FULL PAPER Functional Amyloids www.advancedscience.com Targeting Antitumoral Proteins to Breast Cancer by Local Administration of Functional Inclusion Bodies Mireia Pesarrodona, Toni Jauset, Zamira V. Díaz-Riascos, Alejandro Sánchez-Chardi, Marie-Eve Beaulieu, Joaquin Seras-Franzoso, Laura Sánchez-García, Ricardo Baltà-Foix, Sandra Mancilla, Yolanda Fernández, Ursula Rinas, Simó Schwartz Jr, Laura Soucek, Antonio Villaverde,* Ibane Abasolo, and Esther Vázquez* 1. Introduction Two structurally and functionally unrelated proteins, namely Omomyc and p31, are engineered as CD44-targeted inclusion bodies produced in Bacterial inclusion bodies (IBs) are insol- recombinant bacteria. In this unusual particulate form, both types of protein uble and discrete particles highly enriched by a single protein species, which deposits materials selectively penetrate and kill CD44+ tumor cells in culture, and upon as interdigitated amyloidal fibers and nona- local administration, promote destruction of tumoral tissue in orthotropic myloidal protein forms.[1] They organize mouse models of human breast cancer. These findings support the concept into porous fibrilar networks[2] that confer of bacterial inclusion bodies as versatile protein materials suitable for mechanical stability, in which native or application in chronic diseases that, like cancer, can benefit from a local slow quasinative protein species are embedded.[3] release of therapeutic proteins. These protein clusters, ranging between the nano- and micro-scales, are built up in Dr. M. Pesarrodona,[+] Dr. L. Sánchez-García, Prof. A. Villaverde, Z. V. Díaz-Riascos, S. Mancilla, Y. Fernández, Dr. I. Abasolo Dr. E. Vázquez Functional Validation & Preclinical Research Institut de Biotecnologia i de Biomedicina CIBBIM-Nanomedicine Universitat Autònoma de Barcelona Vall d'Hebron Institut de Recerca (VHIR) Bellaterra, 08193 Barcelona, Spain Universitat Autònoma de Barcelona E-mail: antoni.villaverde@uab.cat; Esther.Vazquez@uab.cat 08035 Barcelona, Spain Dr. M. Pesarrodona, Z. V. Díaz-Riascos, Dr. L. Sánchez-García, Z. V. Díaz-Riascos, Dr. J. Seras-Franzoso, R. Baltà-Foix, S. Mancilla, S. Mancilla, Y. Fernández, Prof. S. Schwartz Jr, Prof. A. Villaverde, Y. Fernández, Prof. S. Schwartz Jr, Dr. I. Abasolo Dr. I. Abasolo, Dr. E. Vázquez Drug Delivery & Targeting CIBBIM-Nanomedicine CIBER de Bioingeniería Vall d’Hebron Institut de Recerca (VHIR) Biomateriales y Nanomedicina (CIBER-BBN) Universitat Autònoma de Barcelona C/ Monforte de Lemos 3-5, 28029 Madrid, Spain 08035 Barcelona, Spain Dr. T. Jauset,[++] Dr. M.-E. Beaulieu,[++] Prof. L. Soucek Dr. A. Sánchez-Chardi Vall d’Hebron Institute of Oncology (VHIO) Departament de Biologia Evolutiva Edifici Cellex Ecologia i Ciències Ambientals Hospital Vall d’Hebron Facultat de Biologia 08035 Barcelona, Spain Universitat de Barcelona Dr. T. Jauset, Dr. M.-E. Beaulieu, Prof. L. Soucek Av. Diagonal 643, 08028 Barcelona, Spain Peptomyc S.L. Dr. L. Sánchez-García, Prof. A. Villaverde, Dr. E. Vázquez Edifici Cellex Departament de Genètica i de Microbiologia Hospital Vall d’Hebron Universitat Autònoma de Barcelona 08035 Barcelona, Spain Bellaterra, 08193 Barcelona, Spain Prof. U. Rinas The ORCID identification number(s) for the author(s) of this article Leibniz University of Hannover can be found under https://doi.org/10.1002/advs.201900849. Technical Chemistry and Life Science Callinstr. 5, 30167 Hannover, Germany © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Prof. U. Rinas Weinheim. This is an open access article under the terms of the Creative Helmholtz Centre for Infection Research Commons Attribution License, which permits use, distribution and Inhoffenstraße 7, 38124 Braunschweig, Germany reproduction in any medium, provided the original work is properly cited. Prof. L. Soucek [+]Presentaddresses: Institute for Research in Biomedicine (IRB Barcelona), Institució Catalana de Recerca i Estudis Avançats (ICREA) The Barcelona Institute of Science and Technology, 08028 Barcelona, Spain 08010 Barcelona, Spain [++]Peptomyc S.L., Edifici Cellex, Hospital Vall d’Hebron, 08035 Prof. L. Soucek Barcelona, Spain Department of Biochemistry and Molecular Biology Universitat Autònoma de Barcelona DOI: 10.1002/advs.201900849 Bellaterra, 08193 Barcelona, Spain Adv. Sci. 2019, 6, 1900849 1900849 (1 of 9) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.advancedscience.com bacterial cells upon expression of a recombinant gene encoding formed by a growth hormone[8]), through appropriate signaling. the IB protein. Upon purification from bacteria, they behave as Whether and how IB proteins released in vivo can penetrate mechanically stable biomaterials,[4] easy to be handled in a diver- neighboring cells and interact with intracellular circuits for sity of platforms, presentations and interfaces. In this regard, IBs biological manipulation and therapeutic impact remains poorly have been explored in the context of tissue engineering as soft explored despite its obvious pharmacological interest. topographies[4] since they are nontoxic to mammalian cells.[5] Being partially composed of functional polypeptides,[6] IB enzymes have found a role in industrial applications as self- 2. Results and Discussion immobilized catalysts.[7] In addition, IBs tend to attach and penetrate mammalian cells without any deleterious effect,[8] via To investigate this issue, we have here designed two mod- macropynocytosis.[9] Since part of the functional IB protein is ular proteins formed by two functional polypeptides (p31 released under physiological conditions (namely cell culture and Omomyc, respectively) involved in cell cycle regulation, media, intracellular environment upon uptake, or in organic with therapeutic potential in antitumoral therapies as tumor- tissues),[9] IBs act as slow release protein platforms, potentially targeting agents. The p31 protein consists of the C-terminal useful in therapeutic approaches (at both cell or organism levels) fragment of p130cas that has shown the ability to promote for protein replacement therapies or protein drug delivery.[8,10,11] apoptosis by disassembling focal adhesion complexes.[16,17] If the IB protein is targeted to a biological marker, for instance, The Omomyc protein is a Myc dominant negative[18] exten- through the incorporation of a tumor-homing peptide, IBs sively validated transgenically and pharmacologically as an can remotely deliver IB proteins through the bloodstream to antitumoral agent.[19–21] Both proteins were tagged with the tumoral tissues upon subcutaneous administration.[12] The tumor-homing peptide FNI/II/V (FN) which binds CD44, a bulk IB material, however, is partially stable in the adminis- glycoreceptor that promotes extracellular cell adhesion through tration site and it remains detectable for weeks long.[13] Then, hyaluronic acid interaction. CD44 is a well-recognized tumoral once implanted, IBs act as local protein depots, mimicking the marker, associated to tumor progression and metastasis,[22,23] natural amyloid repositories of human hormones in the endo- and that has been used to identify, along with low expression crine system,[14] found them listed among examples of the of CD24, the cancer stem cell population in breast cancer.[24] expanding catalogues of nontoxic functional amyloids.[15] In this Therefore, the use of the tumor-homing peptide FN seemed context, extracellular activities of IB proteins, either attached to appropriate to test targeted nanodepots in a tumor type, the IBs or upon release, are responsible for downregulation of cell triple negative breast cancer, that currently lacks targeted thera- surface receptor expression (by the display of a peptidic ligand pies.[25] The modular proteins FN-p31-H6 and Omo-FN-H6 of a cytokine receptor[13]) or for enhanced cell growth (when (Figure 1A) were produced in Escherichia coli as proteolytically Figure 1. Proteins and protein materials. A) Schematic representation of the fusion proteins FN-p31-H6 and Omo-FN-H6, indicating the molecular mass of the products. Control proteins used in the study (FN-GFP-H6 and GFP-H6,[26]) are also included. Box sizes are only approximate. B) Coomassie blue staining of a SDS-PAGE gel loaded with purified proteins. Numbers on the left indicate the molecular masses in kDa of the ladder marker. On the right, arrows indicate the position of the full-length recombinant proteins. M indicates the molecular marker line, and p31 and O indicate FN-p31-H6 and Omo-FN-H6 lanes, respectively. C) Internalization of FN-GFP-H6 in cultured CD44+ MDA-MB-231 and CD44− HepG2 cells, measured as the % of green fluorescent cells. Protein was added at 0.5 × 10−6 m and exposed to cells for 24 h. The percentage of CD44+ cells in each cell line is also indicated as a reference. D) Representative FESEM images of FN-p31-H6 and Omo-FN-H6 IBs at two different magnifications (zoom in the insets). Magnifica- tions are equivalent in each micrograph pair to allow comparative visualization. Magnification bars represent 500 nm. Adv. Sci. 2019, 6, 1900849 1900849 (2 of 9) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.advancedscience.com stable full-length polypeptides (Figure 1B). FN-GFP-H6[26] was longer exposure time, Omomyc IBs showed the highest effi- also produced as a fluorescent reporter, and this protein was cacy already at 48 h post exposure, indicating a faster mecha- found able to penetrate into 55.6% of CD44+ MDA-MB-231 nism of action (Figure 2B). Similar cytotoxic effects of p31 and cells after 24 h of exposure, but not into CD44− HepG2 cells Omomyc IBs on cell viability were observed in multiple CD44+ (7.4%, Figure 1C). This data fully supported the CD44 tar- cell lines,[23] reaching slightly different extents probably due to geting of the FN segment and the further exploration of FN- intrinsic differences in cell survival mechanisms, while GFP- empowered IBs as CD44-targeted agents. Upon production, based IBs remained nontoxic (Figure 2C). Microscopy images an important population of FN-p31-H6 and Omo-FN-H6 was of MDA-MB-231 cultured cells exposed to active and nonac- found in the insoluble cell fraction, aggregated in the form of tive materials, fully confirmed the cytotoxicity promoted by IBs (Figure 1D). After purification from bacterial cell extracts, functionalized IBs (Figure 2D). Indeed, functional IBs caused p31 and Omomyc IBs were clearly distinguishable in shape a clear reduction in tumor sphere size when compared to the and morphology. While the first ones organized in a rod-shape GFP ones. architecture (≈200 × 1000 nm) with a smooth surface, as previ- Since both p31 and Omomyc perform their activities intra- ously described for some nonconventional IBs,[27] Omomyc IBs cellularly, cell uptake of IBs can be inferred from the cytotox- were smaller (≈200 × 500 nm), exhibiting a rough surface and icity exhibited by these materials on cultured cells, in contrast an ellipsoid geometry that is much more common among bac- with the absence of effect shown by control GFP IBs (Figure 2). terial IBs.[28] However, to effectively assess the cell penetrability of cytotoxic To first explore the potential of these IBs to impact tumor IBs, these materials were labeled with AF647. When MDA-MB- cell biology, cultured breast cancer human MDA-MB-231 231 cells were exposed to labeled IBs, intracellular fluorescence cells overexpressing CD44[24,29] were exposed to either p31 or increased in a dose-dependent fashion when cells were exposed Omomyc IBs and appropriate GFP controls. Both p31 and to FNI/II/V functionalized IBs (Figure 3A). However, GFP-H6 Omomyc IBs showed a dose-dependent cytotoxic effect on cell IBs, which lack a cell ligand, did not accumulate in CD44 target viability, not observed when exposing cells to GFP-based IBs cells (Figure 3A). This confirms that cell targeting of IBs for (Figure 2A). The lack of toxicity associated to exposure to GFP specific protein delivery is a feasible concept, not restricted to IBs fully confirmed the protein-associated toxicity of IBs, as the the CXCR4-binding T22 peptide, in which this event has been IB material, per se, is not intrinsically cytotoxic.[1] Moreover, recently described for the first time.[30] Internalization efficacy while p31 IBs showed an increasing cell killing potential with of p31- and Omomyc- based IBs was comparable (Figure 3A,B) Figure 2. Biological impact of IBs on cultured cells. A) Dose-dependent loss of MDA-MB-231 cell viability upon exposure to IBs for 96 h. At 9 × 10−6 m, background reduction of cell viability was observed in cells exposed to GFP-based IBs (not shown). B) Time-dependent loss of MDA-MB-231 cell viability upon exposure to IBs (3 × 10−6 m). C) Killing of different CD44+ cell lines by cytotoxic IB (3 × 10−6 m), upon exposure for 96 h. D) Representa- tive bright field images of MDA-MB-231 tumor-spheres upon addition of 3 × 10−6 m protein and further incubation for 72 h. Qualitative assessment of sphere morphology and integrity when challenged with p31 and Omomyc IBs, compared to an equivalent GFP construct. Magnification bars represent 100 µm. One-way ANOVA and a post-hoc Dunnett test was performed comparing all groups to PBS-treated control cells. p > 0.05 (n.s.); p < 0.05 (*); p < 0.01 (**); p < 0.001 (***). Adv. Sci. 2019, 6, 1900849 1900849 (3 of 9) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.advancedscience.com Figure 3. Cell penetration of CD44-targeted IBs. A) Quantification of protein internalization by fluorescence was quantified with flow cytometry into MDA-MB-231 cells incubated for 24 h at increasing concentration of IBs. B) Kinetics of IB penetration into MDA-MB-231 cells exposed to 3 × 10−6 m IBs. C) Representative confocal microscopy images of protein internalization in MDA-MB-231 after 24 h treatment with 3 × 10−6 m of IBs. Nuclei and membrane cells were labeled with Hoechst (blue) and WGA (light grey) respectively. Magnification bars represent 5 µm. and with similar intracellular distribution (Figure 3C). Further- was detected up to 7 d in the injection site, without apparent more, FNI/II/V-functionalized IBs accumulated inside target loss of emission (Figure 5A), confirming the stability of the pro- cells overtime, a fact that is compatible with moderate or absent tein particles. In this context, p21 overexpression (a cell cycle lysosomal degradation (Figure 3B). In this regard, the confocal regulator usually associated to growth arrest and/or senes- analyses of the internalizing material revealed a punctuated flu- cence) was also clearly observed by immunodetection in tumors orescence pattern indicative of an endosomal route of internali- treated with FN-p31-H6 IBs (Figure 5B). Since the nuclear zation, and a perinuclear localization of most of the engulfed overexpression of p21 has been associated to the triggering of protein particles that was more evident in the case of p31 IBs apoptotic cascades through the blocking of cyclin dependent (Figure 3C). kinases,[31] the presence of apoptotic cells was assessed in BT-20 Because of the high penetrability capacity of CD44-targeted tumors upon single dose administration of FN-p31-H6 IBs, IBs in cultured cells, we decided to evaluate their potential since these cells were the ones with the highest cell growth macroscopic effect in vivo, in mouse cancer models. For a first inhibition in vitro (Figure 2C). As observed in Figure 5C, a sig- screening of antitumoral effect, we chose to test Omomyc IBs, nificant increase in apoptotic cell bodies was clearly visible in which had shown superior cytotoxicity in vitro compared to FN- FN-p31-H6 IBs treated tumors when compared with control p31-H6. Omomyc IBs were injected intratumorally in an ortho- animals, and overexpression of p21 was confirmed by Western topic animal model of human breast cancer and the biological Blot in BT20 cells exposed to both IBs (Figure 5D). impact of the administered materials on the tumor progres- In summary, self-assembling proteins and protein mate- sion was evaluated in repeated dose administration (weekly) rials are of emerging interest because of their intrinsic lack of for 4 weeks. Although the general tumor volume measured toxicity, biodegradability, functional and structural versatility, by caliper was not significantly affected during the test period and because of their suitability for biological fabrication in a (Figure 4A), the proportion of necrotic tumors (Figure 4B) was diversity of microbial cell factories.[32] Some of them, such as clearly higher in the group of animals receiving Omomyc IBs the bovine α-lactalbumin[33] or the hen egg white lysozyme,[34] compared to those in control groups (Figure 4C). This obser- have already proved their utility as the basis of wide spectrum vation indicated destruction of tumoral tissue, which, although antitumoral drugs. Among the diversity of applications of not reflected by any significant change in tumor volume, protein constructs, bacterial IBs have found different prom- was representative of a relevant biological effect. Hence, the ising niches in biomedical research,[35] as delivery systems detected antitumoral effect was indicative of the functional IBs of therapeutic proteins[8,11] or as convenient agents in immu- having an impact on the biology of tumoral target cells. noprophylaxis and immunotherapies.[36] The utility of this In parallel, we continued the characterization of p31-based platform is based on the high mechanical stability of IBs IBs assessing their behavior in in vivo conditions. When these combined with the ability of the particles to release functional IBs were administered in a single dose, the fluorescent material proteins under physiological conditions both in vitro and in Adv. Sci. 2019, 6, 1900849 1900849 (4 of 9) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.advancedscience.com polypeptides with therapeutic value aimed to the local or generic treatment of chronic diseases. A critical step in this direction would be to generate a proof of concept of the healing potential of the biomaterials in clinical contexts. In this regard, we have here proved for the first time that two recombinant proteins with recognized antitumoral effects, empowered with a potent ligand of the tumoral marker CD44 (Figure 1), are able to significantly impact tumor cell biology (Figures 2,4, and 5) through the internalization of these biomaterials in target cells, both in vitro in cell culture (Figure 3) and in vivo in entire organisms (Figure 5). Indeed, we have dem- onstrated here that upon efficient inter- nalization, engineered versions of p31 and Omomyc, formulated as CD44-targeted IBs, promote selective target cell death through their respective impacts in regulatory cell circuits. Omomyc anti-tumor activity is based on its direct interference with Myc func- tion. Myc has been shown to be involved in multiple aspects of tumorigenesis,[39] both at the intracellular and extracellular level, being responsible for cell division, increased cell metabolism, immune tolerance, and survival to treatment of tumor cells. The benefit of inhibiting Myc has been demon- strated by multiple studies,[19] which indi- cated that interfering with Myc function does not only lead to cell growth arrest, but also to energetic crisis, anti-tumor immune reprogramming, and cancer cell death. Omomyc has been instrumental in demon- strating this therapeutic opportunity, first as Figure 4. Antitumoral effect of IBs. A) Effect of Omomyc-based IBs on tumor growth. Balb/c a transgene[20] and more recently as a thera- nude female mice bearing orthotopic tumors of MDA-MB-231 cell line were divided in 5 groups (n = 7–8) and treated intratumorally (i.t.) once a week. One group was treated with Omomyc peutic polypeptide,[40] while also demon- IBs. GFP-based IBs and PBS were included as treatment controls. No significant effect on strating the complete safety of the approach tumor volume between the treatment groups was detected. B) Tumor necrosis was also evalu- for normal tissues. The potential use of ated at the experiment end point. Representative images are shown. C) The percentage of Omomyc as a therapeutic mini-protein has necrotic tumors for the groups treated with either IBs was measured. Omomyc IBs treated mice only emerged recently[40,41] and, to date, this showed an increase in the number of necrotic tumors compared to GFP IB-treated animals. is the first and only report of its application in the IB format, which establishes the fea- vivo in cell culture media,[37] the intracellular environment[9] sibility of its topical administration by intratumoral injection or as locally administered in organs or tissues of entire organ- directly to the tumor site. isms.[13] Both the mechanical stability and capability of pro- P31, so far neglected regarding its potential uses as a thera- tein release are generic IB properties[38] supported by the peutic agent, has been directly linked to the control of p21 expres- particular sponge-like architecture (combining amyloidal and sion and pro-apoptotic activity, especially in triple negative breast nonamyloidal protein forms) of IBs.[3] Protein released from cancers (TNBC).[42] The present study proves that P31 is packable subcutaneous IBs can reach the bloodstream and be remotely as IBs while keeping relevant antitumoral functionalities. This is delivered to target organs, provided the IB protein incorpo- not only pertinent regarding the incorporation of this protein to rates appropriate ligands for cell surface receptors.[12] In addi- the growing catalogue of putative protein drugs, but also because tion, IBs are also nontoxic in vitro when exposed to cultured it indicates that the engineering of antitumoral IBs is a general mammalian cells[4] or upon systemic administration (i.e., concept not restricted to a specific polypeptide. Importantly, the by oral delivery).[8] Therefore, these materials are promising IB format itself is nontoxic, as CD44-targeted GFP IBs do not as implantable protein depots for the release of functional cause any deleterious effects over exposed cells (Figure 2). Adv. Sci. 2019, 6, 1900849 1900849 (5 of 9) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.advancedscience.com Figure 5. In vivo biodistribution and efficacy of FN-p31-H6 IBs. A) Mice bearing orthotopic tumors of HCC1806 cell line were treated intratumorally (i.t.) with p31 IBs labeled with the AF647 fluorochrome. In vivo imaging was performed with IVIS-Spectrum 0, 4, and 7 days after administration (left panel) and quantified (right top panel). No fluorescence was observed outside the tumors (not shown). Ex vivo imaging (right bottom panel) confirmed that fluorescence was retained in the tumors, with higher intensities at the site of injection. B) Representative images of p21 protein expression by immunohistochemistry in tumors of animals treated i.t. with control GFP or p31 IBs, showing that only p31 containing IBs were able to induce p21 expression, in BT-20 orthotropic tumors. Quantitative evaluation of the stained section confirmed that p21 staining was significantly higher in tumors of p31 IBs-treated mice (right panel). C) Representative images of Hoechst staining of control FN-GFP-H6-treated and p31 IBs-treated mice. Arrows indicate the presence of characteristic apoptotic nuclei. Quantification of apoptotic nuclei in control and FN-p31-H6 treated tumors indicated that apoptosis was significantly increased upon treatment with p31 IBs (right panel). Magnification bars in (B,C) represent 100 and 50 µm, respectively. p < 0.01 (**); p < 0.001 (***). D) p21 expression in BT20 cells treated for 4 h with FN containing constructs. 3. Conclusions The FNI/II/V domain was placed at the N- or C-terminus of p31[17] and Omomyc-containing modular protein respectively, considering Since the biological production of IBS is cost-effective, using preliminary results which showed no impact on protein activity. The amino this material appears to be a convenient way of local adminis- acid sequences of the fusion proteins are MWQPPRARITGYIIKYEKP­ GSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKK tration of protein drugs. Considering the increasing spectrum TGGSSRSSSGQYENSEGGWMEDYDYVHLQGKEEFEKTQKELLEKGSI of therapeutic proteins already approved or under development TRQGKSQL­E LQQLKQFERLEQEVSRPIDHDLANWTPAQPLAPGRTG­ for cancer therapies, most of them produced by recombinant GLGPSDRQLLLFYLEQCEANLTTLT­NAVDAFFTAVATNQPPKIFVAHSKFV DNA technologies,[43] their potential packaging as IBs might ILSAHKLVFIGDTLSRQAKAADVRSQVT­HYSNLLCDLLRGIVATTKAAALQ largely expand their spectrum of activities and applicability in YPSPSAAQDMVERVKELGHSTQQFRRVLGQLAAAGGSSRSSSKHHHHHH innovative and personalized medicines, for oncology and in (FN-p31-H6) and MTEENVKRRTHNVL­ERQRRNELKRSFFALRDQIPELENNEK APKVVILKKATAYILSVQAETQKLISEIDLL­RKQNEQLKHKLEQLRNSCAGGSSRSS other fields of human or animal clinics. SWQPPRARITGYIIKYEKPGSPPREVVPRPRPG VTEATITGLEPGTEYTIYVIALK­ NNQKSEPLIGRKKTGGSSRSS­ SKHHHHHH (Omo-FN-H6) respectively. FN-p31-H6 and Omo-FN-H6 genes, together with the biologically 4. Experimental Section inactive control gene FN-GFP-H6 and nontargeted GFP-H6 control gene, were provided by GenScript (Hong Kong, China). Recombinant proteins Protein Design, and IB Production and Purification: Two modular were produced in E. coli strain BL21 (DE3), except Omo-FN-H6 that was proteins were designed to contain the CD44 ligand FNI/II/V fused to produced in BL21 pLys cells. Protein production was usually induced at either p31 (the C-term fragment of p130cas) or Omomyc (the Myc- 37 °C for 3 h upon 1 × 10−3 m IPTG addition. FN-GFP-H6 protein was derived bHLHZip domain mutant), appended with a His-tag (Figure 1). produced overnight at 16 °C. Bacterial IBs were purified upon protein Adv. Sci. 2019, 6, 1900849 1900849 (6 of 9) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.advancedscience.com production; cells were incubated 2 h at 37 °C with a Protease Inhibitor with a Plan Apo 63 × /1.4 (oil HC × PL APO l blue) objective. Hoechst Complete tablet, 10 × 10−3 m PMSF and 1 µg mL−1 lysozyme. Then, 33 342, WGA 594 and Alexa Fluor 647 were excited by a blue diode cultures were treated with 0.1% Triton X-100 for 1 h at room temperature (405 nm), a helium–neon laser (594 nm) and a helium–neon laser (RT). Cycles of freeze/thaw were applied daily to destroy remaining (633 nm) respectively. Z-series were collected at 0.5 mm intervals. bacteria until reaching CFU mL−1 < 10−2. Samples were incubated with Images were processed using Imaris version 6.1.0 software (Bitplane, 0.02% NP-40 for 1 h at 4 °C followed by 1 h incubation with 1 µg mL−1 Zürich, Switzerland). DNAse at 37 °C. IBs obtained after centrifugation at 15 000 g were Flow Cytometry: MDA-MB-231 cells were cultured on a 24 well plate washed twice with PBS, separated in 1 mL aliquots and stored at −80 °C at 80 000 c/well and incubated at 37 °C and 5% CO2 in a humidified in PBS. A milder purification protocol was applied for GFP-H6 IBs atmosphere for 24 h. Cells were incubated with IBs at increasing isolation. After the incubation with PMSF and lysozyme, cells were concentrations and incubation times (internalization kinetics). After lysed using a French Press at 1200 psi. Lysates were treated with 0.1% protein incubation, cells were treated with 1 mg mL−1 trypsin for 15 min Triton X-100 for 1 h at RT followed by 1 h incubation with 1 µg mL−1 to detach cells and remove unbound protein, followed by the addition of DNAses at 37 °C. Cycles of freeze/thaw were applied daily until reaching complete medium and centrifugation at 1400 g for 5 min. Collected cells CFU mL−1 < 10−2. IBs were centrifuged, washed twice in PBS and stored were then resuspended in DPBS. Protein internalization was analyzed at −80°C in PBS in 1 mL samples. using a FACS-Canto system (Becton Dickinson, Franklin Lakes, NJ, IB Protein Quantification: Serial dilutions of 1 mL IB samples were USA), with a 15 W air-cooled red diode laser at 635 nm excitation. Alexa prepared in water with Laemmli buffer and incubated at 98 °C for 45 min. IB fluor 647 emission was measured with an FL4 detector (661/16 nm dilutions together with a soluble GFP-H6 standard of known concentration band pass filter). CD44-positive cells were determined as described.[45] were loaded on SDS PAGE. Protein quality was analyzed by Coomassie Human Cancer Animal Models: All the animal studies were performed blue staining, while protein quantification was calculated by Western Blot in accordance with the ARRIVE guidelines and the 3 Rs (rules of using an anti-His antibody (sc57598, Santa Cruz Biotechnology, Santa Replacement, Reduction, and Refinement). Mice were housed and Cruz, CA, USA). Protein bands were quantified from the standard curve treated following the protocols approved by the Ethical Committee fitting equation of GFP-H6 using the Quantity One software. for the Use of Experimental Animals (CEEA) at the Vall d’Hebron Cell Viability Assay: Cell viability of breast cancer cell lines (MDA-MB- Research Institute (VHIR), Barcelona. MDA-MB-231 cells, classified as 231, MDA-MB-468, BT-20, BT549, and HCC1187) upon treatment with mesenchymal-like,[46] were resuspended in cold PBS at 15 000 000 c mL−1 IBs was measured by the MTT metabolic test (Roche, Basel, Switzerland) and maintained on ice. Before surgery, mice were anesthetized with following manufacturer recommendations. MDA-MB-231, MDA-MB- 2% isoflurane and buprenorphine (0.75 mg kg−1) was administered 468, BT-20, BT549, and HCC1187 cells were plated at 2500 c/well, subcutaneously. 100 µL (1 500 000 c/mouse were injected between the 5000 c/well, 6000 c/well, 1000 c/well, and 6000 c/well respectively for fourth and fifth right mammary fat pads of 8-week-old BALB/c nude 24 h. Cells were incubated with 0.5, 1, and 3 × 10−6 m of IB protein. female mice (n = 40). Tumor size was evaluated 2 times a week by Viability was measured at 48, 72, and 96 h comparing luciferase signal caliper measurements and tumor volume calculated using the following against untreated control cells. formula: volume = (D × d2) / 2, where “D” is the largest diameter and MDA-MB-231 Mammosphere Production: MDA-MB-231 cells were “d” the smallest one. Weight of the mice was measured at least twice seeded at 1000 c/well in low attachment cell culture plates (Nunc, a week. When tumors reached a volume between 100 and 350 mm3, Waltham, MA, USA) and cultured in a defined media without serum[44] at animals were randomized in the different groups (n = 7–8): control 37 °C and 5% CO2. After 5 and 7 d of incubation mammospheres appeared buffer (50 × 10−3 m KH2PO4, 500 × 10−3 m NaCl, 0.8 m Urea, 100 × 10−3 m and cultures were challenged by the addition of 3 × 10−6 m FN-GFP-H6 GuHCl; pH 7.4), PBS, FNI/II/V-GFP-H6, and Omo-FNI/II/V-H6. Protein and FN-p31-H6 IB proteins and Omo-FN-H6 IB protein for 72 h. Sphere stocks were diluted to 4 g L−1 for the treatments. Animals were treated integrity and morphology were assessed by bright field imaging in an intratumorally once a week and the volume/dose of intratumoral Olympus BH2 microscope (Olympus Corporation, Tokyo, Japan). administration was adjusted according to tumor volume (1:10 of the Field Emission Scanning Electron Microscopy: To characterize the tumor volume was inoculated up to a maximum of 50 µL, e.g. a tumor morphometry (size and shape) at ultrastructural level of IBs at nearly of 220 mm3 was treated with 22 µL). Animals were treated up to 4 native state, a rapid and easy method was used. Microdrops of 5 µL of weeks, then euthanized by CO2. diluted samples were deposited for 2 min on silicon wafers (Ted Pella Inc, For biodistribution and efficacy studies, exponentially growing Redding, CA, USA), liquid excess was blotted, air dried and immediately HCC1806 (2.5 × 106) or BT20 (10 × 106) cells, respectively, were monitored without coating in a Field Emission Scanning Electron orthotopically implanted into the fat mammary pad (i. f. m.p.) and Microscope (FESEM) Zeiss Merlin (Carl Zeiss, Oberkochen, Germany) monitored weekly as described above. These cells are classified as operating at 1 kV. IB images were collected with a standard secondary basal like[47] and luminal androgen receptor (LAR),[48] respectively. In electron (SE) detector (4000–40,000x magnifications). biodistribution assays, once the tumors reached an average size of IB Labeling: AlexaFluor 647 NHS (Molecular Probes, Eugene, OR, 150 mm3, mice were intratumorally administered with Alexa 647 labeled USA) was used to label the inclusion bodies so that internalization FN-p31-H6 IBs (200 µg per mouse, n = 3) and fluorescent signal was assays could be normalized and compared at a unique wavelength. monitored in vivo at different time periods post-administration by Conjugation was performed by resuspending IBs at 1 mg mL−1 in PBS means of IVIS Spectrum equipment (Perkin Elmer, Tres Cantos, Spain). with the addition of dye at a dye:protein molar ratio 2:1 and incubating Quantification of the fluorescent signal (in Radiant Efficiency) was for 1 h at RT with agitation. Serial cleaning steps were applied to performed by using the Living Image software (Perkin Elmer). At the remove free dye by centrifuging and resuspending the IBs with H2O. end point, tumors were excised and subjected to fluorescent imaging. Fluorescence units/mass was determined by flow cytometry. Afterward, tissues were snap frozen and stored for further analysis or Confocal Laser Scanning Microscopy: MDA-MB-231 cells were seeded fixed in 4% formaldehyde and processed for histopathological analysis on Mat-Teck culture plate (Mat Teck Corporation, Ashland, MA, USA) at and evaluation. For efficacy assays, mice bearing BT20 tumors with an 200 000 c/well for 24 h. Medium was removed and changed to OptiPRO average size of 200 mm3 (n = 8), were administered with 600 µg of serum-free medium supplemented with L-glutamine and 1 × 10−6 FN-p31-H6 or FN-GFP-H6 IBs. Administration was repeated twice in two m of IBs was added and incubated for 24 h. After incubation, cells week and animals were euthanized one week after the last treatment. were washed with PBS and 3 µg mL−1 WGA 594 (Molecular Probes) Tumor p21 Immunohistochemistry and Apoptosis: The presence of and 0.2 µg mL−1 Hoechst 33 342 (Molecular Probes) were added for p21 antigen in tumor sections was analyzed by pre-treating paraffin 5 min in darkness to visualize the plasma membrane and the nuclei embedded sections with 100 × 10−3 m citrate buffer (pH 9) in a cooker. respectively. Later, cells were washed with PBS and complete medium Sections were incubated with 10% normal goat serum (NGS) in was added. Stained cells were examined using a TCS-SP5 confocal antibody diluent (1% BSA in 100 × 10−3 m Tris buffer) and then of 1:100 laser scanning microscope (Leica Microsystems, Heidelberg, Germany) dilution of anti-p21 antibody (M 7202, Dako, Santa Clara, CA, USA). Adv. Sci. 2019, 6, 1900849 1900849 (7 of 9) © 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.advancedscience.com Secondary antibody consisted in a HRP conjugated system (EnVision+ [1] A. de Marco, N. Ferrer-Miralles, E. Garcia-Fruitos, A. Mitraki, System-HRP Labeled Polymer anti-Mouse), which was later visualized S. Peternel, U. Rinas, M. A. Trujillo-Roldan, N. A. Valdez-Cruz, with DAB and counterstained with Harris haematoxylin. The p21 signal E. Vazquez, A. Villaverde, FEMS Microbiol. Rev. 2019, 43, 53. intensity and extension were scored under the light microscope by two [2] a) L. Wang, S. K. Maji, M. R. Sawaya, D. Eisenberg, R. Riek, PLoS blinded observers. Intensity cores ranged from 0 to 3 (absence, low Biol. 2008, 6, e195; b) L. Wang, Prion 2009, 3, 139. intensity, normal or high, respectively). For each tumor, two independent [3] O. Cano-Garrido, E. Rodriguez-Carmona, C. Diez-Gil, E. Vazquez, sections with p21 staining were evaluated. For the evaluation of the E. Elizondo, R. Cubarsi, J. Seras-Franzoso, J. L. Corchero, U. Rinas, extension of the apoptotic nuclei, sections were stained with Hoescht I. Ratera, N. Ventosa, J. Veciana, A. Villaverde, E. Garcia-Fruitos, (1:500, Sigma-Aldrich, San Luis, MI, USA) mounted in Prolong Acta Biomater. 2013, 9, 6134. (Invitrogen, Carlsbad, CA, USA). Two independent slides per tumor [4] E. Garcia-Fruitos, E. Rodriguez-Carmona, C. Diez-Gil, R. M. Ferraz, and five representative images per slide were analyzed after acquiring E. Vazquez, J. L. Corchero, M. Cano-Sarabia, I. Ratera, N. Ventosa, images at 10x magnification in an Olympus BX61 microscope (Olympus Corporation). In each image, the number of apoptotic nuclei versus the J. Veciana, A. Villaverde, Adv. Mater. 2009, 21, 4249. number of nonapoptotic nuclei was counted by two independent blinded [5] A. Villaverde, E. Garcia-Fruitos, U. Rinas, J. Seras-Franzoso, observers. Results were presented as percentage of apoptotic nuclei. A. Kosoy, J. L. Corchero, E. Vazquez, Microb. Cell Fact. 2012, 11, 76. Anti-p21 (MS-891-B Thermo Scientific) Western Blot was performed [6] N. Gonzalez-Montalban, E. Garcia-Fruitos, A. Villaverde, Nat. Bio- on cell extracts as described[49] using BT20 cells exposed to 9 × 10−6 m technol. 2007, 25, 718. protein for 4 h. Fold increase of p21 was referred to a β-actin control. [7] a) E. Hrabarova, L. Achbergerova, J. Nahalka, Methods Mol. Biol. Statistical Analysis: Quantitative data were expressed as mean and 2015, 1258, 411; b) V. D. Jager, R. Kloss, A. Grunberger, S. Seide, standard error (x̄ ± SE). Depending on the type of data, parametric or D. Hahn, T. Karmainski, M. Piqueray, J. Embruch, S. Longerich, nonparametric statistics were applied to compare divergences between U. Mackfeld, K. E. Jaeger, W. Wiechert, M. Pohl, U. Krauss, Microb. all groups in relation to control or pairwise comparisons. In some data, Cell Fact. 2019, 18, 33. one-way ANOVA followed by a post-hoc Dunnett was used, whereas [8] E. Vazquez, J. L. Corchero, J. F. Burgueno, J. Seras-Franzoso, Kruskall–Wallis and Mann Whitney or Wilcoxon tests were performed A. Kosoy, R. Bosser, R. Mendoza, J. M. Martinez-Lainez, U. Rinas, with the rest of data. Statistical significance levels were represented as E. Fernandez, L. Ruiz-Avila, E. Garcia-Fruitos, A. Villaverde, Adv. n.s. p > 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001. All analyses were Mater. 2012, 24, 1742. performed using SigmaPlot 10.0 software. [9] J. Seras-Franzoso, A. Sanchez-Chardi, E. Garcia-Fruitos, E. Vazquez, A. Villaverde, Soft Matter 2016, 12, 3451. [10] a) M. Liovic, M. Ozir, A. B. Zavec, S. Peternel, R. Komel, Acknowledgements T. Zupancic, Microb. Cell Fact. 2012, 11, 67. b) K. Talafova, E. Hrabarova, D. Chorvat, J. Nahalka, Microb. Cell Fact. 2013, 12, 16. M.P. and T.J. contributed equally to this work. This study has been [11] A. Stamm, S. Strauss, P. Vogt, T. Scheper, I. Pepelanova, Microb. supported by La Fundació Marató TV3 and NanoCanTri (CIBER-BBN) Cell Fact. 2018, 17, 57. to E.V. and I.A., and partially by ISCIII (PI15/00272 and PI1702242 [12] U. Unzueta, M. V. Cespedes, R. Sala, P. Alamo, A. Sanchez-Chardi, co-founded by Fondo Europeo de Desarrollo Regional (FEDER), to M. Pesarrodona, L. Sanchez-Garcia, O. Cano-Garrido, A. Villaverde, E.V. and S.S., respectively), and Agencia Estatal de Investigación (AEI) E. Vazquez, R. Mangues, J. Seras-Franzoso, J. Controlled Release and FEDER (BIO2016-76063-R, AEI/FEDER, UE), AGAUR (2017SGR-229) 2018, 279, 29. and CIBER-BBN (VENOM4CANCER) granted to A.V. Protein production and DLS have been partially performed by the ICTS “NANBIOSIS,” [13] M. V. Cespedes, Y. Fernandez, U. Unzueta, R. Mendoza, more specifically by the Protein Production Platform of CIBER-BBN/IBB J. Seras-Franzoso, A. Sanchez-Chardi, P. Alamo, V. Toledo-Rubio, (http://www.nanbiosis.es/unit/u1-protein-production-platform-ppp/) N. Ferrer-Miralles, E. Vazquez, S. Schwartz, I. Abasolo, and the Biomaterial Processing and Nanostructuring Unit (http://www. J. L. Corchero, R. Mangues, A. Villaverde, Sci. Rep. 2016, 6, 35765. nanbiosis.es/portfolio/u6-biomaterial-processing-and-nanostructuring- [14] S. K. Maji, M. H. Perrin, M. R. Sawaya, S. Jessberger, K. Vadodaria, unit/), respectively. Biodistribution and immunohistochemistry assays R. A. Rissman, P. S. Singru, K. P. Nilsson, R. Simon, D. Schubert, were performed at the ICTS “NANBIOSIS,” specifically by U20/FVPR D. Eisenberg, J. Rivier, P. Sawchenko, W. Vale, R. Riek, Science 2009, (http://www.nanbiosis.es/portfolio/u20-in-vivo-experimental-platform/). 325, 328. L.S.-G. was supported by predoctoral fellowship from AGAUR (2018FI_ [15] a) M. P. Badtke, N. D. Hammer, M. R. Chapman, Sci. Sign- B2_00051). L.S. was supported by the European Research Council (CoG aling 2009, 2, pe43; b) K. Schwartz, A. K. Syed, R. E. 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