Monoclonal antibody 4C5 prevents activation of MMP2 and MMP9 by disrupting their interaction with extracellular HSP90 and inhibits formation of metastatic breast cancer cell deposits

<p>Abstract</p> <p>Background</p> <p>Heat shock protein 90 (HSP90) is a molecular chaperone that is considered a new target for the treatment of cancer. Increasing data reveal an extracellular chaperoning activity for HSP90. Here we investigate the interaction of the secreted isoforms of HSP90 with matrix metalloproteinases (MMP) MMP2 and MMP9. Moreover we examine the role of a monoclonal antibody (mAb) against HSP90, mAb 4C5, regarding these interactions and its value as a potential inhibitor of human breast cancer cell invasion and metastasis.</p> <p>Results</p> <p>Our results showed that both HSP90α and HSP90β are secreted by MDAMB453 human breast cancer cells and interact with MMP2 and MMP9. MAb 4C5, while not affecting the secretion of inactive MMPs, inhibits their activation by disrupting their interaction extracellularly with both isoforms of HSP90. The <it>in vivo </it>studies revealed that mAb 4C5 significantly inhibits the metastatic deposit formation of MDAMB453 cells into the lungs of SCID mice.</p> <p>Conclusion</p> <p>Both isoforms of HSP90 are secreted by MDAMB453 cells and interact with MMP2 and MMP9. MAb 4C5 prevents MMP2 and MMP9 activation, by disrupting their interaction with HSP90. Finally mAb 4C5 significantly inhibits the metastatic deposit formation of MDAMB453 cells, by preventing their extravasation and infiltration in the lung tissue and therefore it could be used as a potential therapeutic agent for cancer metastasis.</p

Supporting data on combined transcriptomic and phosphoproteomic analysis of BMP4 signaling in human embryonic stem cells.

Human embryonic stem cells exhibit great potential as a therapeutic tool in regenerative medicine due to their self-renewal and trilineage differentiation capacity. Maintaining this unique cellular state has been shown to rely primarily on the Activin A / TGFβ signaling pathway. While most conventional culture media are supplemented with TGFβ, in the current study we utilize a modified version of the commercially available mTeSR1, substituting TGFβ for Activin A in order to preserve pluripotency. (1) Cells cultured in ActA-mTesR express pluripotency factors NANOG, OCT4 and SOX2 at comparable levels with cells cultured in TGFβ-mTeSR. (2) ActA-mTeSR cultured cells retain a physiological karyotype. (3) Cells in ActA-mTeSR maintain their trilineage differentiation capacity as shown in the teratoma formation assay. This system can be used to dissect the role of Activin A, downstream effectors and signaling cascades in human embryonic stem cell responses

Heterodimeric IL-15 delays tumor growth and promotes intratumoral CTL and dendritic cell accumulation by a cytokine network involving XCL1, IFN-γ, CXCL9 and CXCL10.

BACKGROUND: Interleukin-15 (IL-15) promotes growth and activation of cytotoxic CD8 METHODS: The antitumor activity of hetIL-15 produced from mammalian cells was tested in mouse tumor models (MC38 colon carcinoma and TC-1 epithelial carcinoma). The functional diversity of the immune infiltrate and the cytokine/chemokine network within the tumor was evaluated by flow cytometry, multicolor immunohistochemistry (IHC), gene expression profiling by Nanostring Technologies, and protein analysis by electrochemiluminescence and ELISA assays. RESULTS: hetIL-15 treatment resulted in delayed primary tumor growth. Increased NK and CD8 CONCLUSIONS: Our results show that hetIL-15 administration enhances T cell entry into tumors, increasing the success rate of immunotherapy interventions. Our study further supports the incorporation of hetIL-15 in tumor immunotherapy approaches to promote the development of antitumor responses by favoring effector over regulatory cells and by promoting lymphocyte and DC localization into tumors through the modification of the tumor chemokine and cytokine milieu

Study of heat shock protein 90 (HSP90) on cancer development

Cancer is a class of diseases in which a group of cells display uncontrolled growth intrusion an and destruction of adjacent tissues (invasion), and sometimes spread to other locations in the body via lymph or blood creating metastases. Heat shock protein 90 (HSP90) is a chaperone protein that is essential for the cell to stabilize the form and the function of a variety of substrates. Moreover, HSP90 interacts with a great number of molecules that are involved in the development and low survival of cancer cells. In order to understand the molecular events regulating neuronal migration and development, we have previously characterized a monoclonal antibody (mAb), named 4C5. This antibody recognizes both HSP90 isoforms. The primary aim of the present study was to evaluate the potential prognostic value of mAb4C5 in malignant melanoma. Interestingly, mAb4C5 reacted positively in all the primary and metastatic melanomas examined, while the cell surface localization of HSP90 was shown immunohistochemically on live B16 F10 melanoma cells using mAb4C5. To assess the participation of cell surface and not intracellular HSP90 in the invasion process of B16 F10 melanoma cells, internalization of mAb4C5 and anti-alpha HSP90 and anti-beta HSP90 antibodies in the culture medium strongly inhibited melanoma cell invasion in vitro. The above described experiments showed that mAB4C5 had the ability to inhibit melanoma cell invasion in vitro, and since it could not be internalized, the inhibition of the invasion could be attributed to the surface pool of HSP90 molecules. In line with these experiments it was shown that mAb4C5 could inhibit melanoma metastasis in vivo using B16 F10 cells and C57/BL mice. As a next step the molecular mechanisms of the surface HSP90 were investigated. This study revealed that both isoforms of HSP90 were secreted from human breast cancer cells (MOAMB453) and that both isoforms interact with MMPs (Matrix metaloproteinases 2 and 9). Furthermore, mAb4C5 could inhibit: a) the activation of these molecules in vitro and b) the metastatic despositions of MOAMB453 cells in vivo mouse models, suggesting that mAb4C5 could be a potential anticancer agent.Ο καρκίνος είναι μια κατηγορία ασθενειών ή διαταραχών που χαρακτηρίζονται από τον ανεξέλεγκτο πολλαπλασιασμό κυττάρων και την δυνατότητα αυτών να διηθούν υποκείμενους ιστούς και να εισβάλουν σε άλλους πιο απομακρυσμένους ιστούς δημιουργόντας μεταστάσεις. Η HSP90, αν και πρωτεΐνη θερμικού σοκ εκφράζεται σε αφθονία στα ευκαρυωτικά κύτταρα υπό φυσιολογικές συνθήκες (απουσία στρες), όπου εκτελεί βασικές λειτουργίες επιπλέον η HSP90 αλληλεπιδρά με έναν μεγάλο αριθμό βιομορίων τα οποία εμπλέκονται στην ανάπτυξη ή/και επιβίωση των καρκινικών κυττάρων. Με σκοπό την κατανόηση των μοριακών μηχανισμών που ρυθμίζουν τη μετανάστευση των νευρικών κυττάρων στο παρελθόν πραγματοποιήθηκε από την ομάδα μας η παραγωγή και ο χαρακτηρισμός ενός μονοκλωνικού αντισώματος του mAb4C5. Το mAb4C5 αναγνωρίζει και τις δύο ισομορφές της HSP90. Πρωταρχικός στόχος της παρούσης εργασίας ήταν η διερεύνηση του ρόλου της HSP90 σε διάφορα καρκινικά φαινόμενα ή και η μελέτη της ενδεχόμενης διαγνωστικής και θεραπευτικής αξίας του αντισώματος mAb4C5 σε μεταστατικούς καρκίνους. Από τα πειράματα μας αρχικά φάνηκε ότι το mAb4C5 έχει την ιδιότητα να δίνει ισχυρή ανοσοϊστοχημική χρώση με όλα τα πρωτογενή και μεταστατικά μελανώματα και αποδείχθηκε η ύπαρξη της HSP90 στην επιφάνεια πληθώρας καρκινικών σειρών τόσο προερχόμενων από καρκινώματα ποντικιών όσο και από ανθρώπινα καρκινώματα. Στη συνέχεια φάνηκε ότι η παρουσία του mAb4C5 στο υλικό καλλιέργειας B16F10 κυττάρων μείωσε σημαντικά τη διηθητική ικανότητα των κυττάρων αυτών λόγω πρόσθεσης του μονοκλωνικού αντισώματος στην επιφανειακή HSP90. Επόμενος στόχος της παρούσας διατριβής ήταν η διερεύνηση των μοριακών μηχανισμών που χαρακτηρίζουν τη συμμετοχή της επιφανειακής HSP90 σε μεταστατικές διαδικασίες. Από τα αποτελέσματα πιστοποιήθηκε τόσο η έκκριση της HSP90a, όσο και της HSP90β από κύτταρα ανθρώπινο καρκίνο μαστού, MDAMB453. Επίσης με πειράματα ανοσοκατακρήμνισης δείχτηκε ότι και οι δύο ισομορφές της HSP90 αλληλεπιδρούν τόσο με την ενεργή όσο και με τις ανενεργές μορφές των MMP2 και MMP9, ενώ διαπιστώθηκε ότι το mAb4C5 αναστέλλει σε μεγάλο βαθμό την ενεργοποίηση αυτών των MMPs και την αλληλεπίδρασή τους με την εκκρινόμενη HSP90. Τέλος, in vivo πειράματα φανέρωσαν ότι το mAb4C5 αναστέλλει με δοσοεξαρτώμενο τρόπο την μεταστατική απόθεση των καρκινικών κυττάρων και αυξάνει, επίσης με δοσοεξαρτώμενο τρόπο την επιβίωση των πειραματόζωων, καθιστώντας το mAb4C5 ως νέο αντιμεταστατικό παράγοντα για μεγάλη ποικιλία καρκίνων

Generation of human induced pluripotent stem cells in defined, feeder-free conditions

Herein, we describe a modified protocol for the generation of human induced pluripotent stem cells (hiPS) and expansion under defined, serum free and feeder free conditions. These cells exhibit a high level of plasticity towards various differentiation pathways both in vitro and in vivo. Ultimately, hiPS-derived lines achieved high standards of three dimensional differentiations on biomaterial scaffolds and promoted in vivo regeneration of complex organs, such as Anterior Cruciate Ligament (in swine ACL-rupture models) and other tissues as well

Heterodimeric IL-15 in Cancer Immunotherapy

Immunotherapy has emerged as a valuable strategy for the treatment of many cancer types. Interleukin-15 (IL-15) promotes the growth and function of cytotoxic CD8+ T and natural killer (NK) cells. It also enhances leukocyte trafficking and stimulates tumor-infiltrating lymphocytes expansion and activity. Bioactive IL-15 is produced in the body as a heterodimeric cytokine, comprising the IL-15 and the so-called IL-15 receptor alpha chain that are together termed “heterodimeric IL-15” (hetIL-15). hetIL-15, closely resembling the natural form of the cytokine produced in vivo, and IL-15:IL-15Rα complex variants, such as hetIL-15Fc, N-803 and RLI, are the currently available IL-15 agents. These molecules have showed favorable pharmacokinetics and biological function in vivo in comparison to single-chain recombinant IL-15. Preclinical animal studies have supported their anti-tumor activity, suggesting IL-15 as a general method to convert “cold” tumors into “hot”, by promoting tumor lymphocyte infiltration. In clinical trials, IL-15-based therapies are overall well-tolerated and result in the expansion and activation of NK and memory CD8+ T cells. Combinations with other immunotherapies are being investigated to improve the anti-tumor efficacy of IL-15 agents in the clinic