scholarly journals Targeting Hypoxia Sensitizes TNBC to Cisplatin and Promotes Inhibition of Both Bulk and Cancer Stem Cells

2020 ◽  
Author(s):  
Andrew Sulaiman ◽  
Sarah McGarry ◽  
Jason Chambers ◽  
Emil Al-Kadi ◽  
Alexandra Phan ◽  
...  
Keyword(s):  
Therapeutic Approach ◽  
Triple Negative ◽  
Ex Vivo ◽  
Ovarian Cancer Cells ◽  
Therapeutic Approaches ◽  
Organotypic Cultures ◽  
Tnbc Patient ◽  
Clinical Dataset ◽  
Medical Need ◽  
Similar Expression Pattern

Development of targeted therapies for triple-negative breast cancer (TNBC) is an unmet medical need. Cisplatin has demonstrated its promising potential for the treatment of TNBC in clinical trials; however, cisplatin treatment is associated with hypoxia that in turn promotes cancer stem cell (CSC) enrichment and drug resistance. Therapeutic approaches to attenuate this may lead to increased cisplatin efficacy in the clinic for the treatment of TNBC. In this report, we analyzed clinical dataset of TNBC and found that TNBC patients possessed higher levels of EGFR and hypoxia gene expression. A similar expression pattern was also observed in cisplatin-resistant ovarian cancer cells. We thus developed a new therapeutic approach to inhibit EGFR and hypoxia by combination of metformin and gefitinib that sensitized TNBC cells to cisplatin and led to the inhibition of both CD44+/CD24- and ALDH+ CSCs. We demonstrated a similar inhibition efficacy on organotypic cultures of TNBC patient samples ex vivo. Since these drugs have already been used frequently in the clinic, this study illustrates a novel, clinically translatable therapeutic approach to treat patients with TNBC.

scholarly journals Targeting Hypoxia Sensitizes TNBC to Cisplatin and Promotes Inhibition of Both Bulk and Cancer Stem Cells

2020 ◽  
Vol 21 (16) ◽  
pp. 5788
Author(s):  
Andrew Sulaiman ◽  
Sarah McGarry ◽  
Jason Chambers ◽  
Emil Al-Kadi ◽  
Alexandra Phan ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Therapeutic Approach ◽  
Triple Negative ◽  
Ex Vivo ◽  
Ovarian Cancer Cells ◽  
Therapeutic Approaches ◽  
Organotypic Cultures ◽  
Tnbc Patient ◽  
Medical Need ◽  
Similar Expression Pattern

Development of targeted therapies for triple-negative breast cancer (TNBC) is an unmet medical need. Cisplatin has demonstrated its promising potential for the treatment of TNBC in clinical trials; however, cisplatin treatment is associated with hypoxia that, in turn, promotes cancer stem cell (CSC) enrichment and drug resistance. Therapeutic approaches to attenuate this may lead to increased cisplatin efficacy in the clinic for the treatment of TNBC. In this report we analyzed clinical datasets of TNBC and found that TNBC patients possessed higher levels of EGFR and hypoxia gene expression. A similar expression pattern was also observed in cisplatin-resistant ovarian cancer cells. We, thus, developed a new therapeutic approach to inhibit EGFR and hypoxia by combination treatment with metformin and gefitinib that sensitized TNBC cells to cisplatin and led to the inhibition of both CD44+/CD24− and ALDH+ CSCs. We demonstrated a similar inhibition efficacy on organotypic cultures of TNBC patient samples ex vivo. Since these drugs have already been used frequently in the clinic; this study illustrates a novel, clinically translatable therapeutic approach to treat patients with TNBC.

scholarly journals Optimizing cisplatin delivery to triple-negative breast cancer through novel EGFR aptamer-conjugated polymeric nanovectors

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Lisa Agnello ◽  
Silvia Tortorella ◽  
Annachiara d’Argenio ◽  
Clarissa Carbone ◽  
Simona Camorani ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Triple Negative Breast Cancer ◽  
Tumor Targeting ◽  
Triple Negative ◽  
Ex Vivo ◽  
Treatment Options ◽  
Negative Control ◽  
Therapeutic Effects ◽  
Metastatic Setting

Abstract Background Management of triple-negative breast cancer (TNBC) is still challenging because of its aggressive clinical behavior and limited targeted treatment options. Cisplatin represents a promising chemotherapeutic compound in neoadjuvant approaches and in the metastatic setting, but its use is limited by scarce bioavailability, severe systemic side effects and drug resistance. Novel site-directed aptamer-based nanotherapeutics have the potential to overcome obstacles of chemotherapy. In this study we investigated the tumor targeting and the anti-tumorigenic effectiveness of novel cisplatin-loaded and aptamer-decorated nanosystems in TNBC. Methods Nanotechnological procedures were applied to entrap cisplatin at high efficacy into polymeric nanoparticles (PNPs) that were conjugated on their surface with the epidermal growth factor receptor (EGFR) selective and cell-internalizing CL4 aptamer to improve targeted therapy. Internalization into TNBC MDA-MB-231 and BT-549 cells of aptamer-decorated PNPs, loaded with BODIPY505-515, was monitored by confocal microscopy using EGFR-depleted cells as negative control. Tumor targeting and biodistribution was evaluated by fluorescence reflectance imaging upon intravenously injection of Cyanine7-labeled nanovectors in nude mice bearing subcutaneous MDA-MB-231 tumors. Cytotoxicity of cisplatin-loaded PNPs toward TNBC cells was evaluated by MTT assay and the antitumor effect was assessed by tumor growth experiments in vivo and ex vivo analyses. Results We demonstrate specific, high and rapid uptake into EGFR-positive TNBC cells of CL4-conjugated fluorescent PNPs which, when loaded with cisplatin, resulted considerably more cytotoxic than the free drug and nanovectors either unconjugated or conjugated with a scrambled aptamer. Importantly, animal studies showed that the CL4-equipped PNPs achieve significantly higher tumor targeting efficiency and enhanced therapeutic effects, without any signs of systemic toxicity, compared with free cisplatin and untargeted PNPs. Conclusions Our study proposes novel and safe drug-loaded targeted nanosystems for EGFR-positive TNBC with excellent potential for the application in cancer diagnosis and therapy.

scholarly journals Deciphering the temporal heterogeneity of cancer-associated fibroblast subpopulations in breast cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Freja Albjerg Venning ◽  
Kamilla Westarp Zornhagen ◽  
Lena Wullkopf ◽  
Jonas Sjölund ◽  
Carmen Rodriguez-Cupello ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Flow Cytometry ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Ex Vivo ◽  
Smooth Muscle Actin ◽  
Mammary Tissue ◽  
Temporal Heterogeneity ◽  
Alpha Smooth Muscle Actin ◽  
Murine Tumours

Abstract Background Cancer-associated fibroblasts (CAFs) comprise a heterogeneous population of stromal cells within the tumour microenvironment. CAFs exhibit both tumour-promoting and tumour-suppressing functions, making them exciting targets for improving cancer treatments. Careful isolation, identification, and characterisation of CAF heterogeneity is thus necessary for ex vivo validation and future implementation of CAF-targeted strategies in cancer. Methods Murine 4T1 (metastatic) and 4T07 (poorly/non-metastatic) orthotopic triple negative breast cancer tumours were collected after 7, 14, or 21 days. The tumours were analysed via flow cytometry for the simultaneous expression of six CAF markers: alpha smooth muscle actin (αSMA), fibroblast activation protein alpha (FAPα), platelet derived growth factor receptor alpha and beta (PDGFRα and PDGFRβ), CD26/DPP4 and podoplanin (PDPN). All non-CAFs were excluded from the analysis using a lineage marker cocktail (CD24, CD31, CD45, CD49f, EpCAM, LYVE-1, and TER-119). In total 128 murine tumours and 12 healthy mammary fat pads were analysed. Results We have developed a multicolour flow cytometry strategy based on exclusion of non-CAFs and successfully employed this to explore the temporal heterogeneity of freshly isolated CAFs in the 4T1 and 4T07 mouse models of triple-negative breast cancer. Analysing 128 murine tumours, we identified 5–6 main CAF populations and numerous minor ones based on the analysis of αSMA, FAPα, PDGFRα, PDGFRβ, CD26, and PDPN. All markers showed temporal changes with a distinct switch from primarily PDGFRα+ fibroblasts in healthy mammary tissue to predominantly PDGFRβ+ CAFs in tumours. CD26+ CAFs emerged as a large novel subpopulation, only matched by FAPα+ CAFs in abundance. Conclusion We demonstrate that multiple subpopulations of CAFs co-exist in murine triple negative breast cancer, and that the abundance and dynamics for each marker differ depending on tumour type and time. Our results form the foundation needed to isolate and characterise specific CAF populations, and ultimately provide an opportunity to therapeutically target specific CAF subpopulations.

scholarly journals Practical classification of triple-negative breast cancer: intratumoral heterogeneity, mechanisms of drug resistance, and novel therapies

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Antonio Marra ◽  
Dario Trapani ◽  
Giulia Viale ◽  
Carmen Criscitiello ◽  
Giuseppe Curigliano
Keyword(s):  
Breast Cancer ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Residual Disease ◽  
Therapeutic Approaches ◽  
Minimal Residual Disease Monitoring ◽  
Anticancer Treatment ◽  
Immune Contexture

Abstract Triple-negative breast cancer (TNBC) is not a unique disease, encompassing multiple entities with marked histopathological, transcriptomic and genomic heterogeneity. Despite several efforts, transcriptomic and genomic classifications have remained merely theoretic and most of the patients are being treated with chemotherapy. Driver alterations in potentially targetable genes, including PIK3CA and AKT, have been identified across TNBC subtypes, prompting the implementation of biomarker-driven therapeutic approaches. However, biomarker-based treatments as well as immune checkpoint inhibitor-based immunotherapy have provided contrasting and limited results so far. Accordingly, a better characterization of the genomic and immune contexture underpinning TNBC, as well as the translation of the lessons learnt in the metastatic disease to the early setting would improve patients’ outcomes. The application of multi-omics technologies, biocomputational algorithms, assays for minimal residual disease monitoring and novel clinical trial designs are strongly warranted to pave the way toward personalized anticancer treatment for patients with TNBC.

scholarly journals CD133 Promotes Adhesion to the Ovarian Cancer Metastatic Niche

2018 ◽  
Vol 11 ◽  
pp. 117906441876788 ◽  
Author(s):  
Lynn Roy ◽  
Alexander Bobbs ◽  
Rachel Sattler ◽  
Jeffrey L Kurkewich ◽  
Paige B Dausinas ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Metastasis ◽  
Ex Vivo ◽  
Surface Protein ◽  
Ovarian Cancer Cells ◽  
Metastatic Niche ◽  
Attractive Therapeutic Target ◽  
Peritoneal Mesothelium

Cancer stem cells (CSCs) are an attractive therapeutic target due to their predicted role in both metastasis and chemoresistance. One of the most commonly agreed on markers for ovarian CSCs is the cell surface protein CD133. CD133+ ovarian CSCs have increased tumorigenicity, resistance to chemotherapy, and increased metastasis. Therefore, we were interested in defining how CD133 is regulated and whether it has a role in tumor metastasis. Previously we found that overexpression of the transcription factor, ARID3B, increased the expression of PROM1 (CD133 gene) in ovarian cancer cells in vitro and in xenograft tumors. We report that ARID3B directly regulates PROM1 expression. Importantly, in a xenograft mouse model of ovarian cancer, knockdown of PROM1 in cells expressing exogenous ARID3B resulted in increased survival time compared with cells expressing ARID3B and a control short hairpin RNA. This indicated that ARID3B regulation of PROM1 is critical for tumor growth. Moreover, we hypothesized that CD133 may affect metastatic spread. Given that the peritoneal mesothelium is a major site of ovarian cancer metastasis, we explored the role of PROM1 in mesothelial attachment. PROM1 expression increased adhesion to mesothelium in vitro and ex vivo. Collectively, our work demonstrates that ARID3B regulates PROM1 adhesion to the ovarian cancer metastatic niche.

scholarly journals Aortic stenosis and diabetes mellitus: An ominous combination

2019 ◽  
Vol 16 (4) ◽  
pp. 310-323 ◽  
Author(s):  
Marko Banovic ◽  
Lavanya Athithan ◽  
Gerry P McCann
Keyword(s):  
Quality Of Life ◽  
Diabetes Mellitus ◽  
Aortic Stenosis ◽  
Therapeutic Approach ◽  
Severe Aortic Stenosis ◽  
Significant Morbidity ◽  
Therapeutic Approaches ◽  
The Relationship ◽  
Insight Into

Aortic stenosis and diabetes mellitus are both progressive diseases which, if left untreated, result in significant morbidity and mortality. There is evidence that the prevalence of diabetes is substantially increased in patients with aortic stenosis and those with diabetes have increased rates of progression from mild to severe aortic stenosis. There are good data supporting the hypothesis that aortic stenosis and diabetes mellitus are associated with diabetes mellitus being detrimental towards the quality of life and survival of patients. Thus, a thorough understanding of the pathogenesis of both of these disease processes and the relationship between them aids in designing appropriate preventive and therapeutic approaches. This review aims to give a comprehensive and up-to-date insight into the influence of diabetes mellitus on patients with degenerative aortic stenosis, as well as the prognosis and therapeutic approach to these patients.

Genomic engineering of adipocytes with CRISPR/Cas9 delivery particles for ex vivo therapeutic approach for type 2 diabetes

2021 ◽  
Author(s):  
Εμμανουέλα Τσαγκαράκη
Keyword(s):  
Type 2 Diabetes ◽  
Therapeutic Approach ◽  
Ex Vivo ◽  
Knock Out ◽  
Genomic Engineering ◽  
Fat Depots

Η παχυσαρκία και ο διαβήτης τύπου 2 (ΔΤ2) σχετίζονται με διαταραχές στην ομοιόσταση της γλυκόζης και των λιπιδίων η οποία ρυθμίζεται από την ινσουλίνη και προκαλεί σοβαρές επιπλοκές συμπεριλαμβανομένων καρδιαγγειακών νοσημάτων και στεατοηπατίτιδας. Ο συστημικός μεταβολισμός της γλυκόζης ρυθμίζεται από τους διακριτούς λιπώδεις ιστούς (fat depots), στις οποίες συμπεριλαμβάνονται δύο κυρίως τύποι λιπώδους ιστού το λευκό λίπος και το φαιό (ή καφέ) λίπος. Το μεν λευκό λίπος αποτελείται από τα «λευκά» λιποκύτταρα και έχει ρόλο αποθήκευσης της ενέργειας σε μορφή λιπιδίων ενώ το φαιό λίπος αποτελείται από τα «φαιά» και «μπεζ» λιποκύτταρα. Tα κύτταρα αυτά καταναλώνουν την αποθηκευμένη ενέργεια για την παραγωγή θερμότητας και εκφράζουν την πρωτεΐνη αποσύζευξης 1 (UCP1) καθώς και πλήθος εκκρινόμενων παραγόντων που ευνοούν τον μεταβολισμό. Έχει περιγραφή στη βιβλιογραφία ότι η μεταμόσχευση φαιού λιπώδους ιστού ή ποντικίσιων ή αθανατοποιημένων ανθρώπινων φαιών λιποκυττάρων σε παχύσαρκους μύες βελτιώνει την ανοχή στη γλυκόζη. Ωστόσο, η εφαρμογή μιας ανάλογης θεραπευτικής παρέμβασης στον άνθρωπο δεν έχει καταστεί δυνατή καθώς η διαθεσιμότητα των πρωτογενών ανθρώπινων φαιών/μπεζ λιποκυττάρων είναι εξαιρετικά περιορισμένη. Στην παρούσα διατριβή, χρησιμοποίησα μεθόδους πολλαπλασιασμού σε μεγάλη κλίματα ανθρώπινων πρόγονων λιποκυττάρων από εξαιρετικά μικρά δείγματα ανθρώπινου λιπώδους ιστού. Στα ανθρώπινα πρόδρομα λιποκύτταρα, παράλληλα με τα ποντικίσια, εφάρμοσα τροποποίηση του γονιδιώματος με τη χρήση ομαδοποιημένων με τακτά μεσοδιαστήματα σύντομων παλινδρομικών μοτίβων (CRISPR). O στόχος αυτής της γονιδιακής τροποποίησης είναι να απενεργοποιήσει γονίδια που φυσιολογικά παρεμποδίζουν τη μετατροπή των λιποκυττάρων από λευκά σε φαιά. Συνεπώς, η απώλεια της λειτουργικότητας αυτών των γονιδίων αναμένεται να προκαλέσει την μετατροπή των λευκών λιποκυττάρων σε φαιά που είναι ωφέλιμα για τον μεταβολισμό της γλυκόζης. Σημαντικοί δευτερεύοντες στόχοι της στρατηγικής της θεραπείας είναι η παράκαμψη της ανοσογονικότητας των συστατικών CRISPR και της γονιδιακής τροποποίησης σε ανεπιθύμητους ιστούς καθώς και η ελαχιστοποίηση της ανεπιθύμητης τροποποίησης σε περιοχές του γονιδιώματος εκτός των στοχευμένων. Στην παρούσα εργασία, ανέπτυξα και μεγιστοποίησα τη μέθοδο ex vivo μεταφοράς στα κύτταρα – στόχους (λιποκύτταρα) των συμπλεγμάτων στρεπτοκοκκικού Cas9 ενζύμου και στου οδηγού sgRNA, ούτως ώστε να επιτυγχάνεται η ταχύτατη διάσπασή τους αμέσως μετά την τροποποίηση του γονιδίου – στόχο. Για τη μεταφορά των συμπλεγμάτων αυτών, χρησιμοποιήθηκε η μέθοδος της ηλεκτροδιάτρησης με αποτελεσματικότητα τροποποίησης που πλησιάζει το 100%. Κατόπιν ελέγχου πλήθους υποψηφίων γονιδίων – στόχων που αναφέρονται στη βιβλιογραφία, εντοπίστηκε ως ο πλέον υποσχόμενος στόχος το γονίδιο της πρωτεΐνης 1 που αναγνωρίζει πυρηνικούς υποδοχείς (NRIP1). Τα λιποκύτταρα στα οποία απενεργοποιήθηκε το Nrip1 γονίδιο (NRIP1 Knock-out, NRIP1KO) επάγουν την έκφραση ενός «φαιού» γονιδιακού προφίλ το οποίο συμπεριλαμβάνει την πρωτεΐνη UCP1 και ορισμένους εκκρινόμενους παράγοντες. Στη συνέχεια, χαρακτήρισα in vitro το φαινότυπο των τροποποιημένων κυττάρων με πλήθος δοκιμασιών όπως έκφραση γνωστών γονιδίων που σχετίζονται με τη θερμογένεση, μιτοχονδριακή αναπνοή, οξέωση λιπιδίων, εκκρινομένους παράγοντες, δοκιμασία κατανάλωση οξυγόνου, έκφραση της πρωτεΐνης UCP1, τόσο στο ποντικίσια όσο και στο ανθρώπινα κύτταρα. Τέλος, τα βελτιστοποιημένα με CRISPR ποντικίσια ή ανθρώπινα «φαιά» λιποκύτταρα εμφυτεύθηκαν σε ποντίκια – λήπτες τα οποία λάμβαναν διατροφή εμπλουτισμένη σε λίπος για την πρόκληση διαβήτη τύπου 2. Τα εμφυτεύματα των τροποποιημένων κυττάρων έδειξαν μικρότερη συσσώρευση σωματικού λίπους μικρότερη συσσώρευση τριγλυκεριδίων στο ηπατικό παρέγχυμα καθώς επίσης βελτίωσαν την ανοχή στη γλυκόζη συγκριτικά με τα ποντίκια – μάρτυρες που έλαβαν εμφυτεύματα μη τροποποιημένων λιποκυττάρων. Παράλληλα, όπως απεδείχθη η παρουσία των συστατικών της CRISPR τροποποίησης ήταν παροδική στα κύτταρα-στόχους καθώς η ενδονουκλεάση Cas9 αποδομείται και δεν είναι ανιχνεύσιμη με ηλεκτροφόρηση ολικής πρωτεΐνης πριν τις εμφυτεύσεις. Τα ευρήματα αυτά υποδεικνύουν μια θεραπευτική στρατηγική για τη βελτίωση της ομοιόστασης του μεταβολισμού μέσω της γονιδιακής τροποποίησης με CRISPR ανθρώπινων λιποκυττάρων χωρίς την έκθεση του ασθενούς στα ανοσογόνα και δυνητικά επιβλαβή ένζυμο Cas9 και οδηγό sgRNA και άλλων οχημάτων μεταφοράς των συστατικών CRISPR.

scholarly journals Targeting of prostate-specific membrane antigen for radio-ligand therapy of triple-negative breast cancer

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Agnieszka Morgenroth ◽  
Ebru Tinkir ◽  
Andreas T. J. Vogg ◽  
Ramya Ambur Sankaranarayanan ◽  
Fatima Baazaoui ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Triple Negative Breast Cancer ◽  
Breast Cancer Cells ◽  
Triple Negative ◽  
Ex Vivo ◽  
Tube Formation ◽  
Huvec Cells ◽  
Specific Membrane

Abstract Background Triple-negative breast cancer has extremely high risk of relapse due to the lack of targeted therapies, intra- and inter-tumoral heterogeneity, and the inherent and acquired resistance to therapies. In this study, we evaluate the potential of prostate-specific membrane antigen (PSMA) as target for radio-ligand therapy (RLT). Methods Tube formation was investigated after incubation of endothelial HUVEC cells in tumor-conditioned media and monitored after staining using microscopy. A binding study with 68Ga-labeled PSMA-addressing ligand was used to indicate targeting potential of PSMA on tumor-conditioned HUVEC cells. For mimicking of the therapeutic application, tube formation potential and vitality of tumor-conditioned HUVEC cells were assessed following an incubation with radiolabeled PSMA-addressing ligand [177Lu]-PSMA-617. For in vivo experiments, NUDE mice were xenografted with triple-negative breast cancer cells MDA-MB231 or estrogen receptor expressing breast cancer cells MCF-7. Biodistribution and binding behavior of [68Ga]-PSMA-11 was investigated in both tumor models at 30 min post injection using μPET. PSMA- and CD31-specific staining was conducted to visualize PSMA expression and neovascularization in tumor tissue ex vivo. Results The triple-negative breast cancer cells MDA-MB231 showed a high pro-angiogenetic potential on tube formation of endothelial HUVEC cells. The induced endothelial expression of PSMA was efficiently addressed by radiolabeled PSMA-specific ligands. 177Lu-labeled PSMA-617 strongly impaired the vitality and angiogenic potential of HUVEC cells. In vivo, as visualized by μPET, radiolabeled PSMA-ligand accumulated specifically in the triple-negative breast cancer xenograft MDA-MB231 (T/B ratio of 43.3 ± 0.9), while no [68Ga]-PSMA-11 was detected in the estrogen-sensitive MCF-7 xenograft (T/B ratio of 1.1 ± 0.1). An ex vivo immunofluorescence analysis confirmed the localization of PSMA on MDA-MB231 xenograft-associated endothelial cells and also on TNBC cells. Conclusions Here we demonstrate PSMA as promising target for two-compartment endogenous radio-ligand therapy of triple-negative breast cancer.

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