Biomedicine & Pharmacotherapy 164 (2023) 114976 Contents lists available at ScienceDirect Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha Lymphocyte infiltration and antitumoral effect promoted by cytotoxic inflammatory proteins formulated as self-assembling, protein-only nanoparticles Carlos Martínez-Torró a, b, 1, Lorena Alba-Castellón b, c, d, 1, Luis Miguel Carrasco-Díaz c, d, Naroa Serna a, b, 2, Laura Imedio c, d, Alberto Gallardo b, e, Isolda Casanova c, d, Ugutz Unzueta b, c, d, Esther Vázquez a, b, f, Ramón Mangues b, c, d, *, Antonio Villaverde a, b, f, * a Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain b CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain c Institut d’Investigació Biomèdica Sant Pau (IIB SANT PAU), Barcelona, Spain d Josep Carreras Leukaemia Research Institute (IJC), Badalona, Spain e Department of Pathology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain f Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain A R T I C L E I N F O A B S T R A C T Keywords: Two human proteins involved in the inflammatory cell death, namely Gasdermin D (GSDMD) and the Mixed Recombinant proteins Lineage Kinase Domain-Like (MLKL) protein have been engineered to accommodate an efficient ligand of the Biomaterials tumoral cell marker CXCR4, and a set of additional peptide agents that allow their spontaneous self-assembling. Nanoparticles Upon production in bacterial cells and further purification, both proteins organized as stable nanoparticles of 46 Pyroptosis and 54 nm respectively, that show, in this form, a moderate but dose-dependent cytotoxicity in cell culture. In Cancer Drug delivery vivo, and when administered in mouse models of colorectal cancer through repeated doses, the nanoscale forms Gasdermin D of tumor-targeted GSDMD and, at a lesser extent, of MLKL promoted CD8+ and CD20+ lymphocyte infiltration in the tumor and an important reduction of tumor size, in absence of systemic toxicity. The potential of these novel pharmacological agents as anticancer drugs is discussed in the context of synergistic approaches to more effective cancer treatments. 1. Introduction categories of materials have been engineered in form of nanoparticles to be loaded with a payload drug, and functionalized with tumor-homing Despite many innovative therapeutic initiatives, cancer therapies are peptides for active and very precise receptor-mediated targeting still largely supported by low molecular weight cytotoxic agents [1–3] through the interaction with cell-surface tumor markers [11–13]. Such that, upon primary tumor resection, are administered systemically to strategies, even though being conceptually promising, have not been prevent or delay tumor relapse and metastasis. Being untargeted, the generically successful [14], as only around 1% of the systemically systemic toxicity and severe side effects linked to chemotherapies [3] administered drug dose reaches the tumor [15]. In part, such failure is push to develop new drug delivery approaches, that being highly se­ attributed to the protein corona, which dramatically reduces the inter­ lective for target cells, could enhance the local cytotoxic effect and activity of the targeting ligands displayed on the surface of carrier reduce undesired off-target toxicities. Selectivity in drug delivery for nanoparticles [16–18]. On the other hand, immunotoxins and cancer was initially based on plain nanoscale carriers [4–6], designed to antibody-drug conjugates, even reaching the clinics, also fail to produce exploit the enhanced retention and permeability (EPR) effect through generically good therapeutic outcomes because of a low uptake in can­ the so-called passive targeting [7–10]. In a step further, several cer tissues [19] and systemic toxicity due to high accumulation and * Corresponding authors at: CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. E-mail addresses: RMangues@santpau.cat (R. Mangues), Antoni.villaverde@uab.cat (A. Villaverde). 1 Equally contributed. 2 Present address: Nanoligent SL. Edifici Eureka. Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain https://doi.org/10.1016/j.biopha.2023.114976 Received 4 April 2023; Received in revised form 24 May 2023; Accepted 30 May 2023 Available online 3 June 2023 0753-3322/© 2023 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). C. Martínez-Torró et al. Biomedicine & Pharmacotherapy 164 (2023) 114976 metabolism in the liver. In this context, some of such approved drugs The production of both constructs was preliminary assayed at 16 ºC have been discontinued [20]. (overnight), 20 ºC (overnight) and 37ºC (3 h). As in all the tested con­ Among the materials tested for cancer nanomedicines, oligomeric ditions the only detected product occurred in the insoluble cell fraction, protein complexes at the nanoscale result from either spontaneous or the proteins were finally produced at 37ºC for three hours to maximize ion-assisted self-assembling [21–26]. These materials benefit from the protein yield and recovery from aggregates. For harvesting, bacterial robust, scalable and repetitive procedures for recombinant protein cultures were centrifuged at 5000 g for 15 min and the pellets washed production that have been developed during the last 40 years and that with PBS and stored at − 80ºC until use. Cell disruption was performed sustain the protein drug industry [27,28]. The multiple and geometri­ by sonication (40% amplitude, 8 min, 1 s ON, 4 s OFF) in a 20% sucrose, cally regular presentation of the homing peptides or other functional 20 mM Tris-HCl buffer. After the centrifugation (15,000 g, 45 min), the protein domains, in a virus-like fashion [29,30], favors cell receptor supernatants were discarded and pellets washed with PBS (15,000 g, 15 binding and internalization what in turn allows a high local concen­ min). Then, the pellets were weighted and 40 mM Tris-HCl, 0.2% N- tration of the payload. Also, the interactivity of these protein-based Lauroyl Sarcosine were added to 50 mL/g. These mixtures were left particles with tumor cells is not shadowed by any protein corona. The overnight in agitation. The next day, samples were centrifuged (15,000 formation of protein coronas around protein nanomaterials has not been g, 45 min), pellets discarded and supernatants kept for further use. observed [31], since protein-protein contacts are exquisitely specific Protein purification was done by immobilized metal affinity chro­ [32,33]. Finally, proteins allow, by recruitment of multiple biologically matography (IMAC). Before such step, the solubilized product was dia­ active domains [34], the construction of nanoparticles that are intrin­ lyzed against 20 mM Tris-HCl, 500 mM NaCl, pH 8.0, in two rounds of sically multi-functional (e.g. a tumor homing peptide, a cytotoxic two hours and one additional overnight round. The soluble protein was domain and a self-assembling tag in the same polypeptide), thus recovered and charged in a HisTrap HP 5 mL column (GE Healthcare). achieving the rising nanomedical concept of self-assembled, self-­ Elution from the columns was achieved by a linear gradient of elution delivered, cell-targeted nanoscale drugs [35] that do not require any buffer (20 mM Tris, 500 mM NaCl, 500 mM imidazole) and the fractions driving, non-drug material as vehicle [36]. that contained the target proteins were dialyzed against 100 mM NaCl, Under this concept, we have recently generated tumor-targeted 20 mM Tris-HCl, pH 8. The engineered proteins were detected by nanoparticles based on plant [37] or microbial toxins [38], Western Blot using an anti-His monoclonal antibody (GenScript) and pro-apoptotic factors [39] or venoms [40]. Being intrinsically cytotoxic, their concentration was determined by Bradford (BioRad). these materials were aimed to tumor reduction and/or metastatic con­ trol. When administering tumor-targeted microbial toxins (namely a 2.2. Dynamic light scattering modified Pseudomonas aeruginosa exotoxin and the diphtheria toxin) to different animal models of human cancers, pyroptotic routes were un­ The size distribution of T22-GSDMDmutS19-H6 and T22- expectedly involved [41,42], and an important and clinically promising MLKLmutS19-H6 particles was determined by dynamic light scattering tumor remission was observed [41,42]. Being both pyroptosis and nec­ (DLS) at 633 nm (Zetasizer Pro, Malvern Instruments). 50 µL of each roptosis inflammatory forms of cell death [43], promoting those routes protein (ranging from 1 mg/mL to 2 mg/mL) were measured in triplicate in cancer cells has been recently proposed as a novel and interesting at 25ºC. Size data were later processed and analyzed in the ZS Explorer therapeutic approach [44–46]. Then, proteins that are directly involved software (Malvern Instruments). in inflammatory cell death cascades might be of great interest as anti­ cancer drugs. In this context, and by exploiting the versatility in the 2.3. Protein stability design and engineering of multidomain proteins, we have designed, produced and functionally characterized two type of protein nano­ T22-GSDMDmutS19-H6 and T22-MLKLmutS19-H6 were incubated particles targeted to the tumoral marker CXCR4. They are based on at a concentration of 1 mg/mL in human serum (Sigma) for 24 h at 37ºC Gasdermin D (GSDMD) and on the Mixed Lineage Kinase Domain-Like without agitation. Samples were extracted at 0, 1, 5 and 24 h. The (MLKL) proteins, which are essential agents in the pyroptotic and nec­ proteolytic stability was determined by Western Blot using a monoclonal roptotic cascades, respectively [47,48]. The results presented here antiHis antibody (GenScript). Protein quantification was assessed with demonstrate an antitumor effect and a clinical potential of these in­ the ImageLab software (Bio-Rad) comparing the proteins at different flammatory proteins formulated as self-assembling, tumor-targeted times with the samples at time 0 (100%). multimeric nanoparticles. 2.4. Cell culture and in vitro cell viability 2. Materials and methods HeLa cells (ATCC, CCL-2) were maintained in MEM alpha medium 2.1. Protein design, production and purification (Gibco) with 10% Fetal Bovine Serum (Gibco) in a humidified atmo­ sphere at 37ºC and 5% CO2. To assess cell viability, HeLa cells were The synthetic genes encoding the modular proteins T22-GSDMDmut- cultured on 96-well plates (3500 cells/well) for 24 h. The next day, the S19-H6 and T22-MLKLmut-S19-H6 were designed in house and pro­ proteins to be tested were diluted with MEM alpha medium until the vided by GeneArt (Thermo Fisher), as subcloned in a pET22b plasmid desired final concentration. Then, 10 µL of the protein dilution were (Novagen). T22-GSDMDmut-S19-H6 contains a mutation (L290D) in the added to the correspondent well. The toxicity of the protein buffer was GSDMD scaffold that prevents the C-terminal auto-inhibition of the N- also assessed as a control. Protein incubations were performed in trip­ terminal effector domain [49]. T22-MLKLmut-S19-H6 carries phos­ licate. Cell viability was determined using CellTiter-Glo Luminiscent phomimetic mutations (T357E and S358D) in the MLKL scaffold to Cell Viability Assay (Promega), in the presence of 1 µg/mL LPS as bypass RIPK3 activation [50]. Both constructions also contain the S19 described [60]. Plates were read in a Victor 3 luminescent plate reader peptide, which stimulates the intracellular delivery [51]. The N-termi­ (Perkin Elmer) at 48 and 72 h. The final data were expressed as the mean nal peptide T22, is a polyphemusin II derivative [52] initially adapted as of the viability percentage ( ± standard deviation) referred to the con­ a blocker of the human immunodeficiency virus (HIV) infection [53], as trol wells in which only MEM alpha was added. it specifically binds the cytokine receptor CXCR4 [54]. Incorporated to protein nanoparticles and imaging agents, T22 promotes their highly 2.5. Animal procedures and histology selective binding and penetration in vitro into CXCR4+ cultured cells [55] and in vivo, in CXCR4+ tumoral cells, with a highly precise tumor Seven-week-old BALB/cByJ females were purchased from Charles biodistribution [56–59]. River (France). Upon arrival, mice were kept in quarantine for 1 week 2 C. Martínez-Torró et al. Biomedicine & Pharmacotherapy 164 (2023) 114976 and maintained in specific pathogen-free conditions with sterile food apparent after 72 h of exposure when the cytotoxicity of the nano­ and water ad libitum. All animal procedures were approved by the particles was more obvious. Hospital de la Santa Creu i Sant Pau Animal Ethics Committee and The systemic context of the immune- and inflammatory-based Catalonia authorities (Authorization number 9721). CT26 cells (ATCC, mechanisms of pyroptosis or necroptosis prompted us to expect an in CRL-2638), which are colon carcinoma mouse cells, were transduced vivo magnified impact of these drugs. Such a biological effect should be with mouse CXCR4 receptor (pLV-EF1A-Puro-mCXCR4, VectorBuilder). more evident than the mild cell killing observed in cell culture, an event Mice were subcutaneously injected with 2.5 × 105 CT26-CXCR4 cells restricted to a mere local context. Before moving to in vivo experiments, into the right flank in 200 µL RPMI 1640 medium (Gibco, Life tech­ the stability of T22-GSDMDmut-S19-H6 and T22-MLKLmut-S19-H6 was nologies). On day 9 post cell inoculation, those animals with palpable determined in human serum during incubation at 37 ºC. As observed, a tumors were randomized into 3 experimental groups (n = 5–6 per slight decrease in the full-length forms of the protein was observed group) and the treatment started. Such mice were treated intravenously throughout the incubation time (Fig. 3A), indicating a half-life of around three times per week either with 8 g/Kg of T22-GSDMD-H6 or T22- 5 h (Fig. 3B). Although this value was not very high, most of the intra­ MLKL-H6 or the equivalent volume of Tris-HCl for control animals. venously injected dose of the T22-driven nanoparticle GFP versions Mice’s well-being, tumor volume (V= length x width2 x 0.5) and mouse accumulate in tumor around 5 h [55]. A half-life around this value was body weight were recorded through the experiment. Twenty-four hours then considered as promising and sufficient to observe a biological effect after the fifth dose, animals were euthanized and tumors were collected, of the proteins. fixed in 4% formaldehyde and embedded in paraffin. For histological To assess such expected effect of both nanoparticles in vivo, an analysis, 4 µm sections were stained with hematoxylin and eosin and the immunocompetent mouse model of aggressive, CXCR4+ colorectal necrotic area and tumor-infiltrated lymphocytes (TILs) were quantified cancer was developed, through the implantation of CXCR4+ CD26 cells by a pathologist. Representative images were acquired using an (Fig. 3C) in immunocompetent animals. The resulting tumors were, as Olympus DP73 digital camera. For immunostaining, 4 µm paraffin expected, CXCR4+ (Fig. 3D). The animals were treated with repeated embedded sections were dewaxing and submitted to an antigen retrieval intravenous doses of each protein once the tumors were palpable (9 days procedure using a high pH buffer (PTLink, Agilent Technologies). CD3, post cell implantation). The selected tentative dosage was 8 µg of each CD8, CD20 (ready to use antibodies, Agilent Technologies) and CXCR4 nanoparticle per g of animal weight, administered intravenously 3 times (1:200, Abcam) staining were performed in a DAKO Autostainer Link48 per week, with a total of 5 doses. (Agilent Technologies) following manufacturer’s directions. Stained As observed (Fig. 4A), the weight of the treated animals remained slides were scanned and quantified using Panoramic Scan II and Den­ unmodified in comparison with the buffer-treated group, indicative of sitoQuant image analysis center from 3DHISTECH Ltd. Representative absence of systemic toxicity associated to the administration of both images were acquired using Slide Viewer software. protein nanoparticles. Notably, the treatment with T22-GSDMDmut- S19-H6 significantly reduced tumor growth since its implantation 2.6. Cell cytometry compared with the buffer-treated group (Fig. 4B, adjusted P value = 0.008, Tukey post-hoc test following an ANOVA test), while T22- CXCR4 membrane expression in CT26-CXCR4 was confirmed by MLKLmut-S19-H6 also showed a similar therapeutic trend (adjusted P MACSQuant® Analyzer 10 flow cytometer (Miltenyi Biotech) using PE value = 0.095). This antitumoral effect was evident along the experi­ rat anti-mouse CXCR4 antibody. The isotype PE rat IgG2b antibody was mental time and very pronounced at 13, 15 and 19 days post- used as a negative control (Biolegend). implantation (Fig. 4B). Unfortunately, the scattering of data within the control group rendered a few comparative points in which the differ­ 3. Results ences resulted in the border of significance, while the global healing effect was evident along the treatment time (Fig. 4B). In the last sam­ Modified versions of human GSDMD and MLKL have been engi­ pling analysis (20 days post implantation), the tumor size was similar in neered as multidomain proteins (Fig. 1), intended as building blocks for all the groups, suggesting a loss of antitumoral activity while the drug self-assembling as nanoscale oligomers. The occurrence of C-terminal was still administered (Fig. 4B). Interestingly, the histological analysis of polyhistidine tails (here an hexahistidine, H6) [21,61] combined with the tumors at day 20 (Fig. 5A, B) indicated infiltration of TILs in tumor the cationic peptide T22 (a specific ligand of CXCR4 [62] and an effi­ tissue at a statistic significant level, for the animals treated with T22- cient tumor homing peptide [58]), are expected to promote the forma­ GSDMDmut-S19-H6 nanoparticles and also for those treated with T22- tion of regular oligomers assisted by cations from the media [63–65]. MLKLmut-S19-H6 nanoparticles (Fig. 5C). Moreover, at day 20, there This category of H6-driven nanoparticles is structurally robust and sta­ was also a trend towards an increase in necrotic tumor area in ble during circulation in blood [31], what make them excellent drugs or nanoparticle-treated mice, compared to control mice, again in the drug vehicles for nanomedical purposes [57,59]. Both modular proteins border of statistical significance (Fig. 5C). were well produced in recombinant Escherichia coli and recovered upon To more precisely assess lymphocyte infiltration in tumor, CD3, CD8 purification as discrete bands with the predicted migration and molec­ and CD20 lymphocyte subpopulations, relevant to the emerging im­ ular mass (Fig. 1). Also, both protein constructs organized, as expected, munotherapies of cancer [68–70], were analyzed in tumors of treated as monodisperse nanoparticle populations, sizing around 46 nm animals, through immunohistochemistry. As observed (Fig. 6 A, B), tu­ (T22-GSDMDmut-S19-H6) and 54 nm (T22-MLKLmut-S19-H6) respec­ mors of animals treated with T22-GSDMDmut-S19-H6 or tively (Fig. 1). When exposing these oligomeric materials to denaturing T22-MLKLmut-S19-H6, exhibit an increased number of infiltrated plus chelating conditions they disassembled into the forming monomers B-lymphocytes (CD20) and cytotoxic T-Lymphocyte (CD8) when (Fig. 1). compared with buffered treated mice. CD3, a pan T lymphocyte marker The potential cytotoxicity of these nanoparticles was tested over did not show differences, suggesting a specific anti-tumor immune cell cultured HeLa (CXCR4+, [66]) cells. A moderate but significant reduc­ recruitment. tion of the cell viability was observed at 72 h in both cases, ranging between 15% and 20% depending on the protein concentration (Fig. 2). 4. Discussion Since these data were in the range of previous reports about the effect of plain unformulated pyroptotic proteins in cell culture [67], we thus Pyroptosis and necroptosis are related types of programmed cell confirmed the retention of the biological activity of both products death [71] characterized by the activation of inflammatory caspases, formulated as nanoscale oligomeric forms. The observed deleterious including caspase-1, caspase-4, caspase-5, and caspase-11 [72], which effect over cultured cells was dose-dependent (Fig. 2), a fact especially lead to the release of pro-inflammatory cytokines and 3 C. Martínez-Torró et al. Biomedicine & Pharmacotherapy 164 (2023) 114976 Fig. 1. Modular organization of building blocks and characterization of the resulting nanoparticles. Modular design and amino acid sequence of T22-GSDMDmut- S19-H6 (up) and T22-MLKLmut-S19-H6 (bottom). The CXCR4-ligand T22 (green), a peptide spacer (dark yellow) and two furin-cleavable sites (orange) are included in the construct, that also harbors the endosomal escape peptide S19 (purple) and the end terminal H6 tag (blue). In red, there are indicated the point mutations over the original sequences known to enhance the biological activity of either GSDMD [49] or MLKL [50]. Linked to each construction map, the western blot detection of purified protein is shown, in which protein bands were visualized using an anti-His antibody. The size of the soluble protein nanoparticles and their monomeric forms (upon disassembling with 2 mM EDTA + 2% SDS), were determined by DLS. PI stands for polydispersity index. 4 C. Martínez-Torró et al. Biomedicine & Pharmacotherapy 164 (2023) 114976 Fig. 2. Cytotoxicity of protein nanoparticles on cultured cells. HeLa cells were incubated with increasing concentrations of GSDMD- (up) or MLKL-based (down) nanoparticles together with 1 µg/mL LPS for 48 or 72 h. PBS and LPS 1 µg/mL were used as controls. Significance was assessed with an ANOVA test and the adjusted p-value (p in the plot) was calculated using a Tukey post-hoc test comparing the different protein concentrations with the PBS control. damage-associated molecular patterns (DAMPs) [73,74]. They are key to their poor recognition by the immune system and their limited players in the regulation of both inflammation and immune responses, responsiveness to conventional treatments [82]. and pyroptotic and necrotic events have been associated with several One of the most promising strategies for inducing inflammatory cell pathological conditions, including cancer [73,75–77]. Both pyroptosis death in cancer would be the targeting to tumor cells of pore-forming and necroptosis are lytic cell death types that upon induced in cancer inflammatory proteins, specifically gasdermin D (GSDMD) [83]. Gas­ cells release cytokines and DAMPs that trigger inflammation. These dermins are a family of pore-forming proteins that are involved in events attract circulating lymphocytes to cancer tissues. The resulting various cellular processes, including cell death. In particular, GSDMD activation of the antitumoral immunogenic cell death is then a potential has been shown to be a key mediator of pyroptosis, as its activation leads new approach for cancer treatment [78–81]. This is of special interest to the formation of membrane pores and subsequent release of inflam­ regarding ‘cold tumors’, which are highly relevant in human clinics due matory signals [84]. On the other hand, MLKL induces necroptosis in 5 C. Martínez-Torró et al. Biomedicine & Pharmacotherapy 164 (2023) 114976 Fig. 3. Stability of GSDMDmutS19-H6 and T22-MLKLmutS19-H6 in human serum. A. Western blot analysis of both proteins incubated in human serum at 37ºC. The full length forms (red arrow) were quantified for further analysis. B. Declining of full length protein forms throughout incubation time. The horizontal line indicates 50% of the initial protein amount. C. Histogram of CXCR4-membrane expression detected by flow cytometry in transduced CT26 cells. Cells incubated with the PE- isotype antibody were considered as negative. D. CXCR4 expression in subcutaneous CT26-CXCR4 tumor grown in BALB/c mice. Scale bar: 50 µm. several cancer cell lines, and its expression has been found to be lymphocyte infiltration in tumors (Fig. 5), what can be seen as turning downregulated in various types of cancer, suggesting that it may func­ ‘cold’ tumors into ‘hot’ tumors [82,93,94]. As far as we know, this is the tion as a tumor suppressor [85]. MLKL expression and cancer progres­ first report in which such activity is described for a pore-forming in­ sion have been shown as inversely correlated [86–88]. This whole set of flammatory protein. In agreement with this concept, tumors from observations places both GSDMD and MLKL as potential protein-based treated mice exhibited an increased number of infiltrated B-lymphocytes anticancer drugs candidates. (CD20) and also of cytotoxic T-lymphocytes (CD8). Interestingly, a In previous studies, we were successful in promoting the self- general T-lymphocyte increase was not detected (no changes in of CD3+ assembling of cytotoxic proteins (namely venoms, toxins or pro- cell infiltration), suggesting an antitumoral specific recruitment within apoptotic factors) as both scaffolding and functional building blocks of the tumor when compared with control mice (Fig. 6 A,B). Cytotoxic tumor-targeted protein-only nanoparticles [89]. This fact prompted us lymphocytes are observed as antitumoral lymphocytes, as they partici­ to engineer GSDMD and MLKL (Fig. 1) for this purpose through minor pate in antigen recognition and correlate with better prognosis in several amino acid sequence modifications. The assembling of the materials as cancers, including colorectal cancer [95,96]. In this tumor type, CD20 nanoparticles was successful (Fig. 1) and the cytotoxicity in vitro was infiltration has been particularly associated with improved survival. The observed at the expected levels (Fig. 2). The particles proved to be proteins tested here efficiently induce the activation of the antitumor sufficiently stable in human serum (Fig. 3) to be moved towards in vivo adaptive immune system mediated by B and T lymphocytes. testing for systemic administration in repeated doses. MLKL showed a Regarding the antitumoral effect, that was marked the first two mild tendency towards an antitumoral effect once administered in an weeks upon stating the treatment, the functional nanoparticle T22- animal model of colorectal cancer (Fig. 4B), while GSDMD-based GSDMDmut-S19-H6 promoted an important reduction of tumor nanoparticles promoted a clear reduction of tumor growth several growth followed by a relapse (Fig. 4B). This particular outcome would days upon starting the administration regimen (Fig. 4B). The fact that need further analyses at the cell and tissue level to discriminate between systemic toxicities were not observed (Fig. 4 A) demonstrated the pre­ appearing of resistance or other issues. However, even if transient, the cise T22-mediated selective delivery of the nanoparticles into CXCR4+ cancer tissue destruction and the consequent lymphocyte recruitment tumor cells, as previously seen in related T22-empowered oligomeric (Figs. 5 and 6) initiated by the pyroptotic window might be of great vehicles used for the delivery of highly cytotoxic small molecular weight clinical value when used in combination with synergistic treatments. In drugs [90–92]. On the other hand, both proteins induced a potent the clinics, it has been generically observed a correlation between the 6 C. Martínez-Torró et al. Biomedicine & Pharmacotherapy 164 (2023) 114976 Fig. 4. Impact of protein nanoparticles on tumor size in a colorectal cancer mouse model. A murine colorectal carcinoma cell line was implanted in several mice (n = 16); the animals were divided into three groups depending on the treatment: a saline buffer (n = 6), T22-GSDMDmutS19-H6 (n = 5) and T22-MLKLmutS19-H6 (n = 5). A. Mouse body eight evolution for every group. Data are the mean for each time point and the bars represent the standard deviation. B. Tumor growth was analyzed at day 9, 13, 15, 19 and 20 post-implantation. Two mice (one for each nanoparticle type) had a disproportionately higher tumor size at each measure and were not considered for statistical analysis. These mice are highlighted in the figure with a double circle. Statistical significance was assessed with an ANOVA test and a Tukey post-hoc test. 7 C. Martínez-Torró et al. Biomedicine & Pharmacotherapy 164 (2023) 114976 Fig. 5. Lymphocyte infiltration and necrotic tumor area in mice treated with protein nanoparticles. A. Tumor-infiltrating Lymphocytes (TIL) in hematoxylin and eosin (H&E) stained sections. Arrows point to TILs. Scale bar represents 50 µm in the general view and 20 µm in the zoomed view. B. Necrotic areas in H&E-stained tumor sections. The necrotic tissue is at the left and the viable tumor is at the right. Scale bar represents 50 µm. C. Percentage of TIL (left) and necrotic area (right) in each condition. Statistical significance was assessed with a Kruskal-Wallis test and a Dunn post-hoc test (the p value was adjusted using the Benjamini- Hochberg procedure). intensity of TILs infiltration and a low tumor volume, limited metastatic TILs infiltration and tumor recurrence was only observed in large progression and good prognosis [97,98]. However, some authors have (>5 cm in diameter) tumors but not in smaller ones [100]. In fact, a reported, such as in the case of meningioma, that the infiltration of TILs diversity of controversial outcomes from the crosstalk between inflam­ is linked with a tumor size reduction for large tumors but in contrast, it is matory processes and tumor development have been also repeatedly associated to a slight increase in the tumor size in small tumors [99]. stressed [101,102], also depending on the sub-populations of involved Also, in Non-Small-Cell Lung cancer, an inverse correlation between lymphocytes [103]. Among these observations, tumors might develop 8 C. Martínez-Torró et al. Biomedicine & Pharmacotherapy 164 (2023) 114976 Fig. 6. Characterization of tumor infiltrating lymphocytes. A. Representative images of immunohistochemical staining against CD3, CD8 and CD20 proteins in tumor sections. Scale bar: 50 µm. B. Quantification of positive staining per mm2 of alive tumor tissue. Data are shown as mean±SEM. Statistical significance was assessed with a Kruskal-Wallis test and a Dunn post-hoc test (P value was adjusted using the Benjamini-Hochberg Procedure). 9 C. Martínez-Torró et al. Biomedicine & Pharmacotherapy 164 (2023) 114976 tolerance to the immune response through upregulation of PD-L1, an administration, Supervision, Resources, Writing – original draft, Writing immune checkpoint [104]. In the trends of using combined drug for­ – review & editing. mulations in immunotherapy [105], inhibitors of PD-L1 have been developed for such type of synergistic approaches [106]. The presented data demonstrate that the pyroptotic inflammatory Declaration of Competing Interest cell death induced by T22-GSDMDmut-S19-H6 nanoparticles (Figs. 2, 4) was able to release DAMPs and cytokines capable of recruiting TILs In this study, we have developed a new prototype of nano-organized (Fig. 5 C, 6) which, according to the expected outcome [67,107], had an antitumoral drugs based on two different inflammatory proteins, namely impact on the tumor volume size (Fig. 5). Importantly, the cellular Gasdermin D, involved in the pyroptotic cell death pathway and MLKL, events triggered by GSDMD and more moderately by MLKL promote a involved in the necrotic cell death pathway. The effectivity of these new lytic cell death that could engage the antitumor activity of TILs and, drugs has been demonstrated both in vitro and particularly in vivo, in an therefore, inducing immunogenic cell death. The observed cell death animal model of human colorectal cancer. In this model, by the type is in stark contrast to the classical induction of apoptosis by administration of nanoparticles based on the above-mentioned proteins, chemotherapy, which is not immunogenic [108]. In fact, and because of we have observed lymphocyte infiltration in tumor, necrosis and tumor such inflammatory character, even a low level of pyroptosis in tumors volume reduction upon systemic administration, in absence of detect­ appears to be sufficient for recruiting the actors of the immune system able side effects. The novelty of the approach, the easy and scalable relevant to potent anticancer activities [67]. Thus, this novel approach biofabrication of the drugs in cell factories and their potential applica­ based on tumor-targeted pro-inflammatory proteins might be especially tion in nanomedicine makes the study of interest for innovative treat­ suited for the treatment of tumors resistant to chemotherapy by an ments for cancer. artificially induced upregulation of anti-apoptotic proteins [109,110]. In the particular case of colorectal cancer, it is believed that immuno­ Data Availability therapy resistance may be related to insufficient tumor antigen pre­ sentation, tumor antigen presentation damage, T cell exclusion and Data is available on the following link: https://doi. immunosuppressive signaling in the tumor microenvironment [111]. org/10.34810/data735. In the context of the multifaceted nature of human cancer and the increasingly recognized need of synergistic treatments to reach efficient Acknowledgements therapeutic effects [112–115], the induction of a potent pyroptotic event by the treatment with functional protein-only drugs might create The authors are indebted to Agencia Española de Investigación (AEI) new opportunities for a potent multimodal approach to cancer treat­ for granting projects on the construction of protein materials of clinical ment. In this context, the use of oligomeric, virus-like nanoparticles interest (PID2019-105416RB-I00/AEI/10.13039/501100011033 and based on self-assembling, self-delivering concepts [116] might not only PDC2022-133858-I00 to EV and PID2020-116174RB-I00 to AV), to provide precision targeting but also a high concentration of the active AGAUR for projects SGR 2021 00092 to AV and SGR 2021 01140 to RM, drug at the local site of action. and to ISCIII for projects P21/00150 to RM and PI20/00400 to UU co- funded by European Regional Development Fund (ERDF, “a way to 5. Conclusions make Europe”). We also appreciate the support from CERCA Pro­ gramme, Generalitat de Catalunya and from CIBER -Consorcio Centro de The inflammatory proteins GSDMD and the MLKL have been Investigación Biomédica en Red- (CB06/01/0014), Instituto de Salud genetically engineered for their assembly as regular and stable nano­ Carlos III, Ministerio de Ciencia e Innovación. A.V. received an ICREA particles of around 50 nm that are targeted to the tumoral marker ACADEMIA award, the CERCA program and ISCIII CIBER (CB06/01/ CXCR4. Both drug formulations induce a moderate target cell destruc­ 1031) support to RM, and a Postdoctoral fellowship from AECC (Spanish tion in vitro but an important antitumoral effect, in absence of side Association Against Cancer, POSTD20070ALBA) to L.A.C. U.U was toxicity, when intravenously administered in an animal model of colo­ supported by Miguel Servet contract (CP19/00028) from ISCIII co- rectal cancer. The therapeutic impact involves arrest of tumor growth, funded by European Social Fund (ESF investing in your future). We necrosis and specific infiltration of TILs in tumor tissue at a statistically would like to thank the ISCIII for funding CIBER-BBN and the Ministry of significant level. Importantly, both tested proteins induce the activation Science for funding ICTS Platform Nanbiosis. The in vivo study of tumor of the antitumor adaptive immune system mediated by B and T lym­ cell growth was done at the nanotoxicology Unit of the ICTS-141007 phocytes. The observed effects point out the exploration and further Nanbiosis Platform, (http://www.nanbiosis.es/portfolio/u18-nano­ development of tumor-targeted pro-inflammatory proteins as suited toxicology-unit/). The production of the therapeutic proteins was novel drugs for the primary or synergistic treatment of cancer through assisted, in part, by the Protein Production (PPP) Unit of the ICTS the exploitation of immunogenic cell death routes. Nanbiosis Platform of the CIBER-BBN/IBB (http://www.nanbiosis.es/ unit/u1-protein-production-platform-ppp/). 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