Christiane Cantin

Glycoproteomic analysis of two mouse mammary cell lines during transforming growth factor (TGF)-β induced epithelial to mesenchymal transition

BackgroundTGF-β acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. However, later in tumor development TGF-β can become tumor promoting through mechanisms including the induction of epithelial-to-mesenchymal transition (EMT), a process that is thought to contribute to tumor progression, invasion and metastasis. To identify EMT-related breast cancer therapeutic targets and biomarkers, we have used two proteomic approaches to find proteins that change in abundance upon the induction of EMT by TGF-β in two mouse mammary epithelial cell lines, NMuMG and BRI-JM01.ResultsPreliminary experiments based on two-dimensional electrophoresis of a hydrophobic cell fraction identified only 5 differentially expressed proteins from BRI-JM01 cells. Since 3 of these proteins were glycoproteins, we next used the lectin, wheat germ agglutinin (WGA), to enrich for glycoproteins, followed by relative quantification of tryptic peptides using a label-free LC-MS based method. Using these approaches, we identified several proteins that are modulated during the EMT process, including cell adhesion molecules (several members of the Integrin family, Fibronectin, Activated leukocyte cell adhesion molecule, and Neural cell adhesion molecule 1) and regulators of cellular signaling (Tumor-associated calcium signal transducer 2, Basigin).ConclusionInterestingly, despite the fact that TGF-β induces similar EMT phenotypes in NMuMG and BRI-JM01 cells, the proteomic results for the two cell lines showed only minimal overlap. These differences likely result in part from the conservative cut-off values used to define differentially-expressed proteins in these experiments. Alternatively, it is possible that the two cell lines may use different mechanisms to achieve an EMT transition.

Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-β and Neu pathway signaling studies: identification of a novel model for TGF-β-induced epithelial-to-mesenchymal transition

IntroductionThis report describes the isolation and characterization of three new murine mammary epithelial cell lines derived from mammary tumors from MMTV (mouse mammary tumor virus)/activated Neu + TβRII-AS (transforming growth factor [TGF]-β type II receptor antisense RNA) bigenic mice (BRI-JM01 and BRI-JM05 cell lines) and MMTV/activated Neu transgenic mice (BRI-JM04 cell line).MethodsThe BRI-JM01, BRI-JM04, and BRI-JM05 cell lines were analyzed for transgene expression, their general growth characteristics, and their sensitivities to several growth factors from the epidermal growth factor (EGF) and TGF-β families (recombinant human EGF, heregulin-β1 and TGF-β1). The BRI-JM01 cells were observed to undergo a striking morphologic change in response to TGF-β1, and they were therefore further investigated for their ability to undergo a TGF-β-induced epithelial-to-mesenchymal transition (EMT) using motility assays and immunofluorescence microscopy.ResultsWe found that two of the three cell lines (BRI-JM04 and BRI-JM05) express the Neu transgene, whereas, unexpectedly, both of the cell lines that were established from MMTV/activated Neu + TβRII-AS bigenic tumors (BRI-JM01 and BRI-JM05) do not express the TβRII-AS transgene. The cuboidal BRI-JM01 cells exhibit a short doubling time and are able to form confluent monolayers. The BRI-JM04 and BRI-JM05 cell lines are morphologically much less uniform, grow at a much slower rate, and do not form confluent monolayers. Only the BRI-JM05 cells can form colonies in soft agar. In contrast, all three cell lines form colonies in Matrigel, although the BRI-JM04 and BRI-JM05 cell lines do so more efficiently than the BRI-JM01 cell line. All three cell lines express the cell surface marker E-cadherin, confirming their epithelial character. Proliferation assays showed that the three cell lines respond differently to recombinant human EGF and heregulin-β1, and that all are growth inhibited by TGF-β1, but that only the BRI-JM01 cell line undergoes an EMT and exhibits increased motility upon TGF-β1 treatment.ConclusionWe suggest that the BRI-JM04 and BRI-JM05 cell lines can be used to investigate Neu oncogene driven mammary tumorigenesis, whereas the BRI-JM01 cell line will be useful for studying TGF-β1-induced EMT.

Development of reverse phase protein microarrays for the validation of clusterin, a mid-abundant blood biomarker

BackgroundMany putative disease blood biomarkers discovered in genomic and proteomic studies await validation in large clinically annotated cohorts of patient samples. ELISA assays require large quantities of precious blood samples and are not high-throughput. The reverse phase protein microarray platform has been developed for the high-throughput quantification of protein levels in small amounts of clinical samples.ResultsIn the present study we present the development of reverse-phase protein microarrays (RPPMs) for the measurement of clusterin, a mid-abundant blood biomarker. An experimental protocol was optimized for the printing of serum and plasma on RPPMs using epoxy coated microscope slides and a non-denaturing printing buffer. Using fluorescent-tagged secondary antibodies, we achieved the reproducible detection of clusterin in spotted serum and plasma and reached a limit of detection of 780 ng/mL. Validation studies using both spiked clusterin and clinical samples showed excellent correlations with ELISA measurements of clusterin.ConclusionSerum and plasma spotted in the reverse phase array format allow for reliable and reproducible high-throughput validation of a mid-abundant blood biomarker such as clusterin.

Molecular imaging of breast tumors using a near‐infrared fluorescently labeled clusterin binding peptide

Several reports have shown that secreted clusterin (sCLU) plays multiple roles in tumor development and metastasis. Here, we report on a 12‐mer sCLU binding peptide (designated P3378) that was identified by screening a phage‐display peptide library against purified human sCLU. Differential resonance perturbation nuclear magnetic resonance using P3378 and a scrambled control peptide (designated P3378R) confirmed the P3378‐sCLU interaction and demonstrated that it was sequence specific. P3378 and P3378R peptides were conjugated to an Alexa680 near infrared fluorophore (NIRF) and assessed for their tumor homing abilities in in vivo time‐domain fluorescence optical imaging experiments using living 4T1 tumor bearing BALB/c mice. When injected in separate animals, both peptides accumulated at the tumor site, however the NIRF‐labeled P3378 peptide was retained for a significant longer period of time than the P3378R peptide. Similar observations were made after simultaneously injecting the same tumor‐bearing animal with a peptide mixture of P3378 DyLight (DL)680 and the P3378R‐DL800. Coinjection of P3378‐DL680 with excess unlabeled P3378 blocked tumor accumulation of fluorescent signal while excess P3378R control peptide did not confirming the sequence specificity of the tumor accumulation. Finally, ex vivo fluorescence microscopy of these tumors confirmed the presence of P3378‐DL680 in the tumor and its colocalization with CLU. These results confirm the tumor targeting specificity of the P3378 CLU‐binding peptide and suggest its usefulness for the in vivo monitoring of solid tumors secreting detectable levels of CLU.

Abstract B058: Development of AVID200, a novel and highly potent TGF-beta neutralizing immunotherapy

Introduction: Elevated TGF-beta ligand markedly augments cancer progression primarily by suppressing the immune system in the tumor microenvironment, in particular by suppressing T-cell recruitment and/or activation. We developed AVID200, a novel decoy receptor trap that potently blocks TGF-beta and induces T-cell infiltration into tumors. This promotes the “T-cell-inflamed” tumor state, which is expected to render tumors sensitive to immune checkpoint inhibitors and other immunotherapeutics. Experimental Procedures: AVID200 is a computationally designed novel class of avidity-enhanced receptor-ectodomain-based trap that binds and neutralizes TGF-beta 1 and 3 with low pM potency. Several trap formats have been produced and tested, with each format exhibiting varying characteristics, including differing circulating half-lives and in vitro blocking potencies. AVID200 was evaluated for efficacy in in vivo studies using the syngeneic 4T1 triple negative breast cancer (TNBC) tumor model. Additionally, ex vivo studies were performed on CD4+ and CD8+ T-cells harvested from the draining lymph nodes of treated animals. Results: In efficacy studies using the syngeneic 4T1 TNBC model, novel TGF-beta traps were shown to promote significant T-cell infiltration into tumors. This infiltration resulted in reduced primary tumor growth as well as significant reductions in metastatic lesions. Additionally, ex vivo studies revealed that trap treatment decreased T-cell apoptosis, promoted T-cell proliferation in response to tumor cell lysates in the presence of dendritic cells, as well as increased the capacity of T-cells to specifically lyse 4T1 tumor cells. Conclusion: AVID200 is a novel computationally-designed TGF-beta trap that neutralizes TGF-beta 1 and 3 with pM potency and markedly promotes the “T-cell-inflamed” tumor state. Combination studies with immune checkpoint inhibitors are ongoing. Citation Format: Maureen D. O9Connor-McCourt, Anne E.G. Lenferink, John Zwaagstra, Traian Sulea, Jason Baardsnes, Catherine Collins, Christiane Cantin, Lucie Couture, Limei Tao, Yves Durocher, Renu Singh, James Koropatnick. Development of AVID200, a novel and highly potent TGF-beta neutralizing immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B058.

Abstract B024: Single-chain traps targeting transforming growth factor-beta (TGF-beta) home to tumors and reduce tumor growth and metastasis by counteracting TGF-beta-mediated immunosuppression

The TGF-beta superfamily includes a large number of ligands with physiological and pathological significance. In particular, elevated TGF-beta in the tumor microenvironment markedly augments cancer progression by inducing metastasis and angiogenesis and by suppressing the immune system. Therapeutic agents targeting TGF-beta, such as antibodies or IgG-Fc-fused receptor ectodomains, have been developed, however these agents are relatively large molecules, which may restrict tumor penetration. We have computationally designed single-chain traps that are comprised of tandemly-fused TGF-beta receptor domains. These traps are approximately one third the size of monoclonal antibodies which would potentially facilitate a better tumor penetration. The homobivalent TGF-beta RII-RII traps (i.e. T22d35 and T22d60) neutralize TGF-beta isoforms 1 and 3 and the heterovalent TGF-beta RI-RII based traps (i.e. T12d and T122bt) are pan-specific and neutralize all three isoforms (TGF-beta 1, 2 and 3). Both the T22d35 and T12d trap blocked TGF-beta 1 and 3, induced epithelial mesenchymal transition (EMT) and motility of mouse mammary tumor cells (JM01). Heterovalent T12d also blocked TGF-beta2 effects on these phenotypes. In vivo comparison of T22d35 trap and the pan-neutralizing TGF-beta antibody 1D11 indicated that T22d35 treatment but not 1D11 reduced the growth of established primary 4T1 mammary tumors, suggesting better neutralization of TGF-beta due to a better tumor penetration of the T22d35 trap (Mol. Cancer Ther. 11:1477, 2012). Trap treatment significantly increased T lymphocyte infiltration and cytotoxic activity within the tumors. Biodistribution studies demonstrated that the T22d35 trap, although eliminated rapidly from the circulating blood and other tissues (within 24 hours), localized and was retained within primary 4T1 tumors. Furthermore, a single injection of trap per week reduced 4T1 metastatic lesions by more than 80%, relative to saline controls, indicating that a short-term trap exposure in the host is sufficient to trigger long-lasting effects. Together, our results indicate that the TGF-beta traps readily home to tumors, antagonize immunosuppression and reduce metastatic spread. Citation Format: John Zwaagstra, Traian Sulea, Anne E.G. Lenferink, Jason Baardsnes, Catherine Collins, Christiane Cantin, Lucie Couture, Limei Tao, Yves Durocher, Maureen O9Connor-McCourt. Single-chain traps targeting transforming growth factor-beta (TGF-beta) home to tumors and reduce tumor growth and metastasis by counteracting TGF-beta-mediated immunosuppression. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B024.

Abstract 560: Development of a novel class of traps that potently block transforming growth factor-beta (TGF-beta) thereby counteracting TGF-beta mediated immunosuppression and promoting T-cell infiltration into tumors

Introduction: Elevated TGF-β ligand markedly augments cancer progression primarily by suppressing the immune system in the tumor microenvironment, in particular by suppressing T-cell recruitment and/or activation. We developed a novel class of decoy receptor traps to potently block TGF- β and induce T-cell infiltration into tumors. This promotes the “T-cell-inflamed” tumor state, which is expected to render tumors sensitive to immune checkpoint inhibitors and other immunotherapeutics. Experimental Procedures: We have computationally designed a class of avidity-enhanced receptor-ectodomain-based traps which bind and neutralize TGF-β. Several trap formats have been produced and tested, with each format exhibiting varying characteristics, including differing circulating half-lives and in vitro blocking potencies (from nM to pM). Representative therapeutic candidates from the different trap formats were evaluated for efficacy in in vivo studies using the syngeneic 4T1 triple negative breast cancer (TNBC) tumor model. Additionally, ex vivo studies were performed on CD4+ and CD8+ T-cells harvested from the draining lymph nodes of treated animals. Results: In efficacy studies using the syngeneic 4T1 TNBC model, novel TGF-β traps were shown to promote significant T-cell infiltration into tumors. This infiltration resulted in reduced primary tumor growth as well as significant reductions in metastatic lesions. Additionally, ex vivo studies revealed that trap treatment decreased T-cell apoptosis, promoted T-cell proliferation in response to tumor cell lysates in the presence of dendritic cells, as well as increased the capacity of T-cells to specifically lyse 4T1 tumor cells. Conclusion: Novel computationally-designed TGF-β traps are capable of promoting the “T-cell-inflamed” tumor state. Combination studies in which this novel class of anti-TGF-β immunotherapy is combined with immune checkpoint inhibitors are ongoing. Citation Format: Maureen D. O’Connor-McCourt, Anne E.G. Lenferink, John Zwaagstra, Traian Sulea, Jason Baardsnes, Catherine Collins, Christiane Cantin, Yves Durocher, Renu Singh, James Koropatnick. Development of a novel class of traps that potently block transforming growth factor-beta (TGF-beta) thereby counteracting TGF-beta mediated immunosuppression and promoting T-cell infiltration into tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 560.

Identification of genes that are differentially expressed in response to EGF receptor signaling in A549 lung cancer cells using a combination of suppression subtractive hybridization and microarrays

Identification of genes that are differentially expressed in response to EGF receptor signaling in A549 lung cancer cells using a combination of suppression subtractive hybridization and microarrays