Selective depletion of metastatic stem cells as therapy for human colorectal cancer
2018; Springer Nature; Volume: 10; Issue: 10 Linguagem: Inglês
10.15252/emmm.201708772
ISSN1757-4684
AutoresMaría Virtudes Céspedes, Ugutz Unzueta, Anna Aviñó, Alberto Gallardo, Patricia Álamo, Rita Sala, Alejandro Sánchez‐Chardi, Isolda Casanova, M. A. Mangues, Antonio López‐Pousa, Ramón Eritja, Antonio Villaverde, Esther Vázquez, Ramón Mangues,
Tópico(s)Immunotherapy and Immune Responses
ResumoResearch Article6 September 2018Open Access Transparent process Selective depletion of metastatic stem cells as therapy for human colorectal cancer María Virtudes Céspedes María Virtudes Céspedes Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Santa Creu i Sant Pau, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Search for more papers by this author Ugutz Unzueta Ugutz Unzueta Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Santa Creu i Sant Pau, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Search for more papers by this author Anna Aviñó Anna Aviñó CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Institute for Advanced Chemistry of Catalonia (IQAC), CSIC, Barcelona, Spain Search for more papers by this author Alberto Gallardo Alberto Gallardo CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Department of Pathology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Search for more papers by this author Patricia Álamo Patricia Álamo Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Santa Creu i Sant Pau, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Search for more papers by this author Rita Sala Rita Sala Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Santa Creu i Sant Pau, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Search for more papers by this author Alejandro Sánchez-Chardi Alejandro Sánchez-Chardi Servei de Microscòpia, Universitat Autònoma de Barcelona, Barcelona, Spain Search for more papers by this author Isolda Casanova Isolda Casanova Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Santa Creu i Sant Pau, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Search for more papers by this author María Antònia Mangues María Antònia Mangues Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Santa Creu i Sant Pau, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Department of Pharmacy, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Search for more papers by this author Antonio Lopez-Pousa Antonio Lopez-Pousa CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Department of Medical Oncology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Search for more papers by this author Ramón Eritja Ramón Eritja orcid.org/0000-0001-5383-9334 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Institute for Advanced Chemistry of Catalonia (IQAC), CSIC, Barcelona, Spain Search for more papers by this author Antonio Villaverde Corresponding Author Antonio Villaverde [email protected] orcid.org/0000-0002-2615-4521 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Barcelona, Spain Search for more papers by this author Esther Vázquez Esther Vázquez CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Barcelona, Spain Search for more papers by this author Ramón Mangues Corresponding Author Ramón Mangues [email protected] orcid.org/0000-0003-2661-9525 Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Santa Creu i Sant Pau, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Search for more papers by this author María Virtudes Céspedes María Virtudes Céspedes Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Santa Creu i Sant Pau, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Search for more papers by this author Ugutz Unzueta Ugutz Unzueta Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Santa Creu i Sant Pau, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Search for more papers by this author Anna Aviñó Anna Aviñó CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Institute for Advanced Chemistry of Catalonia (IQAC), CSIC, Barcelona, Spain Search for more papers by this author Alberto Gallardo Alberto Gallardo CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Department of Pathology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Search for more papers by this author Patricia Álamo Patricia Álamo Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Santa Creu i Sant Pau, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Search for more papers by this author Rita Sala Rita Sala Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Santa Creu i Sant Pau, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Search for more papers by this author Alejandro Sánchez-Chardi Alejandro Sánchez-Chardi Servei de Microscòpia, Universitat Autònoma de Barcelona, Barcelona, Spain Search for more papers by this author Isolda Casanova Isolda Casanova Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Santa Creu i Sant Pau, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Search for more papers by this author María Antònia Mangues María Antònia Mangues Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Santa Creu i Sant Pau, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Department of Pharmacy, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Search for more papers by this author Antonio Lopez-Pousa Antonio Lopez-Pousa CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Department of Medical Oncology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Search for more papers by this author Ramón Eritja Ramón Eritja orcid.org/0000-0001-5383-9334 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Institute for Advanced Chemistry of Catalonia (IQAC), CSIC, Barcelona, Spain Search for more papers by this author Antonio Villaverde Corresponding Author Antonio Villaverde [email protected] orcid.org/0000-0002-2615-4521 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Barcelona, Spain Search for more papers by this author Esther Vázquez Esther Vázquez CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Barcelona, Spain Search for more papers by this author Ramón Mangues Corresponding Author Ramón Mangues [email protected] orcid.org/0000-0003-2661-9525 Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Santa Creu i Sant Pau, Barcelona, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain Search for more papers by this author Author Information María Virtudes Céspedes1,2,‡, Ugutz Unzueta1,2,‡, Anna Aviñó2,3, Alberto Gallardo2,4, Patricia Álamo1,2, Rita Sala1,2, Alejandro Sánchez-Chardi5, Isolda Casanova1,2, María Antònia Mangues1,2,6, Antonio Lopez-Pousa2,7, Ramón Eritja2,3, Antonio Villaverde *,2,8,9, Esther Vázquez2,8,9,‡ and Ramón Mangues *,1,2,‡ 1Institut d'Investigacions Biomèdiques Sant Pau, Hospital de Santa Creu i Sant Pau, Barcelona, Spain 2CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain 3Institute for Advanced Chemistry of Catalonia (IQAC), CSIC, Barcelona, Spain 4Department of Pathology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain 5Servei de Microscòpia, Universitat Autònoma de Barcelona, Barcelona, Spain 6Department of Pharmacy, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain 7Department of Medical Oncology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain 8Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain 9Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Barcelona, Spain ‡These authors contributed equally to this work ‡These authors contributed equally to this work as senior authors *Corresponding author. Tel: +34 935813086; E-mail: [email protected] *Corresponding author. Tel: +34 935537918; E-mail: [email protected] EMBO Mol Med (2018)10:e8772https://doi.org/10.15252/emmm.201708772 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Selective elimination of metastatic stem cells (MetSCs) promises to block metastatic dissemination. Colorectal cancer (CRC) cells overexpressing CXCR4 display trafficking functions and metastasis-initiating capacity. We assessed the antimetastatic activity of a nanoconjugate (T22-GFP-H6-FdU) that selectively delivers Floxuridine to CXCR4+ cells. In contrast to free oligo-FdU, intravenous T22-GFP-H6-FdU selectively accumulates and internalizes in CXCR4+ cancer cells, triggering DNA damage and apoptosis, which leads to their selective elimination and to reduced tumor re-initiation capacity. Repeated T22-GFP-H6-FdU administration in cell line and patient-derived CRC models blocks intravasation and completely prevents metastases development in 38–83% of mice, while showing CXCR4 expression-dependent and site-dependent reduction in foci number and size in liver, peritoneal, or lung metastases in the rest of mice, compared to free oligo-FdU. T22-GFP-H6-FdU induces also higher regression of established metastases than free oligo-FdU, with negligible distribution or toxicity in normal tissues. This targeted drug delivery approach yields potent antimetastatic effect, through selective depletion of metastatic CXCR4+ cancer cells, and validates metastatic stem cells (MetSCs) as targets for clinical therapy. Synopsis Control of metastatic spread is an unmet need. This study reports on the therapeutic effect of a nanoconjugate (NC) that selectively delivers a drug to metastatic stem cells overexpressing surface CXCR4 receptor (CXCR4+ MetSCs) (Targeted drug delivery) after its intravenous injection in colorectal cancer (CRC) mouse models. Selective NC internalization, leading to a high genotoxic damage and elimination of CXCR4+ CRC cells, in a CXCR4-dependent way, both in vitro and in vivo. Reduction of tumor re-initiation capacity and CXCR4+ tumor emboli intravasation in colonic peri-tumoral vessels after NC treatment. Potent prevention of metastases, in cell line and patient-derived orthotopic CRC mouse models, yielding a high percent of metastasis-free mice after repeated-dose therapy, as compared to the free drug. Metastatic sites with high CXCR4 expression show higher response to the NC and higher reduction in CXCR4+ cancer cell fraction at the end of treatment. Absence of accumulation or toxicity in normal tissues and high therapeutic index achieved by the NC, which exploits the high CXCR4 overexpression in target MetSCs in comparison to non-tumor cells. Introduction Control of metastatic spread remains an unmet medical need. In colorectal cancer (CRC), as in other tumor types, adjuvant therapy controls metastases and prolongs survival at the expense of high toxicity; however, metastases remain the primary cause of death (Schrag, 2004; Mehlen & Puisieux, 2006; Spano et al, 2012; Riihimäki et al, 2016). There is an urgent need to develop less toxic and more effective antimetastatic agents. To achieve this goal, preclinical and clinical drug development should shift its focus from primary tumor to metastasis control, using metastatic cancer models and evaluating promising drugs in patients with limited or non-metastatic disease (Steeg & Theodorescu, 2008; Steeg, 2016). This is relevant because metastases differ from primary tumors in their mutational or gene expression profiles (Rhodes & Chinnaiyan, 2005; Vignot et al, 2015) and response to drugs (Takebayashi et al, 2013; Chen et al, 2015). Metastatic stem cells (MetSCs) are a subset of cancer stem cells (CSCs) that, in addition to self-renewal and differentiation capacities, have trafficking functions (Steeg, 2016; Brabletz et al, 2005; Sleeman & Steeg, 2010; Oskarsson et al, 2014). In CRC, CXCR4 receptor enhances metastatic dissemination and confers poor patient prognosis (Kim et al, 2005, 2006; Schimanski et al, 2005), a finding similar to other cancers (Müller et al, 2001; Balkwill, 2004; Kucia et al, 2005; Schimanski et al, 2006; Hermann et al, 2007; Sun et al, 2010). Moreover, CXCR4-overexpressing (CXCR4+) cells have metastasis-initiating capacity (MICs) in CRC (Croker & Allan, 2008; Zhang et al, 2012), whereas CXCR4 RNAi-mediated downregulation or blockade of membrane localization inhibits hepatic and lung metastases (Murakami et al, 2013; Wang et al, 2014). These findings support a MetSC function for CXCR4+ CRC cells. Nevertheless, the formal proof for MetSCs clinical relevance will only come by demonstrating that their selective targeting and elimination leads to antimetastatic effect. Nanomedicine pursues targeted drug delivery, which aims at increasing anticancer effect while reducing toxicity (Das et al, 2009). We here use targeted drug delivery to CXCR4+ MetSCs in an attempt to achieve their selective elimination. We produced the drug nanoconjugate T22-GFP-H6-FdU by covalently binding a protein nanoparticle, which selectively targets CXCR4+ cancer cells (Unzueta et al, 2012a; Céspedes et al, 2016) to Floxuridine (FdU), a cytotoxic drug used to treat CRC liver metastases (Shi et al, 2015). We here demonstrate selective T22-GFP-H6-FdU biodistribution to tumor and metastatic foci in cell line- and patient-derived CRC models. We also observed its internalization and selective FdU delivery in CXCR4+ MetSCs, leading to their depletion. After repeated T22-GFP-H6-FdU administration, and in contrast to free oligo-FdU, we achieved highly significant activity in the prevention and regression of metastases in the absence of toxicity, supporting the clinical relevance of developing drugs that selectively target MetSCs to achieve metastasis control. Results Development of T22-GFP-H6-FdU, a nanoconjugate that targets CXCR4+ CRC cells The previous demonstration of MIC capacity for CXCR4-overexpressing (CXCR4+) CRC cells (Croker & Allan, 2008; Zhang et al, 2012), and its inhibition by CXCR4 downregulation (Murakami et al, 2013; Wang et al, 2014), identifies these cells as MetSCs (Oskarsson et al, 2014). On this basis, we generated a CXCR4-targeted nanoconjugate to evaluate its capacity to achieve antimetastatic effect by selectively eliminating CXCR4+ CRC cells. The structure and physico-chemical characterization of this new T22-GFP-H6-FdU nanoconjugate are described in Fig 1A–C, and Appendix Figs S1 and S2, which contains T22 (a ligand that targets the CXCR4 receptor), a green fluorescent protein (allowing its in vivo monitoring) and oligo-FdU, an oligonucleotide of a drug active against CRC (Shi et al, 2015), which allows to load a high number of drug molecules into the nanoconjugate. Figure 1. T22-GFP-H6-FdU nanoconjugate synthesis and selective internalization and killing of CXCR4+ CRC cells in vitro A. The nanoconjugate contains a fusion protein [T22-GFP-H6—composed of the peptide T22 as a CXCR4 ligand, a green fluorescent protein and a histidine tail—bound to the payload drug (Unzueta et al, 2012a)]. B. Three to four pentameric oligonucleotides (approximately 20 molecules) of the antitumor drug 5-Fluoro-2′-deoxyuridine (FdU), named oligo-FdU, are conjugated to the T22-GFP-H6 targeting vector using a linker. C. T22-GFP-H6-FdU chemical synthesis: T22-GFP-H6 is first covalently bound to the 6-Maleimidohexanoic acid N-hydroxysuccinimide ester linker through its amino groups in the external lysines (Hermanson, 2013). The thiol-functionalized oligo-FdU (oligo-(FdU)5-SH; see Appendix Fig S1) is then reacted with T22-GFP-H6 functionalized with maleimide (Michael reaction). D. High and constitutive expression of CXCR4 in the membrane of SW1417 CRC cells as measured by flow cytometry. E. Lack of human SDF-1α release from cultured SW1417 CRC cells, as measured by ELISA, whereas human control 1BR3.G fibroblasts express high SDF-1α levels, after 48 or 72 h of growth in culture (mean ± s.e.m., N = 2 experiment in duplicate). F. Nanoconjugate internalization in CXCR4-overexpressing (CXCR4+) SW1417 CRC cells after 1-h exposure at 1 μM, as measured by fluorescence emission using flow cytometry (mean ± s.e.m., N = 3 experiments in duplicate). Significant difference at **P = 0.002 between the T22-GFP-H6-FdU and the T22-GFP-H6-FdU + AMD3100 groups, Mann–Whitney U-test. G. Intracellular trafficking of T22-GFP-H6-FdU in CXCR4+ SW1417 cells by confocal microscopy after exposure at 1 μM for 24 h. The green staining corresponds to GFP-containing nanoconjugates, and the red staining corresponds to plasma cell membranes stained with a red dye (CellMask™), whereas cell nucleus was stained in blue with Hoechst. The insets show detail of the intracellular localization of nanostructured, fluorescent entities, in an isosurface representation within a three-dimensional volumetric x-y-z data field. H. Linearized T22-GFP-H6-FdU dose–response trend line representation compared with unconjugated free oligo-FdU exposure. Antitumor effect was measured as CXCR4+ SW1417 cell viability by MTT after 72-h exposure as the described concentrations (mean ± s.e.m., N = 3 experiments in duplicate). I. Reduction in cell viability determined by optical microscope images of SW1417 cells exposed to 1 μM T22-GFP-H6-FdU for 72 h, as compared to T22-GFP-H6 or free oligo-FdU (N = 3 experiments in duplicate; Scale bar, 100 μm). Download figure Download PowerPoint T22-GFP-H6-FdU was synthesized by functionalizing oligo-FdU with thiol (Fig 1C and Appendix Fig S1A), which was subsequently conjugated to the previously described T22-GFP-H6 protein nanoparticle (Unzueta et al, 2012a) once bound to a chemical linker (Fig 1C). We physico-chemically characterized the HS-oligo-FdU. The functionalized pentamer FdU-HEG-SH was quantified by absorption at 260 nm and confirmed by MALDI mass spectrometry (MALDI-TOF), yielding a MW of 1,976.2, being the expected MW 1,974.0. The control pentanucleotide (free oligo-FdU) characterized by mass spectrometry (MALDI-TOF) yield a MW of 1,476.5, being the expected MW: 1,478.1. The analysis of the conjugation products was performed by MALDI-TOF spectra identifying the peaks corresponding to one or two molecules of pentaoligonucleotides of FdU bound to the nanoparticle with the MW indicated in Appendix Figs S1 and S2. The T22-GFP-H6-FdU size was determined by dynamic light scattering, being 14.6 + 0.14, as compared to 13.4 + 0.11 for the control T22-GFP-H6 nanoparticle, a size consistent with that determined by transmission electron microscopy. This product had an approximate FdU/nanoparticle (DNR) ratio of 20 (Appendix Fig S2), and maintained its capacity for self-assembling (Unzueta et al, 2012a; Rueda et al, 2015; Appendix Fig S2D). The determined size was higher than the renal filtration cutoff (6–7 nm) ensuring a high re-circulation time in blood, a requirement for effective targeted drug delivery (Unzueta et al, 2012b, 2015). T22-GFP-H6-FdU selectively internalizes and kills CXCR4+ CRC cells in vitro Following, we used the human SW1417 CRC cell line to assess if the loaded oligo-FdU conferred cytotoxic activity to the nanoparticle while maintaining its CXCR4 targeting capacity, provided that drug conjugation can alter protein conformation and function (Goswami et al, 2013). We first determined that this cell line constitutively expresses membrane CXCR4 (Fig 1D) while lacking SDF-1α expression (Fig 1E). Then, we demonstrated T22-GFP-H6-FdU capacity to internalize in CXCR4+ SW1417, as measured by fluorescence emission using flow cytometry (Fig 1F), and to accumulate and traffic into its cytosol as observed by confocal microscopy (Fig 1G). The nanoconjugate maintains also its dependence on CXCR4 for internalization, since AMD3100, a CXCR4 antagonist, was able to downregulate CXCR4 receptor in the membrane and completely blocked nanoconjugate internalization (Fig 1F). In addition, T22-GFP-H6-FdU induced significantly higher cytotoxicity than free oligo-FdU in the same cells, as measured by cell viability (Fig 1H) or phase-contrast microscopy (Fig 1I). We confirmed CXCR4-dependent nanoconjugate internalization and higher cytotoxicity than free oligo-FdU in human CXCR4+ HeLa cells (Appendix Fig S3A–D). T22-GFP-H6-FdU selectively targets CXCR4+ CRC cells in vivo Once CXCR4-dependence for T22-GFP-H6-FdU in vitro activity was established, we investigated whether the nanoconjugate could achieve targeted drug delivery after its intravenous administration in the subcutaneous (SC) CXCR4+ SW1417 CRC model. We assayed its selectivity and CXCR4 dependence regarding tumor tissue uptake, internalization in CXCR4-overexpressing MetSCs (target cells), intracellular release of the cytotoxic drug FdU, and selective CXCR4+ MetSC killing (Fig 2A). Figure 2. Selective biodistribution and receptor-dependent uptake of T22-GFP-H6-FdU in CXCR4+ cells in vivo A. Approach to achieve targeted drug delivery and selective killing of metastatic stem cells: CXCR4-nanoconjugate interaction triggers CXCR4-mediated internalization in MetSCs, in primary tumors and metastatic foci, followed by FdU release to the cytosol and diffusion to the nucleus to induce double-strand breaks leading to selective killing of CXCR4+ cells. B. Selective T22-GFP-H6-FdU nanoconjugate biodistribution in subcutaneous CXCR4+ SW1417 CRC tumor tissue 5 h after a 100 μg single intravenous dose, as measured by fluorescence emission using IVIS Spectrum 200 (N = 5/group). Biodistribution is similar to that achieved by the T22-GFP-H6 targeting vector and undetectable after Buffer or free oligo-FdU treatment (N = 5 mice/group). C. Co-localization (yellow merged) of the T22-GFP-H6-FdU (green) and the CXCR4 receptor (red) and release of T22-GFP-H6-FdU into the cytosol in CXCR4+ tumor cells 5 h after a 100 μg dose of nanoconjugate, as measured by dual anti-GFP/anti-CXCR4 immunofluorescence (IF). DAPI (blue nuclear staining). Fluorescence emission was measured in the green and red channels using the ImageJ software and expressed as mean area (A) ± s.e.m (μm2) (N = 10, 2 tumor fields × 5 mice; 200×). Note the significant (P = 0.003) increase in the area occupied by the green dots (nanoconjugate released to the cell cytosol) in T22-GFP-H6-FdU-treated tumors, compared to free oligo-FdU-treated control tissues. Scale bar, 50 μm. D. Administration of the CXCR4 antagonist AMD3100 completely blocks T22-GFP-H6-FdU tumor biodistribution, as measured by fluorescence emission. Fluorescence is not detected in Buffer or free oligo-FdU controls (N = 5 tumor fields/group). E. The uptake of T22-GFP-H6-FdU observed in CXCR4+ SW1417 tumor tissues is almost completely blocked by prior AMD3100 administration, as quantified using the anti-GFP IHC H-score (mean ± s.e.m., N = 5 tumor fields/group). Comparison of T22-GFP-H6 uptake between groups: (B: Buffer; F: free oligo-FdU; T-F: T22-GFP-H6-FdU; T-F+A: T22-GFP-H6-FdU+AMD3100). P-values for statistical differences B vs. T-F, **P = 0.000; F vs. T-F, **P = 0.000; T-F vs. TFA, **P = 0.004. Mann–Whitney U-test. F. Representative images of T22-GFP-H6-FdU uptake and AMD3100 competition by anti-GFP immunostaining, which quantitation is reported in panel (E). Scale bar, 50 μm. Download figure Download PowerPoint T22-GFP-H6-FdU showed selective tumor uptake, as measured by fluorescence emission, 5 h after the injection of a 100 μg dose in mice (Fig 2B) as previously demonstrated for T22-GFP-H6 (Céspedes et al, 2016). Moreover, T22-GFP-H6-FdU selectively internalized into CXCR4+ tumor cells as determined by their co-localization (merged yellow color) in the cell membrane, using dual anti-GFP and anti-CXCR4 immunofluorescence, as well as the detection of released nanoconjugate into the CXCR4+ cell cytosol (green dots; Fig 2C). In addition, administering the CXCR4 antagonist AMD3100 to mice prior to the nanoconjugate completely blocked its tumor uptake (Fig 2D) as well as its internalization in CXCR4+ cancer cells (Fig 2E and F). Therefore, the nanoconjugate achieves not only selective tumor biodistribution, but also its specific internalization into target CXCR4+ cancer cells, in a CXCR4-dependent manner. T22-GFP-H6-FdU achieves targeted drug delivery leading to selective depletion of CXCR4+ cancer cells in CRC tumors We next used the same SC SW1417 CRC model to assess if the selective internalization into the cytosol of CXCR4+ target cancer cells achieved by the nanoconjugate led to selective FdU delivery. We also evaluated whether the delivered FdU could induce DNA damage and caspase-3-dependent cell death, triggering the specific elimination of CXCR4+ tumor cells. To that aim, we used γ-H2AX IHC to measure the generation of DNA double-strand breaks (DSBs), since they mediate FdU antitumor activity (Longley et al, 2003). Five hours after T22-GFP-H6-FdU treatment, the number of cells containing DSBs foci in tumors (22.8 ± 1.4) was significantly higher (P = 0.02) than after free oligo-FdU treatment (13.4 ± 0.7), whereas cells containing DSBs in control T22-GFP-H6 or Buffer-treated tumors were barely detectable (Fig 3A and B). Figure 3. T22-GFP-H6-FdU-induced depletion of CXCR4-overexpressing cancer cells in tumor tissue A. Representative images of CXCR4 overexpression in subcutaneous tumor tissue, showing similar CXCR4 levels among compared groups (N = 5/group; Buffer, T22-GFP-H6-FdU, T22-GFP-H6, and free oligo-FdU) before treatment (upper panels). Representative images of DNA double-strand break induction and caspase-3 activation (measured with anti-γ-H2AX or anticleaved caspase-3 by IHC) 5 h post-administration (middle panels). Apoptotic induction (Hoechst staining, 24 h post-administration, lower panels). Note the higher number of cells positive for DSBs, caspase-3 activation, and apoptosis induction in the T22-GFP-H6-FdU as compared to free oligo-FdU. Black or white arrows indicate dead cells. Scale bar, 50 μm. B. Quantitation of the number of cells containing DSBs or active caspase-3 in IHC-stained tumor sections 5 h post-treatment and the number of condensated or disaggregated nuclei (by Hoechst staining) 24 h post-treatment in tumor sections of 10 high-power fields (400× magnification) using the Cell∧D software (N = 50; 10 tumor fields/mice; 5 mice/group). Data expressed as mean ± s.e.m. Parameter comparison between groups: (B: Buffer; T: T22-GFP-H6; F: free oligo-FdU; T-F: T22-GFP-H6-FdU). P-values for statistical differences: γ-H2AX staining quantitation: B vs. T, #P = 0.001; B vs. F, #P = 0.000; B vs. T-F, #P = 0.000; T vs. T-F, **P = 0.001; F vs. T-F, *P = 0.02. Cleaved caspase-3 quantitation: B vs. F, *P = 0.034; B vs. T-F, **P = 0.009; T vs. T-F, **P = 0.003; F vs. T-F, *P = 0.012. Hoechst staining quantitation: B vs. F, **P = 0.01; B vs. T-F, **P = 0.001; T vs. T-F, **P = 0.000; F vs. T-F, *P = 0.032. Mann Whitney U-test. Download figure Download PowerPoint T22-GFP-H6-FdU induction of DSBs indicated its capacity to release FdU in target cells to reach the nucleus and incorporate into DNA to induce DNA damage. In addition, the number of cleaved caspase-3-positive cells signaling for apoptosis (IHC measured using anticleaved caspase-3 antibody) 5 h after T22-GFP-H6-FdU treatment (10.1 ± 1.0) was significantly higher (P = 0.03) than after free oligo-FdU (5.2 ± 0.9) treatment (Fig 3A and B). Moreover, increased DSB-positive cells led to higher antitumor activity, since the number of cell dead bodies, measured by Hoechst staining, which identify nuclear condensation or defragmentation, in tumor tissue 24 h after T22-GFP-H6-FdU injection was significantly (P = 0.03) higher (13.9 ± 0.5) than free oligo-FdU (7.1 ± 0.6), T22-GFP-H6 (3.0 ± 0.3), or Buffer (1.9 ± 0.4) treatment (Fig 3A and B). Following, we analyzed the fraction of CXCR4+ cancer cells (CXCR4+ CCF) remaining in tumor tissue, along time, after a single 100 μg T22-GFP-H6-FdU dose, as compared to free oligo-FdU, using the SC CXCR4+ SW1417 CRC model in NOD/SCID mice. Before treatment, both groups showed a similar CXCR4+ CCF in tumor tissue (Fig 4A and B); however, after T22-GFP-H6-FdU treatment, the CXCR4+ CCF was reduced at 24 h and reached its valley at 48 h (Fig 4A and B). In contrast, the CXCR4+ CCF in tumor tissue after an equimolecular dose of free oligo-FdU remained similar to its basal level along time. Taken together, these results indicate that T22-GFP-H6-FdU achieves selective biodistribution to tumor tissue and FdU delivery to target CXCR4+ cancer cells, a
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