND: Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions. We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer. METHODS: We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses. RESULTS: We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is ß1-integrin/FAK mediated. CONCLUSION: Cancer cell invasiveness can be affected by alterations in the tumor microenvironment. Disruption of FAP activity and ß1-integrins may abrogate the invasive capabilities of pancreatic and other tumors by disrupting the FAP-directed organization of stromal ECM and blocking ß1-integrin dependent cell-matrix interactions. This provides a novel preclinical rationale for therapeutics aimed at interfering with the architectural organization of tumor-associated ECM. Better understanding of the stromal influences that fuel progressive tumorigenic behaviors may allow the effective future use of targeted therapeutics aimed at disrupting specific tumor-stromal interactions.


Subject(s)
Adenocarcinoma/pathology , Extracellular Matrix Proteins/metabolism , Extracellular Matrix/physiology , Fibroblasts/enzymology , Gelatinases/physiology , Membrane Proteins/physiology , Neoplasm Invasiveness/pathology , Neoplasm Proteins/physiology , Pancreatic Neoplasms/pathology , Serine Endopeptidases/physiology , Tumor Microenvironment/physiology , Adenocarcinoma/enzymology , Animals , Blotting, Western , Breast Neoplasms/pathology , Cell Culture Techniques , Cell Line, Tumor/enzymology , Cell Line, Tumor/pathology , Cell Movement , Collagen Type I/metabolism , Endopeptidases , Extracellular Matrix/ultrastructure , Fibronectins/metabolism , Fibronectins/ultrastructure , Focal Adhesion Kinase 1/physiology , Gelatinases/genetics , Humans , Integrin beta1/physiology , Membrane Proteins/genetics , Mice , Mice, Inbred ICR , Mice, SCID , NIH 3T3 Cells/enzymology , Pancreatic Neoplasms/enzymology , Recombinant Fusion Proteins/physiology , Serine Endopeptidases/genetics , Time-Lapse Imaging , Transplantation, Heterologous
13.
Methods Mol Biol ; 539: 49-57, 2009.
Article in English | MEDLINE | ID: mdl-19377971

ABSTRACT

Expression of a given protease and of the endogenous inhibitors that regulate protease activity can be readily determined at the transcript level by using whole genome microarray chips. In the case of proteases and protease inhibitors, however, determining which cells are expressing them is often critical to understanding the functional roles of the proteases. For example, in cancer many of the proteases are derived from cells that are found in the microenvironment surrounding the tumor, e.g., fibroblasts and inflammatory cells. Proteases from both fibroblasts and inflammatory cells have been implicated in malignant progression. Therefore, it is important to recognize the origin of these molecules if one is to develop effective therapies. In this regard, mouse transgenic models and xenograft models in which human tumor cells are implanted in mice are useful tools. To profile human and mouse proteases, protease inhibitors, and protease interactors, we have developed in partnership with Affymetrix a custom, single platform, dual species chip: the Hu/Mu ProtIn chip. The Hu/Mu ProtIn chip has been validated for its ability to identify human and mouse transcripts in single species specimens and to identify and distinguish between human and mouse transcripts in dual species specimens such as xenografts. In the latter specimens, the Hu/Mu ProtIn chip has enabled us to identify host (mouse) proteases that play a protective role in development of lung tumors. Here we outline a protocol for using the Hu/Mu ProtIn chip to profile proteases, protease inhibitors, and protease interactors in tissues and cells.


Subject(s)
Peptide Hydrolases/analysis , Peptide Hydrolases/genetics , Protein Array Analysis/methods , Animals , Cells, Cultured , Gene Expression Profiling/methods , Humans , Mice , Protease Inhibitors/analysis , RNA/genetics , RNA/isolation & purification , Transplantation, Heterologous
14.
J Med Chem ; 52(2): 358-68, 2009 Jan 22.
Article in English | MEDLINE | ID: mdl-19093877

ABSTRACT

Fibroblast activation protein (FAP) is a cell-surface serine protease highly expressed on cancer-associated fibroblasts of human epithelial carcinomas but not on normal fibroblasts, normal tissues, and cancer cells. We report herein a novel FAP-triggered photodynamic molecular beacon (FAP-PPB) comprising a fluorescent photosensitizer and a black hole quencher 3 linked by a peptide sequence (TSGPNQEQK) specific to FAP. FAP-PPB was effectively cleaved by both human FAP and murine FAP. By use of the HEK293 transfected cells (HEK-mFAP, FAP(+); HEK-vector, FAP(-)), systematic in vitro and in vivo experiments validated the FAP-specific activation of FAP-PPB in cancer cells and mouse xenografts, respectively. FAP-PPB was cleaved by FAP, allowing fluorescence restoration in FAP-expressing cells while leaving non-expressing FAP cells undetectable. Moreover, FAP-PPB showed FAP-specific photocytotoxicity toward HEK-mFAP cells whereas it was non-cytotoxic toward HEK-Vector cells. This study suggests that the FAP-PPB is a potentially useful tool for epithelial cancer detection and treatment.


Subject(s)
Antigens, Neoplasm/physiology , Biomarkers, Tumor/physiology , Neoplasms, Glandular and Epithelial/diagnosis , Neoplasms, Glandular and Epithelial/drug therapy , Photochemotherapy , Serine Endopeptidases/physiology , Animals , Blotting, Western , Cell Line , Chromatography, High Pressure Liquid , Endopeptidases , Fibroblasts/cytology , Flow Cytometry , Gelatinases , Humans , Immunohistochemistry , Membrane Proteins , Mice , Microscopy, Confocal , Neoplasms, Glandular and Epithelial/pathology
15.
Clin Exp Metastasis ; 26(4): 299-309, 2009.
Article in English | MEDLINE | ID: mdl-19082919

ABSTRACT

The roles of proteases in cancer are dynamic. Furthermore, the roles or functions of any one protease may differ from one stage of cancer to another. Proteases from tumor-associated cells (e.g., fibroblasts, inflammatory cells, endothelial cells) as well as from tumor cells make important contributions to 'tumor proteolysis'. Many tumors exhibit increases in expression of proteases at the level of transcripts and protein; however, whether those proteases play causal roles in malignant progression is known for only a handful of proteases. What the critical substrate or substrates that are cleaved in vivo by any given protease is also known for only a few proteases. Therefore, the recent development of techniques and reagents for live cell imaging of protease activity, in conjunction with informed knowledge of critical natural substrates, should help to define protease functions. Here we describe live cell assays for imaging proteolysis, protocols for quantifying proteolysis and the use of such assays to follow the dynamics of proteolysis by tumor cells alone and tumor cells interacting with other cells found in the tumor microenvironment. In addition, we describe an in vitro model that recapitulates the architecture of the mammary gland, a model designed to determine the effects of dynamic interactions with the surrounding microenvironment on 'tumor proteolysis' and the respective contributions of various cell types to 'tumor proteolysis'. The assays and models described here could serve as screening platforms for the identification of proteolytic pathways that are potential therapeutic targets and for further development of technologies and imaging probes for in vivo use.


Subject(s)
Diagnostic Imaging , Neoplasms/diagnosis , Neoplasms/metabolism , Peptide Hydrolases/metabolism , Animals , Humans
16.
Neoplasia ; 10(4): 314-29, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18392133

ABSTRACT

Overexpression of p21-activated kinase 1 (PAK1) occurs during the progression of human breast cancer. We have investigated the role of PAK1 in the premalignant progression of the MCF10 series of human breast epithelial cell lines. Levels of PAK1 expression and activation increased with premalignant progression, and expression of dominant-negative (DN) PAK1 reduced both cell proliferation and migration/invasion. In three-dimensional (3D) overlay cultures in reconstituted basement membrane, the MCF10 series produced an in vitro model for premalignant progression. MCF10AneoT cells formed a hyperplastic morphology in which some spheroids developed abnormal lumens. The MCF10.AT1 line exhibited an atypical hyperplastic morphology of abnormal spheroid clusters that did not form lumens. The MCF10.DCIS cells exhibited dysplastic growth. Expression of DN-PAK1 promoted lumen formation in 3D-cultured MCF10A, NeoT, and AT1 structures, suggesting partial reversion of the premalignant phenotype, but did not affect the atypical budding of AT1 structures or the dysplastic growth of ductal carcinoma in situ structures. Aberrant proteolysis is another important characteristic of breast cancer progression and invasion. DN-PAK1 or knock-down of PAK1 reduced pericellular proteolysis of DQ-collagen IV in the 3D cultures. Treatment of cells with an inhibitor of Rac1 also reduced pericellular proteolysis, and this reduction was reversed by the expression of activated PAK1. Our conclusion is that overexpressed and activated PAK1 may be a key coordinator of aberrant cell survival and proteolysis in breast cancer progression.


Subject(s)
Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Cell Survival/physiology , Extracellular Matrix/metabolism , Peptide Hydrolases/metabolism , p21-Activated Kinases/metabolism , Blotting, Western , Carcinoma, Intraductal, Noninfiltrating/enzymology , Carcinoma, Intraductal, Noninfiltrating/pathology , Cell Movement , Cell Proliferation , Cells, Cultured , Collagen Type IV/metabolism , Disease Progression , Epithelial Cells/enzymology , Epithelial Cells/pathology , Female , Genes, Dominant , Humans , Imaging, Three-Dimensional , Models, Biological , Neoplasm Invasiveness , Phosphorylation , Precancerous Conditions/enzymology , Precancerous Conditions/pathology , Protease Inhibitors/pharmacology , Protein Processing, Post-Translational , p21-Activated Kinases/antagonists & inhibitors , p21-Activated Kinases/genetics
17.
Cancer Biol Ther ; 6(11): 1691-9, 2007 Nov.
Article in English | MEDLINE | ID: mdl-18032930

ABSTRACT

PURPOSE: Fibroblast Activation Protein (FAP) is a tumor fibroblast protease that has been shown to potentiate colorectal cancer growth. The clinical impact of FAP inhibition was tested using Val-boroPro (Talabostat), the first clinical inhibitor of FAP enzymatic activity, in a phase II study of patients with metastatic colorectal cancer. METHODS: Patients with metastatic colorectal cancer who had previously received systemic chemotherapies were treated with single agent Val-boroPro 200 microg p.o. BID continuously. Eligibility included measurable disease, performance status of 0 to 2, and adequate organ function. Laboratory correlates evaluated the pharmacodynamic effects of Val-boroPro on FAP enzymatic function in the peripheral blood. RESULTS: Twenty-eight patients (median age 62; 12 males, 16 females) were enrolled in this study. There were no objective responses. Six of 28 (21%) patients had stable disease for a median of 25 weeks (range 11-38 weeks). Laboratory analysis demonstrated significant, although incomplete inhibition of FAP enzymatic activity in the peripheral blood. CONCLUSION: This phase II trial of Val-boroPro demonstrated minimal clinical activity in patients with previously treated metastatic colorectal cancer. However it provides the initial proof-of-concept that physiologic inhibition of FAP activity can be accomplished in patients with colorectal cancer, and lays the groundwork for future studies targeting the tumor stroma.


Subject(s)
Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/antagonists & inhibitors , Boronic Acids/therapeutic use , Colorectal Neoplasms/drug therapy , Dipeptides/therapeutic use , Adult , Aged , Aged, 80 and over , Antigens, Neoplasm/blood , Biomarkers, Tumor/blood , Boronic Acids/adverse effects , Boronic Acids/blood , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Dipeptides/adverse effects , Dipeptides/blood , Endopeptidases , Female , Gelatinases , Humans , Immunohistochemistry , Male , Membrane Proteins , Middle Aged , Neoplasm Metastasis , Serine Endopeptidases/blood , alpha-2-Antiplasmin/analysis
18.
Nat Chem Biol ; 1(4): 203-9, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16408036

ABSTRACT

Protease activity is tightly regulated in both normal and disease conditions. However, it is often difficult to monitor the dynamic nature of this regulation in the context of a live cell or whole organism. To address this limitation, we developed a series of quenched activity-based probes (qABPs) that become fluorescent upon activity-dependent covalent modification of a protease target. These reagents freely penetrate cells and allow direct imaging of protease activity in living cells. Targeted proteases are directly identified and monitored biochemically by virtue of the resulting covalent tag, thereby allowing unambiguous assignment of protease activities observed in imaging studies. We report here the design and synthesis of a selective, cell-permeable qABP for the study of papain-family cysteine proteases. This probe is used to monitor real-time protease activity in live human cells with fluorescence microscopy techniques as well as standard biochemical methods.


Subject(s)
Cysteine Endopeptidases/chemistry , Fluorescent Dyes/chemistry , Molecular Probes/chemistry , Molecular Probes/metabolism , Animals , Cathepsins/chemistry , Cathepsins/genetics , Cysteine Endopeptidases/metabolism , Fluorescent Dyes/metabolism , Mice , Molecular Structure , NIH 3T3 Cells , Recombinant Proteins/chemistry , Recombinant Proteins/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...