A versatile drug delivery system targeting senescent cells
2018; Springer Nature; Volume: 10; Issue: 9 Linguagem: Inglês
10.15252/emmm.201809355
ISSN1757-4684
AutoresDaniel Muñoz‐Espín, Miguel Rovira, Irene Galiana, Cristina Giménez, Beatriz Lozano‐Torres, Marta Pàez‐Ribes, Susana Llanos, Selim Chaib, Maribel Muñoz‐Martín, Álvaro C. Ucero, Guillermo Garaulet, Francisca Mulero, Stephen G. Dann, Todd VanArsdale, David J. Shields, Andrea Bernardos, José Ramón Murguía, Ramón Martı́nez-Máñez, Manuel Serrano,
Tópico(s)RNA Interference and Gene Delivery
ResumoResearch Article16 July 2018Open Access Source Data A versatile drug delivery system targeting senescent cells Daniel Muñoz-Espín Corresponding Author Daniel Muñoz-Espín [email protected] orcid.org/0000-0002-0550-9514 Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain CRUK Cambridge Centre Early Detection Programme, Department of Oncology, Hutchison/MRC Research Centre, University of Cambridge, Cambridge, UK Search for more papers by this author Miguel Rovira Miguel Rovira orcid.org/0000-0002-1391-8465 Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Cellular Plasticity and Disease Group, Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Irene Galiana Irene Galiana Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Valencia, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain Search for more papers by this author Cristina Giménez Cristina Giménez Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Valencia, Spain Search for more papers by this author Beatriz Lozano-Torres Beatriz Lozano-Torres Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Valencia, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain Search for more papers by this author Marta Paez-Ribes Marta Paez-Ribes CRUK Cambridge Centre Early Detection Programme, Department of Oncology, Hutchison/MRC Research Centre, University of Cambridge, Cambridge, UK Search for more papers by this author Susana Llanos Susana Llanos Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Selim Chaib Selim Chaib Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Cellular Plasticity and Disease Group, Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Maribel Muñoz-Martín Maribel Muñoz-Martín Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Cellular Plasticity and Disease Group, Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Alvaro C Ucero Alvaro C Ucero Genes, Development and Disease Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Guillermo Garaulet Guillermo Garaulet Molecular Imaging Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Francisca Mulero Francisca Mulero Molecular Imaging Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Stephen G Dann Stephen G Dann Oncology R&D Group, Pfizer Worldwide Research & Development, Pfizer Inc., La Jolla, CA, USA Search for more papers by this author Todd VanArsdale Todd VanArsdale Oncology R&D Group, Pfizer Worldwide Research & Development, Pfizer Inc., La Jolla, CA, USA Search for more papers by this author David J Shields David J Shields Oncology R&D Group, Pfizer Worldwide Research & Development, Pfizer Inc., La Jolla, CA, USA Search for more papers by this author Andrea Bernardos Andrea Bernardos Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Valencia, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain Search for more papers by this author José Ramón Murguía José Ramón Murguía Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Valencia, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain Search for more papers by this author Ramón Martínez-Máñez Ramón Martínez-Máñez Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Valencia, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain Departamento de Química, Universitat Politècnica de València, Valencia, Spain Search for more papers by this author Manuel Serrano Corresponding Author Manuel Serrano [email protected] orcid.org/0000-0001-7177-9312 Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Cellular Plasticity and Disease Group, Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain Search for more papers by this author Daniel Muñoz-Espín Corresponding Author Daniel Muñoz-Espín [email protected] orcid.org/0000-0002-0550-9514 Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain CRUK Cambridge Centre Early Detection Programme, Department of Oncology, Hutchison/MRC Research Centre, University of Cambridge, Cambridge, UK Search for more papers by this author Miguel Rovira Miguel Rovira orcid.org/0000-0002-1391-8465 Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Cellular Plasticity and Disease Group, Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Irene Galiana Irene Galiana Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Valencia, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain Search for more papers by this author Cristina Giménez Cristina Giménez Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Valencia, Spain Search for more papers by this author Beatriz Lozano-Torres Beatriz Lozano-Torres Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Valencia, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain Search for more papers by this author Marta Paez-Ribes Marta Paez-Ribes CRUK Cambridge Centre Early Detection Programme, Department of Oncology, Hutchison/MRC Research Centre, University of Cambridge, Cambridge, UK Search for more papers by this author Susana Llanos Susana Llanos Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Selim Chaib Selim Chaib Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Cellular Plasticity and Disease Group, Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Maribel Muñoz-Martín Maribel Muñoz-Martín Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Cellular Plasticity and Disease Group, Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Alvaro C Ucero Alvaro C Ucero Genes, Development and Disease Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Guillermo Garaulet Guillermo Garaulet Molecular Imaging Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Francisca Mulero Francisca Mulero Molecular Imaging Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Stephen G Dann Stephen G Dann Oncology R&D Group, Pfizer Worldwide Research & Development, Pfizer Inc., La Jolla, CA, USA Search for more papers by this author Todd VanArsdale Todd VanArsdale Oncology R&D Group, Pfizer Worldwide Research & Development, Pfizer Inc., La Jolla, CA, USA Search for more papers by this author David J Shields David J Shields Oncology R&D Group, Pfizer Worldwide Research & Development, Pfizer Inc., La Jolla, CA, USA Search for more papers by this author Andrea Bernardos Andrea Bernardos Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Valencia, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain Search for more papers by this author José Ramón Murguía José Ramón Murguía Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Valencia, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain Search for more papers by this author Ramón Martínez-Máñez Ramón Martínez-Máñez Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Valencia, Spain CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain Departamento de Química, Universitat Politècnica de València, Valencia, Spain Search for more papers by this author Manuel Serrano Corresponding Author Manuel Serrano [email protected] orcid.org/0000-0001-7177-9312 Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Cellular Plasticity and Disease Group, Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain Search for more papers by this author Author Information Daniel Muñoz-Espín *,1,2,‡, Miguel Rovira1,3,‡, Irene Galiana4,5, Cristina Giménez4, Beatriz Lozano-Torres4,5, Marta Paez-Ribes2, Susana Llanos1, Selim Chaib1,3, Maribel Muñoz-Martín1,3, Alvaro C Ucero6, Guillermo Garaulet7, Francisca Mulero7, Stephen G Dann8, Todd VanArsdale8, David J Shields8, Andrea Bernardos4,5, José Ramón Murguía4,5, Ramón Martínez-Máñez4,5,9 and Manuel Serrano *,1,3,10 1Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain 2CRUK Cambridge Centre Early Detection Programme, Department of Oncology, Hutchison/MRC Research Centre, University of Cambridge, Cambridge, UK 3Cellular Plasticity and Disease Group, Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain 4Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Valencia, Spain 5CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain 6Genes, Development and Disease Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain 7Molecular Imaging Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain 8Oncology R&D Group, Pfizer Worldwide Research & Development, Pfizer Inc., La Jolla, CA, USA 9Departamento de Química, Universitat Politècnica de València, Valencia, Spain 10Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain ‡These authors contributed equally to this work *Corresponding author. Tel: +44 1223763337; E-mail: [email protected] *Corresponding author. Tel: +34 934020287; E-mail: [email protected] EMBO Mol Med (2018)10:e9355https://doi.org/10.15252/emmm.201809355 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 Senescent cells accumulate in multiple aging-associated diseases, and eliminating these cells has recently emerged as a promising therapeutic approach. Here, we take advantage of the high lysosomal β-galactosidase activity of senescent cells to design a drug delivery system based on the encapsulation of drugs with galacto-oligosaccharides. We show that gal-encapsulated fluorophores are preferentially released within senescent cells in mice. In a model of chemotherapy-induced senescence, gal-encapsulated cytotoxic drugs target senescent tumor cells and improve tumor xenograft regression in combination with palbociclib. Moreover, in a model of pulmonary fibrosis in mice, gal-encapsulated cytotoxics target senescent cells, reducing collagen deposition and restoring pulmonary function. Finally, gal-encapsulation reduces the toxic side effects of the cytotoxic drugs. Drug delivery into senescent cells opens new diagnostic and therapeutic applications for senescence-associated disorders. Synopsis Senescent cells are present in many diseases where they play an active pathological role. A common feature of senescent cells is their high content of lysosomes. Here, it is reported a pharmacological vehicle with lysosomal tropism that preferentially releases drugs into senescent cells. Drugs encapsulated with galacto-oligosaccharides (gal-encapsulation) are released into cells after digestion with lysosomal β-galactosidase and this happens more efficiently in senescent cells. After intravenous injection, gal-encapsulated drugs preferentially deliver their cargo into pathological tissues with high content of senescent cells. Gal-encapsulated doxorubicin ameliorates lung fibrosis in mice, reducing collagen and recovering normal breathing, and this is in contrast to free doxorubicin. When xenograft tumors in mice are treated with chemotherapy, a fraction of tumor cells undergo senescence, and concomitant treatment with gal-encapsulated doxorubicin results in full tumor regression. Gal-encapsulation prevents the exposure of non-pathological tissues to drugs and therefore reduces their associated toxicities, as it is shown for doxorubicin cardiotoxicity and for navitoclax-induced thrombocytopenia. Introduction Severe or unrepairable cellular damage often triggers a stereotypic cellular response known as senescence. This cellular program is controlled by relatively well-understood pathways that stably block cell proliferation and are conserved across vertebrates (Muñoz-Espín & Serrano, 2014). The main purpose of cellular senescence is to prevent the proliferation of damaged cells and, at the same time, to trigger tissue repair through the secretion of a complex mixture of extracellular factors, known as senescence-associated secretory phenotype or SASP. Specifically, senescence-initiated tissue repair involves the recruitment of inflammatory cells, the dismissal of senescent cells by phagocytic and immune cells, and the activation of stem/progenitor features in non-damaged surrounding cells (Krizhanovsky et al, 2008; Demaria et al, 2014; Yun et al, 2015; Mosteiro et al, 2016; Chiche et al, 2017; Ritschka et al, 2017). Even during vertebrate development, embryos use senescence in a damage-independent manner to initiate specific tissue remodeling processes (Muñoz-Espín et al, 2013; Storer et al, 2013). Upon persistent damage or during aging, senescent cells accumulate, probably due to an inefficient clearance by immune cells, and this accumulation may lead to chronic inflammation and fibrosis (Muñoz-Espín & Serrano, 2014). Indeed, evidence in mice indicates that the accumulation of senescent cells actively contributes to multiple diseases and aging (Muñoz-Espín & Serrano, 2014). In this regard, genetic ablation of senescent cells delays and ameliorates some aging-associated diseases, reverts long-term degenerative processes associated with chemotherapy, and extends longevity (Baker et al, 2016; Childs et al, 2016; Demaria et al, 2017). Importantly, senescent cells present vulnerabilities to particular small molecule inhibitors, known as "senolytics", that trigger apoptosis preferentially in senescent cells (Zhu et al, 2015). For example, the combination of dasatinib (a tyrosine kinase inhibitor of broad specificity) and quercetin (a flavonol with antioxidant and estrogenic activity) has preferential toxicity over senescent cells compared to control cells (Zhu et al, 2015). Also, the survival of senescent cells is highly dependent on elevated levels of the BCL-2 family of anti-apoptotic factors, and accordingly, senescent cells are hyper-sensitive to apoptosis induced by navitoclax, a BCL-2 family inhibitor (Chang et al, 2016; Yosef et al, 2016; Zhu et al, 2016; Pan et al, 2017). Similarly, inactivation of the transcription factor FOXA4 with a peptide derivative preferentially eliminates senescent cells over non-senescent ones (Baar et al, 2017). These pharmacological treatments reduce the number of senescent cells in vivo and show therapeutic activity against senescence-associated diseases and aging (Zhu et al, 2015, 2016; Chang et al, 2016; Roos et al, 2016; Yosef et al, 2016; Baar et al, 2017; Pan et al, 2017; Schafer et al, 2017). Senescent cells in vitro are characterized by high levels of lysosomal β-galactosidase activity, known as senescence-associated β-galactosidase (SAβGal; Dimri et al, 1995; Muñoz-Espín & Serrano, 2014; Kurz et al, 2000; Lee et al, 2006). In addition to β-galactosidase, senescent cells present high levels of most tested lysosomal hydrolases (Knaś et al, 2012). Indeed, senescent cells show a remarkable accumulation of lysosomes, together with abnormal endosomal traffic and autophagy (Cho et al, 2011; Narita et al, 2011; Ivanov et al, 2013; Udono et al, 2015; Tai et al, 2017). Interestingly, damaged or diseased tissues generally contain cells that are positive for SAβGal, while normal healthy tissues are negative for this marker (Sharpless & Sherr, 2015). Here, we have explored the possibility of using lysosomal β-galactosidase as a vulnerable trait of senescent cells that can be exploited to deliver tracers or drugs preferentially to diseased tissues with high content of senescent cells. Our approach is based on the encapsulation of diagnostic or therapeutic agents with β(1,4)-galacto-oligosaccharides and their delivery to lysosomes via endocytosis. We show that this delivery strategy is effective in vivo, and it is therapeutic in the context of cancer chemotherapy and also against pulmonary fibrosis. Moreover, our encapsulation system has the added value of reducing the systemic toxicities of cytotoxic drugs. Results Validation of gal-encapsulation to target senescent cells in vitro Sugar-coated beads (~100 nm diameter), based on a silica porous scaffold known as MCM-41 (Kresge et al, 1992), are efficiently internalized into cells by endocytosis, targeted to the lysosomes, and eventually released by exocytosis (Slowing et al, 2006, 2011; Tao et al, 2009; Bernardos et al, 2010; Hocine et al, 2010; Yanes et al, 2013; Aznar et al, 2016). In a previous work (Agostini et al, 2012), we designed beads pre-loaded with a fluorophore and then coated with a layer of galacto-oligosaccharides of mixed lengths (referred to as GosNP). We observed that β-galactosidase can digest the sugar coating of the beads, thus allowing the diffusion of the cargo out of the silica scaffold. Interestingly, fluorophore-loaded GosNP efficiently release their content within senescent cells, in agreement with the high levels of β-galactosidase activity of these cells (Agostini et al, 2012). Here, we have improved this system by using a homogeneous coating mostly consisting of a 6-mer galacto-oligosaccharide (referred to as GalNP) (Fig 1A and Appendix Fig S1A). Using this strategy, we have encapsulated rhodamine B [GalNP(rho)], doxorubicin [GalNP(dox)], and navitoclax [GalNP(nav)], and enzymatic digestion of the three types of beads with fungal β-galactosidase demonstrated efficient release of their respective cargos (Appendix Fig S1B–E). In general, 100 mg of drug is encapsulated per gram of beads and, upon digestion in vitro, ~30 mg of drug is released per gram of beads (Appendix Tables S1–S3). Targeting of senescent cells with GalNP(rho) was validated in three human cancer cell lines treated with palbociclib (a selective CDK4/CDK6 inhibitor approved in combination with letrozole or fulvestrant for hormone receptor+/HER2− metastatic breast cancer; Fig 1B and Appendix Fig S1F and G). Of note, the three cell lines used, SK-MEL-103, NCI-H226, and UT-SCC-42B, have an active retinoblastoma pathway (RB1-proficient; Ikediobi et al, 2006; Giefing et al, 2011; Massaro et al, 2017) and undergo senescence upon treatment with palbociclib (Fig 1B and Appendix Fig S1F). In these cellular models, the fluorophore was released more efficiently in senescent cells compared to control cells, demonstrating the functionality of the GalNP encapsulation method. Co-staining of cells with a lysosomal marker indicated that a substantial fraction of rhodamine was present in lysosomes (Appendix Fig S1H). Figure 1. Release of gal-encapsulated fluorophores in xenografts A. GalNP beads are based on a mesoporous silica scaffold (MCM-41) that can be loaded with different cargoes encapsulated by a coat of 6-mer β(1,4)-galacto-oligosaccharides. Cellular uptake of the GalNP beads occurs via endocytosis and, after fusion with lysosomal vesicles, the beads are released by exocytosis. The high lysosomal β-galactosidase activity of senescent cells allows a preferential release of the cargo by a β-galactosidase-mediated hydrolysis of the cap. B. SK-MEL-103 melanoma cells were treated with palbociclib (1 μM) for 1 week, and senescence induction was assessed by SAβgal staining. Next, cultures were exposed to GalNP(rho) (50 μg/ml, for 16 h). Pictures show representative images illustrating rhodamine release by confocal microscopy. Cells were co-stained with Calcein, and nuclei were stained with Hoechst. Graphs to the right show the rhodamine intensity relative to cell surface in senescent cells and non-senescent (control) cells. Each assay was repeated at least three times with similar results. Scale bar: 50 μm. C. Subcutaneous tumor xenografts of SK-MEL-103 melanoma cells in athymic female nude mice. Upon tumor formation, mice were treated daily with palbociclib (oral gavage, 100 mg/kg) during 7 days. The left panel picture shows representative whole tissue portions of tumors after SAβGal staining. The right panel shows sections of control and palbociclib-treated tumors processed for SAβGal staining, and Ki67 and phosphorylated Rb (p-Rb) immunohistochemistry. This experiment has been repeated at least two times with similar results. Scale bar: 50 μm. D. Mice bearing SK-MEL-103 xenografts, control or treated with palbociclib for 7 days, as in (C), were tail vein injected with 200 μl of a solution containing GalNP(rho) (4 mg/ml). At 6 h post-injection, mice were sacrificed, tumors were collected, and fluorescence was analyzed by an IVIS spectrum imaging system. The graph indicates the average difference in tumor radiance between GalNP-injected control and palbociclib-treated groups. The inset shows the absolute values of radiance (p/s/cm2/sr × 106) for each group. The corresponding differences are highlighted in black or red. Values are expressed as mean ± SD, and statistical significance was assessed by the two-tailed Student's t-test. Download figure Download PowerPoint Release of gal-encapsulated fluorophores in xenografts To evaluate the release of gal-encapsulated fluorophores in vivo, we employed tumor xenografts treated with senescence-inducing chemotherapy. Subcutaneous xenografts were generated using SK-MEL-103 melanoma cells and NCI-H226 lung squamous carcinoma cells. Upon tumor formation, mice were treated daily with palbociclib for 7 days, and this resulted in high levels of intratumoral senescence, as inferred from elevated SAβGal activity, absence of the proliferative marker Ki67, and reduction in phosphorylated Rb (Fig 1C). In a first approach, palbociclib-treated mice carrying SK-MEL-103 xenografts were given a single intravenous injection of GalNP(rho) and fluorescence was analyzed 6 h later. Palbociclib-treated tumors were strongly autofluorescent (Fig 1D). Importantly, however, rhodamine was detectable above background in mice treated with GalNP(rho), and the signal attributed to rhodamine was higher in tumors treated with palbociclib compared to non-treated tumors (Fig 1D). Fluorescence was not detected in other organs at 6 h post-injection, including liver, spleen, and lungs (Appendix Fig S1I and see below Fig 2B). To avoid the detection of autofluorescence, we used beads loaded with indocyanine green (see Appendix Fig S1E), a fluorophore that emits in the far-red spectrum and therefore is minimally affected by the autofluorescence of palbociclib-senescent cells. Confirming the rhodamine release data, SK-MEL-103 and NCI-H226 xenografts treated with palbociclib and gal-encapsulated indocyanine green showed much higher fluorescence compared to tumors treated with a single agent alone (Appendix Fig S1J and K). Figure 2. Release of gal-encapsulated fluorophores in fibrotic lungs A. C57BL/6 male mice were subjected to a single intratracheal administration bleomycin (1.5 U/kg) and analyzed 2 weeks later. Pictures at the left correspond to representative lungs after whole tissue SAβGal staining. The right panel shows sections of control and bleomycin-treated lungs processed for SAβGal and Masson's trichrome staining to detect collagen fibers (stained in blue). Scale bar: 50 μm. B. Control and bleomycin-treated mice, as in (A), were tail vein injected with 200 μl of a solution containing GalNP(rho) (4 mg/ml). At 6 h post-injection, mice were sacrificed and the lungs were analyzed by an IVIS spectrum imaging system, as in Fig 1D. The graph indicates the average difference in lung radiance between GalNP-injected control and bleomycin-treated groups. The inset shows the absolute values of radiance (p/s/cm2/sr × 106) for each group. The corresponding differences are highlighted in black or red. Values are expressed as mean ± SD, and statistical significance was assessed by the two-tailed Student's t-test. C. Control and bleomycin-treated mice were injected with GalNP(rho), as in (B), and lung sections were analyzed 6 h post-injection by confocal microscopy. Pictures correspond to representative sections of control and bleomycin-treated lungs. The graph shows % of rhodamine+ cells in fibrotic areas from bleomycin-treated mice compared to normal lung tissue from control mice. Values are expressed as mean ± SD, and statistical significance was assessed by the two-tailed Student's t-test. Scale bar: 25 μm. D. Lung cell suspensions from control and bleomycin-treated mice, as in (B), were analyzed by flow cytometry. The upper panels show representative dot plots of rhodamine staining in CD45−CD31− cells. The gating strategy is shown in detail in Appendix Fig S2C using as example the panel corresponding to the bleomycin-treated lung shown here. Values in boxes correspond to the mean ± SEM, and statistical significance was assessed by the two-tailed Student's t-test. The lower panels show representative dot plots of rhodamine+ cells (after exclusion of CD45+ and CD31+ cells) separated in EpCAM− (fibroblasts) and EpCAM+ (epithelial cells) subpopulations. Values in boxes correspond to the mean ± SEM of these two populations. Statistical significance between the fibroblast:epithelial ratio of positivity was assessed by the two-sided Fisher exact test (P = 0.008). E. GSEA plots of published signatures of SASP and SIR (senescence-inflammatory response) (Lasry & Ben-Neriah, 2015) against the ranked list of differential expression between Rho+ and Rho− cells (all CD45−CD31−) from bleomycin-treated mice (n = 3), at 2 weeks post-bleomycin, as in (D). Download figure Download PowerPoint Release of gal-encapsulated fluorophores in pulmonary fibrosis Cellular senescence is abundant in pulmonary fibrosis, both in humans and in mice, and actively contributes to the pathological manifestations of this disease (Aoshiba et al, 2003, 2013; Hecker et al, 2014; Pan et al, 2017; Schafer et al, 2017). We wondered whether our senescence delivery system would also work in a mouse model of pulmonary fibrosis. Intratracheal instillation of bleomycin in mice produced full-blown lung fibrosis in a period of 2 weeks, accompanied by focal areas of SAβGal activity and strong collagen deposition as indicated by Masson's trichrome staining (Fig 2A). Two weeks post-bleomycin administration, mice were intravenously injected with GalNP(rho) and 6 h later fluorescence was measured in the lungs. In this in vivo senescence model, autofluorescence was less prominent than in the case of palbociclib-treated tumors. Importantly, rhodamine release occurred preferentially in fibrotic lungs compared to healthy lungs (Fig 2B). Moreover, confocal microscopy indicated that Rho+ cells were more abundant in fibrotic lung lesions compared to non-fibrotic lungs (Fig 2C). The differential fluorescence observed between fibrotic and healthy lungs could conceivably reflect, a
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