Efficient in vivo anti-tumoral effect of the anti-CEA, granulysin-based immunotoxin MFE23GRNLY in a lung adenocarcinoma model. Combination with a Bcl-xL-selective BH3 mimetic

Efficient in vivo anti-tumoral effect of the anti-CEA, granulysin-based immunotoxin MFE23GRNLY in a lung adenocarcinoma model. Combination with a Bcl-xL-selective BH3 mimetic | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficient in vivo anti-tumoral effect of the anti-CEA, granulysin-based immunotoxin MFE23GRNLY in a lung adenocarcinoma model. Combination with a Bcl-x L -selective BH3 mimetic Raquel Ibáñez-Pérez, Ana Pilar Tobajas, Patricia Guerrero-Ochoa, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5446761/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract MFE23GRNLY is an immunotoxin formed by the recombinant fusion of granulysin and the single chain fraction variable (scFv) of the anti-CEA antibody MFE23. Granulysin is a protein stored in the granules of human cytotoxic T lymphocytes (CTL) and NK cells with antimicrobial and antitumoral activity. Previous work from our group demonstrated that recombinant MFE23GRNLY preserves the cytotoxic activity of GRNLY and the specific binding to CEA, targeting efficiently the antitumoral activity of GRNLY towards CEA-expressing tumors after systemic injection in vivo . In the present work we observed that the lung adenocarcinoma A549 expressed CEA and was sensitive to the immunotoxin in vitro . We have then demonstrated the efficacy of MFE23GRNLY against this tumor in vivo, broadening the application of the immunotoxin to the treatment of lung tumors. Furthermore, we attempted tu unveil MFE23GRNLY mechanism of action compared to GRNLY on HeLa-CEA and on A549 cells. We demonstrated that MFE23GRNLY treated human tumor cells showed signs of ER stress and autophagosome accumulation. Remarkably, the general caspase inhibitor Z-VAD-fmk prevented MFE23GRNLY-induced death of HeLa-CEA cells but not of A549 cells, a feature that correlated with high levels of expression of the anti-apoptotic protein Bcl-x L in A549 cells. Accordingly, the combination of the BH3 mimetic A1155463, inhibitimg the activity of the anti-apoptotic member of the Bcl-2 family Bcl-x L , with MFE23GRNLY, showed a potent in vitro synergistic effect with MFE23GRNLY in A549 cells. However, when we combined both agents in vivo in athymic mice xenotransplanted with A549 cells, the synergistic effect was not observed, although the anti-tumoral potential of the systemic injection of the immunotoxin was again confirmed. immunotoxin scFv CEA granulysin apoptosis Bcl-xL Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction MFE232GRNLY is an immunotoxin resulting from the fusion of GRNLY, a cytolytic human protein, and the scFv of MFE23, an anti-carcinoembryonic antigen (CEA) antibody, described in a previous work if our group (1). The carcinoembryonic antigen (CEA), also known as CEACAM5 (2) was originally described in colorectal cancer (CRC) (3) as one of the first tumor-associated antigens, although it is also expressed at lower levels in normal tissues. Serum levels of CEA are useful in the prognosis, clinical control and monitoring the response to treatment of CRC (4). The expression of CEACAM5 in at least a fraction of primary lung tumors, especially in small-cell lung cancer, has also been described (5, 6). Although CEA is secreted from tumor cells, it has been shown that the antitumoral effects of bispecific anti-CEA x anti-CD3 antibodies (7) or of anti-CEA targeted CAR T cells (8) are not inhibited by soluble CEA. On the other hand, antibody-drug conjugates or immunotoxins based on CEA targeting are being explored in clinical and preclinical assays especially for CRC treatment The cytotoxic 9 kDa isoform of granulysin (GRNLY) is present in the granules of activated human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells (9). This protein shows cytolytic activity against a wide variety of microbes, such as bacteria, fungi and protozoan parasites and its physiological role is related with killing intracellular bacteria such as M. Tuberculosis in concert with perforin (10-14). It also acts against protozoan parasites in concert with granzyme B (15). Our group demonstrated that GRNLY was able to induce apoptotic cell death in several types of tumor cell lines in vitro (16, 17) and also on ex vivo samples from B-CLL patients, without showing toxicity against peripheral mononuclear cells (PBMC) from healthy donors (16). Afterwards, we demonstrated its antitumor potential by intratumoral injection in in vivo xenotransplantation models of human tumors in athymic mice (18, 19). In order to achieve tumor targeting after systemic injection, we developed the anti-CEA MFE23GRNLY immunotoxin and demonstrated its increased efficiency in eliminating CEA-expressing tumors in vivo , specifically the colon carcinoma HT-29 and the cervix carcinoma HeLa transfected with CEA, HeLa-CEA cells (1). In line with this, we have also developed anti-MUC1-Tn granulysin-based immunotoxins and demonstrated their in vivo anti-tumoral potential (20), although these immunotoxins had lower stability or yield of production than MFE23GRNLY. In the present work, we have first demonstrated the efficiency of MFE23GRNLY against the CEA-expressing human lung carcinoma A549 and analysed the mechanism of cell death induced by the immunotoxin compared with GRNLY alone in HeLa-CEA and A549 cells, finding interesting connections between the type and extent of cell death induced in the lung model and the expression of Bcl-x L , that have been also explored in vivo . Methods Cell culture A549 lung adenocarcinoma, HT-29 colon adenocarcinomas, HeLa cervix carcinoma and HeLa cervix carcinoma stably transfected with CEA (HeLa-CEA cells) were cultured in DMEM medium (Gibco, Barcelona) supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin (all of them from Pan Biotech, Aidenbach, Germany) and GlutaMAX (Invitrogen, Barcelona), at 37ºC and 5% CO 2 using standard procedures. All cell lines were routinely tested for mycoplasma contamination by PCR. Fungi culture Pichia pastoris was grown for 24h at 30°C with agitation at 250 rpm in BMGY medium (1% yeast extract, 2% peptone, 1.34% YNB, 1% glycerol, 400 μg/L biotin, and 0.1 M potassium phosphate, pH 6.0). Then, BMGY medium was changed to BMM medium (1.34% YNB, 0,5% methanol, 400 μg/L biotin, 0,03% Anti-foam 204, 1% casamino acids and 0.1 M potassium phosphate, pH 6.0) and incubated at 18°C and 250 rpm for 24h. Subsequently, 1% methanol was added and yeast cultured for 48h. Expression and purification of recombinant granulysin and of the granulysin-MFE23 immunotoxin in Pichia pastoris The P. pastoris strain SMD1168 transfected with GRNLY or with MFE23GRNLY was cultured in BMGY medium at 30 °C in a shaking incubator (250 rpm) overnight. The following day, the culture was transferred to BMMY medium at and incubated overnight at 18 °C in a shaking incubator (250 rpm) overnight for induction. For protein secretion induction, the culture was supplemented with 1.5 % methanol (v/v) every 24 h for 2 days. Subsequently, supernatants were collected and proteins were purified using by Ni 2+ affinity chromatography (Ni-NTA agarose, Qiagen). Finally, the elution buffer was replaced by PBS by dialysis. Flow cytometry analysis of CEA binding Analysis of the binding of both MF23 or MF23GRNLY to the CEA antigen on the surface of living cells was performed as follows: 10 5 cells per well were placed in a 96-well roundbottom plate and incubated with MF23 or with MFE23GRNLY (0,2 μM) in PBS supplemented with 5% FBS for 30 min at 4°C followed by incubation with a mouse anti-histidine tag antibody (1:500; Genscript, Netherlands) and goat anti-mouse antibody bound to APC (1:500; Biolegend, San Diego) or FITC (1:500; Caltag, Barcelona). After each incubation step, cells were washed with in PBS supplemented with 5% FBS. CEA-binding specificity was determined on CEA-positive HeLa-CEA cells and compared to HeLa cells (negative for CEA expression), using a FACScalibur flow cytometer (BD Biosciences, Madrid). Western blot After SDS-electrophoresis, the proteins were transferred to a nitrocelulose membrane. Then, membranes were blocked overnight at 4ºC with agitation with TBS-T buffer (10 mM Tris / HCl, pH 8.0, 0.12 M NaCl, 0.1% Tween-20, 0.05% thimerosal) containing 5% non-fat milk. Afterwards, membranes were washed with TBS-T buffer and incubated with primary antibodies against diferent proteins (see Table 1), diluted in TBS-T containing 5% BSA. After that, membranes were washed again with TBS-T and incubated with 0.2 µg/mL of the secondary antibody conjugated with peroxidase (Sigma). Finally, proteins levels were determined by chemiluminescence using the peroxidase substrate Pierce ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA). Table 1. Antibodies used for western blot. Antibody Company β-Actin Sigma BCL-xL/S (2H12) Santa Cruz Biotechnology CHOP Santa Cruz Biotechnology EIF2α-P (Ser51) Cell Signaling Technology LC3B Sigma MCL1 (S-19) Santa Cruz Biotechnology Noxa (114C307) Santa Cruz Biotechnology p62 (D-3) Santa Cruz Biotechnology PUMA (G-3) Santa Cruz Biotechnology α-Tubuline Sigma In vitro cytotoxicity assays 3x10 4 suspension cells or 1.5x10 4 adherent cells were seeded per well in 96-well plates. Adherent cells were seeded in the plates 24h before the experiment begins and medium was refreshed just before the incubation with the cytotoxic proteins. Subsequently, GRNLY or MFE23GRNLY were added at the indicated concentrations. In control wells, equal volumes of PBS were added. In some experiments, cells were preincubated for 1h in the presence or absence of 100 µM Z-VAD-fmk or 20 µM NEC-1 alone or in combination followed by the incubation with GRNLY or MFE23GRNLY for 24 h. In other experiments cells were preincubated for 1h in the presence or absence of 100 µM Z-VAD-fmk or 2 µM A-1155463 alone or in combination, followed by the incubation with GRNLY or MFE23GRNLY for 48h, with Z-VAD-fmk refreshing evey 24h. Then, cells were incubated at 37ºC with Annezin-V and t-AAD in annexin-binding buffer (140 mM NaCl, 2.5 mM CaCl 2 ,10 mM Hepes/NaOH, pH 7.4) for 15 min at room temperature and cell death analysed by flow cytometry using a FACSCalibur (BD Bioscience, Madrid). Autophagosome detection by CYTO-ID® Autophagy Detection Kit Cells (5x10 4 per well) were seeded in 48-well plates and were treated with GRNLY or MFE23GRNLY for 12h. Then, cells were stained with CYTO-ID® Autophagy Detection Kit (Enzo Life Sciences, Farmingdale, USA) as per manufacturer’s specifications and analysed by flow cytometry. In addition, fluorescence microscopy images were taken as follows: 5x10 4 cells were seeded per well in 24 well plates and were treated with GRNLY or MFE23GRNLY for 12h. Then, cells were simultaneously stained with CYTOID® Autophagy Detection Kit and Hoechst 33342 (Thermo Fisher, Barcelona) nuclear dye and photographed in a E600/E400 Nikon fluorescence microscope equipped with a digital camera (DXM1200F, Nikon). I n vivo experiments Immune-deficient athymic mice, Swiss nu/nu strain (Charles River, Barcelona), six-week-old males, were used in the present study. All procedures were carried out under Project Licence PI38/23 approved by the Ethic Committee for Animal Experiments from the University of Zaragoza. The care and use of animals were performed accordingly with the Spanish Policy for Animal Protection RD53/2013, which meets the European Union Directive 2010/63 on the protection of animals used for experimental and other scientific purposes. Mice were kept under specific standard pathogen-free conditions (average ambient temperature 24°C, 12/12-h light/dark cycle) with water and food provided ad libitum throughout the study. GRNLY and MFE23GRNLY experiments 2 x 10 6 A549 tumor cells were subcutaneously injected in athymic mice. Treatments were administered when tumors reached approximatively 50 mm 2 in size. GRNLY and MFE23GRNLY groups received in total 8 intraperitoneal injections every 2 days at the concentration of 10 nmol diluted in PBS before euthanasia. Control group received intraperitoneal injections of PBS following the same treatment schedule as GRNLY and MFE23GRNLY. Tumor growth was daily measured using a precision caliper and tumor area was calculated by collecting the width and length of the tumor. Tumors from euthanized mice were surgically excised, measured, weighed and stored in a preservation solution (10% buffered formalin) to perform histological analysis. MFE23GRNLY and A-1155463 combination experiments 5 x 10 6 A549 tumor cells were subcutaneously injected in nude mice. Treatments were administered when tumors reached approximately 35 mm 2 in size. MFE23GRNLY-treated groups received 10 intraperitoneal injections 3 times a week (5 nmol/dose diluted in PBS). A-1155463-treated groups received 6 intraperitoneal injections (260 nmol/dose in 5% DMSO, 10% ethanol, 20% Cremophor® RH-40 and 65% dextrose (5% in water)), twice a week. Control group received intraperitoneal injections of PBS following the same schedule than the MFE23GRNLY-treated group. Tumor growth was monitored by measuring the tumor 3 times a week with a precision caliper by measuring the width and length of the tumor. Tumors from euthanized mice were surgically excised, measured, weighed and stored in a preservation solution (10% buffered formalin) to perform histological analysis. Histological studies Hematoxylin-eosin staining Tissue for light microscopy was fixed in 4% formaldehyde and embedded in paraffin using routine procedure. Sections 5 μm thick were cut from the tissue blocks and deparaffinized by immersion in two changes of xylene for 5 min each. After that, tissue sections were rehydrated by immersion in decreasing concentrations of ethanol (immersion in separated baths of 100%, 95% and 70% of ethanol for 2 min/each bath). The nuclei of the cells in tissue sections were stained by immersing in GILL II Hematoxylin for 3 min. Sections were rinsed in HCl 0.1% for 5 seconds and then, in tap water for 3-5 min. Cell cytoplasm was stained by immersing in 0.5% of eosin containing 0.2% of glacial acetic acid, for 3 min and dipped once quickly in water for few seconds. After eosin staining, sections were dehydrated by immersion in ascending alcohol solutions (70%, 95% and absolute alcohol 100%) for 5 seconds/each. Finally, all sections were cleaned by xylene for 5 seconds, dried at room temperature and mounted with DPX using glass coverslips Fluorescence study of apoptotic nuclei and immunohistochemistry for detection of activated caspase-3 and NK cell infiltration Tissue for fluorescence microscopy was fixed in 4% formaldehyde and embedded in paraffin using routine procedure. Sections of tumor samples (4 μm thick) were deparaffinized in xylene, hydrated through a graded series of ethanol, and then immersed in 3% hydrogen peroxide in 100% methanol for 30 min to inhibit endogenous peroxidase activity. For antigen retrieval, the sections were boiled in 10 mM citrate buffer, pH 6.0 for 30 min. Staining of nuclei was performed using DAPI and detected using a fluorescence microscope (E600/E400, Nikon) equipped with a digital camera (DXM1200F, Nikon). The detection of activated caspase-3 or of NK cell infiltration was performed by immunohistochemistry using rabbit polyclonal antibodies either anti-human cleaved caspase-3 antibody (Cell Signalling, Barcelona) or anti-NKp46 (Biorbyt, Cambridge, UK). After rinsing the samples with PBS-Tween 20 for 2 times (2 min each), the sections were incubated with 200 μl blocking solution (5% rabbit or horse serum diluted in PBS) for 1hr at room temperature, and then incubated overnight at 4°C in humid chambers with the primary antibodies at 1/200 dilution. Labeling was revealed using DAB staining. Isotype control staining was also performed, giving no substantial signal in the experimental conditions described. Statistical analysis Computer-based statistical analysis was performed using GraphPad Prism 4.0 program (GrandPath Software Inc). Results are shown as mean±SD. Statistical significance was evaluated by using two-way ANOVA for in vitro assays and multiple t-test analysis, using the Holm-Sidak method or the Welch’s t-test analysis for in vivo assays. Differences were considered significant if p <0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Results Binding of GRNLY and MFE23GRNLY to HeLa-CEA and A549 cells In our previous study, we demonstrated the specific binding of MFE23GRNLY to the purified CEACAM5 antigen by ELISA, showing a similar titration pattern than the MFE23 scFv. We also showed that it bound to the antigen expressed on the surface of CEACAM5 + cells, such as the colon adenocarcinoma HT-29 or HeLa cells transfected with CEA (HeLa-CEA cells) (1). To confirm CEACAM5 expression on the surface of lung adenocarcinoma A549 cells, we performed a flow cytometry assay using the MFE23 scFv moiety alone. As shown in Fig 1A, HeLa cells proved to be negative for CEACAM5 expression, while HeLa-CEA cells showed a high surface expression of the antigen, with A549 cells showing an intermediate, but significant level of expression. Then, we tested the binding of MFE23GRNLY to the lung adenocarcinoma A549, and compared with HeLa-CEA staining as a positive control and with HeLa cells as a negative control (Fig 1B). In these experiments, we also tested GRNLY binding to the surface of these cells, since it is a protein that interacts with membrane phospholipids (21). In HeLa cells, the binding of the immunotoxin was indistinguishable from that of GRNLY alone, indicating no contribution of the antibody moiety to this binding, due to the absence of CEA expression on the surface of these cells. On the contrary, a substantial shift of the labelling was observed in HeLa-CEA cells incubated with the immunotoxin, showing a 3-fold increase in MFI compared to GRNLY. A similar situation was observed in the case of A549 cells, further confirming the presence of the CEACAM5 antigen on their surface. Dose-response of GRNLY and MFE23GRNLY on CEA-expressing tumor cell lines Next, we performed complete dose-response cytotoxicity studies on HeLa-CEA and A549 cells, to calculate the corresponding IC 50 (Fig 2A). As previously shown (1), MFE23GRNLY was more cytotoxic than GRNLY alone on HeLa-CEA cells, with an IC 50 of 6 µM vs. 25 µM. A similar situation was observed for A549 cells, with an IC 50 of 4 µM for MF23GRNLY vs. 10 µM for GRNLY alone (Fig 2B). Altogether, these data would suggest the potential use of MFE23GRNLY not only for the treatment of colon or gastric tumors, but also for CEACAM5 + lung tumors. In vivo experiment on athymic mice xenografted with A549 tumor cells To test this hypothesis, we performed in vivo experiments using systemic administration of the treatments in a model of A549 cells xenotransplanted in athymic mice (Fig 3A). When recombinant granulysin was administered systemically (i.p.), it did not result in a significant reduction in tumor growth (Fig 3B). However, administration of the immunotoxin clearly reduced tumor growth, being this reduction statistically significant from the 1st injection onwards (Fig 3C). Relative to the control group, mean tumor growth was reduced around 50% by the immunotoxin at the end of the experiment. This reduction was also observed when the size (Fig 3D) or weight (Fig 3E) of the resected tumors were analysed. Next, we performed histological studies on tissue sections obtained from resected tumors. Hematoxylin/eosin staining (H/E in Figure 4) showed that untreated tumors presented a compact tissue structure with high cellularity (upper left panel), while tumors from GRNLY-treated mice showed somewhat lower cellularity (middle left panel). By contrast, in the case of tumors from MFE23GRNLY-treated mice a dramatic loss of cellularity was observed, with most of the sections occupied by unstained stroma (lower left panel). Upon DAPI labeling, untreated tumors displayed nuclei with non-condensed chromatin (Fig 4, DAPI column, upper middle panel), a phenotype that was the predominant in tumors from mice treated by systemic GRNLY injection, although some cells with condensed chromatin were also observed (arrows, Fig 4, bottom middle panel). Resected tumors from MFE23GRNLY-treated mice showed some cell with condensed chromatin and fragmented nuclei (indicated with arrows in the lower panel). However, the major phenotype in this case corresponded to cells with perinuclear-condensed chromatin (indicated by circles in the same image). This nuclear morphology was also observed in our previous in vivo study using HeLa-CEA cells (1), being characteristic of the caspase-independent apoptosis-inducing factor (AIF)-mediated cell death (22). Finally, active caspase-3 immunochemistry analysis provided some limited staining in tumors from GRNLY-treated mice (Fig 4, caspase-3 column, middle right panel), while a relative increase was observed in the case of tumors derived from MFE23GRNLY-treated mice (lower right panel). MFE23GRNLY induces ER stress Once demonstrated the in vivo efficacy of MFE23GRNLY against A549-derived tumors, we sought to determine the cell death mechanism elicited by the immunotoxin on tumor cells. Alterations in the endoplasmic reticulum (ER) folding environment are known to cause the accumulation of misfolded proteins, leading to ER stress. To deal with this and to restore normal ER functions, cells activate the unfolded protein response (UPR) via the induction of protein kinase RNA-like endoplasmic reticulum kinase (PERK). The interaction of the chaperone BiP (Binding immunoglobulin Protein) with misfolded proteins releases PERK, which phosphorylates eIF2α, inhibiting protein synthesis and inducing the selective translation of Activating Transcription Factor 4 (ATF4). ATF4 subsequently induces the upregulation of proapoptotic factor C/EBP homologous protein (CHOP), a biomarker of the ER stress response (23). Recombinant GRNLY induces an alteration in plasma membrane structure resulting in intracelullar [Ca 2+ ] increase (21, 22, 24-26), which in turn could induce ER stress. Hence, we analysed the expression of proteins involved in the PERK pathway in tumor cell lines treated with MFE23GRNLY or with GRNLY. We clearly observed in both A549 and HeLa-CEA cells a down-modulation of BIP chaperone expression and an increase in CHOP levels upon MFEGRNLY incubation. In the case of GRNLY, we observed a similar pattern for BIP expression, although we did not detect an increase in CHOP expression in A549 cells. Altogether, these data indicate that tumor cells treated with GRNLY, and especially with MFE23GRNLY, suffer activation of the PERK pathway and ER stress (Fig 5). In addition, an increase in eIF2α phosphorylation was observed in A549 cells treated with GRNLY or MFE23GRNLY (Fig 5A), but not in HeLa-CEA cells (Fig 5B). MFE23GRNLY induces incomplete authophagy Autophagy is a fundamental cell survival mechanism that allows cells to adapt to metabolic stress through the degradation and recycling of intracellular components to generate macromolecular precursors and produce energy.We first explored the formation of autophagosomes upon treatment with GRNLY or MFE23GRNLY using the Cyto-ID flow cytometry technique (27). As shown in Fig 6A , GRNLY and the immunotoxin clearly induced autophagosome formation on HeLa-CEA and A549 cells, albeit the immunotoxin was more active at the same concentration. Autophagy induction could be also clearly detected by fluorescence microscopy in A549 cells, showing the formation of multiple cytoplasmic punctate structures corresponding to autophagosomes (Fig 6B) . We also analysed the lipidation of the LC3 protein and the expression levels of p62 as other hallmarks of autophagy induction, that can be detected by immunoblot. The lipidation of LC3 was detected by immunoblot through the conversion of LC3-I to LC3-II. As shown in Fig 6C, MFE23GRNLY increased the level of LC3BII, while keeping constant the levels of p62 in both HeLa-CEA and A549 cells. These results suggests that autophagy was initiated as demonstrated by the formation of autophagosomes, but it was not completed, since p62 was not induced neither finally degraded. This observation is compatible with the phenotype of cells observed by phase-contrast microscopy after being treated with GRNLY or MFE23GRNLY, in which abundant cytoplasmic punctate structures, compatible with autophagosome accumulation, were clearly detected before the completion of cell death (Supplementary Fig 1). In addition, blocking the initiation of autophagy by 3-Methyladenine (3-MA), a widely used inhibitor of class III PI3K, in HeLa-CEA cells treated with GRNLY or MFE23GRNLY, resulted in an increase of the percentage of cell death (Supplementary Figure 2). Therefore, these data indicate that autophagy activation induced by GRNLY or MFE23GRNLY treatments seems to be a protective process put in place by cells to cope with stress and manage to survive. MFE23GRNLY death mechanism is dependent on the cell line analysed It was previously described that GRNLY induced cell death mainly through the mitochondrial apoptotic pathway (22, 25, 28, 29), which results in the caspase-dependent phosphatidylserine (PS) exposure on the external membrane hemileaflet and final membrane permeabilization. PS exposure can be analysed by Annexin-V staining and membrane permeabilization by 7-AAD nuclear staining using flow cytometry. In the subsequent cytotoxicity experiments we have used the IC 50 values for GRNLY and for the immunotoxin calculated for each molecule in the second section of Results (see Fig 2). To study the mechanism of cell death induction by MFE23GRNLY as compared with GRNLY we used inhibitory molecules of known death pathways. The general caspase inhibitor Z-VAD-fmk significantly prevented death in HeLa-CEA cells induced by MFE23GRNLY, although only partially (from 50% to 30%), while the necroptosis inhibitor necrostatin-1 (NEC-1) had no effect either alone or in combination with Z-VAD-fmk (Fig 7A). Similar results were observed with GRNLY, although in this case NEC-1 also partially prevented cell death, suggesting that GRNLY is able to partially induce necroptotic death in these cells. In our previous work, a minor necroptotic component was also observed in GRNLY-induced cell death in other cell lines, especially if caspases were inhibited (16). Remarkably, in A549 cells treated with MFE23GRNLY or with GRNLY (Fig 7B), no effect on cell death was oberved when combined with Z-VAD-fmk or NEC-1, suggesting the activation of a caspase-independent cell death pathway different to necroptosis. Willing to understand the cell death mechanism in A549 cells, we analysed the expression of pro- and anti-apoptotic proteins of the Bcl-2 family before and after treatment with the same concentrations of the recombinant cytotoxic proteins. The expression of pro-apoptotic BH3-only proteins like PUMA and Noxa were increased in MFE23GRNLY-treated A549 cells. However, anti-apoptotic proteins like Mcl-1 and Bcl-x L were also substantially increased (Fig 8A). In the case of HeLa-CEA cells, an increase in Puma expression, but also in Mcl-1, was clearly observed (Fig 8A). Connecting these results with the observed induction of ER stress, it has been described that ER stress promotes p47 and/or p53 activity, being Puma and Noxa targets of these transcription factors (30, 31). In Suppl. Fig 3, it can be observed that, especially after MFE23GRNLY treatment of A549 cells, an increase in the expression of p47 is clearly observed. It was previously described that GRNLY was able, in addition to activate the mitochondrial apoptotic pathway leading to caspase activation, to induce the translocation of AIF from mitochondria to the nucleus, a process that leads to caspase-independent cell death (22, 32). AIF is sytnthesized as a precursor of about 67 kDa and it is then imported to mitochondria, where it is processed to a 62/64 kDa mature form. Upon exposition to cell death-inducing agents, AIF is processed into a 57 kDa and lower molecular weight forms, which are translocated to the cytoplasm and finally to the nucleus (33, 34). In Suppl Fig 4, we show that, especially in the case of A549 cells treated with MFE23GRNLY, a processing of AIF to truncated isoforms with MW lower than 62 kDa took place, suggesting the efficient activation of this pathway. These results suggest that although A549 cells increased expression of pro-apoptotic proteins levels in the response to the treatments, they were protected from apoptosis leading to the execution of an alternative cell death pathway. Remarkably, if analyzing the basal expression of anti-apoptotic members of the Bcl-2 family in several tumor cell lines, we found that basal Bcl-x L expression was very high in A549 cells, while the expression of this protein was moderate in HeLa-CEA cells, inversely correlating with the sensitivity to Z-VAD-fmk inhibition of cell death in these cell lines (Fig 8B). We reasoned that the protection from caspase-dependent cell death observed in A549 cells could be due to the high basal Bcl-x L expression detected in this particular cell line and that it could be reversed by the BH3 mimetic Bcl-x L inhibitor A1155463. As shown in Fig 9A, the combination of A1155463 with GRNLY or with MFE23GRNLY greatly increased cell death in this cell line, and, in addition, this increase was completely abrogated by the general caspase inhibitor Z-VAD-fmk. The effect observed was synergic, since there were statistically significant differences when comparing the expected additive cell death with the result obtained (Fig 9B). It should be noted that the synergy of A1155463 with GRNLY or MFE23GRNLY was achieved at a low 2 µM concentration, that is not toxic for A549 cells, with overt cytotoxicity obtained only at a 10-fold higher concentration, 20 µM (see Fig 9C). In vivo experiment combining MFE23GRNLY with the BH3 mimetic A1155463 We next explored this remarkable synergy in the same in vivo model described in Figures 3 and 4. In order to achieve the same synergistic effect observed in vitro , we reduced the dose of MFE23GRNLY used in the previous experiments from 10 to 5 nmol per injection (9.4 mg/Kg). In the case of A1155463, we also lowered the 10 mg/Kg dose reported in previous in vivo studies (35-37) to a dose of 7 mg/Kg (260 nmol per injection). In addition, we followed a 72h injection schedule to give time to the animals to recover from its platelet toxicity, as reported in previous studies (35, 37). Hence, for the 10 MFE23GRNLY injections performed in the study, following the described 48h schedule, mice in the A1155463 groups received 6 injections of the agent, as indicated in the protocol scheme shown in Figure 10A. As shown in Figure 10B, the BH3 mimetic had no effect on the growth of tumors generated by the A549 lung adenocarcinoma. However, when the recombinant immunotoxin was administered systemically (i.p.), the effect was similar to that observed in the previous experiments, clearly reducing tumor growth, being this reduction statistically significant from the 5th injection onwards (Fig 10C). Relative to the control group, mean tumor growth was reduced more than 40% by the immunotoxin at the end of the experiment. When analyzing the combined effect of A1155463 and MFE23GRNLY (Fig 10D), the result was highly variable across the animals included in this experimental group. While in four mice the combination had a similar effect than MFE23GRNLY alone or even in two of these mice the combined effect was higher, in two other mice, the combination was much less effective than MFE23GRNLY alone. Remarkably, when we analysed the volume and weight of resected tumors after the end of the experiment, the variability described above was not reproduced. In this analysis, both the immunotoxin and its combination with the BH3 mimetic significantly reduced the volume and weight of the tumors by 50% (Figs 10E and 10F). In any case, the synergic effect observed in vitro between both agents was not reproduced in vivo , and the effect of the combination was equivalent to the effect obtained with the immunotoxin alone. Additionally, we performed histological studies on tissue sections obtained from resected tumors (Figure 11A). Hematoxylin/eosin staining showed that tumors from the control group or from the group treated with A1155463 presented a compact tissue structure with high cellularity, while tumors from MFE23GRNLY-treated mice, alone or in combination with A1155463 showed a great cellularity loss, with big sections occupied by unlabeled stroma. Upon DAPI labeling, untreated tumors or tumors treated by A1155463 displayed nuclei with non-condensed chromatin. In the case of resected tumors from MFE23GRNLY-treated mice, some cells with condensed chromatin and/or fragmented nuclei were observed (indicated with arrows), being in fact more abundant the cells with the AIF-related perinuclear-condensed chromatin (indicated with circles). When combined with A1155463, DAPI labeling showed a rather increased number of cells with condensed chromatin and fragmented nuclei (arrows) relative to cells with the mentioned AIF-related nuclear phenotype (circles). Finally, active caspase-3 immunochemistry showed no labelling in control or A1155463-treated mice (upper panels), a limited labeling in the case of tumors from MFE23GRNLY-treated mice (lower-middle panel), and a somewhat increased labeling in the case of tumors from mice treated with the combination of the immunotoxin with the BH3 mimetic (lower panel). Results regarding NK cell infiltration, obtained by anti-NKp46 immunochemistry, are shown in Fig 11B. No NK cell infiltration was observed in control or A1155463-treated mice, while a clear NK infiltration was observed in all samples from the MFE23GRNLY experimental group. In the case of tumors treated with the combination of A1155463 with MFE23GRNLY, while in those mice in which tumor growth was no apparently affected by the treatment (see Figure 10D; indicated as “high-volume tumors” in the lower panels of Fig 11B), no NK infiltration was observed, the infiltration was present in the tumor which size was even smaller than that of the mean of the MFE23GRNLY treated group (see Fig 10D; indicated as “small-volume tumor” in the lower panels of Fig 11B). Discussion We have previously demonstrated in vitro and in vivo that the immunotoxin MFE23GRNLY notably increases the cytotoxic activity of GRNLY against CEA-expressing human tumor cell lines. Now, we report molecular events that take place when tumor cells were treated with this immunotoxin and which could help to understand its death-inducing mechanism. ER is an essential site for the regulation of apoptotic pathways and it has also been recently recognized as an important component of autophagic signaling (38). Autophagy is a mechanism which enables the elimination of unfolded or misfolded proteins under ER stress conditions (39). Hence , ER stress and autophagy are intimately connected under cell stress conditions and their cooperation can result in both survival or death induction (38). If the overload of unfolded or misfolded proteins in the ER is not resolved, the prolonged UPR will induce ER stress-associated apoptosis (40), probably through CHOP expression (41). In addition, CHOP is involved not only in apoptosis induction, but also in the suppression of autophagy interacting with LC3B (41, 42). ER stress is also connected with the mitochondrial apoptotic pathway through the increase in expression and activation of several BH3-only pro-apoptotic members of the Bcl-2 family such as Puma and Noxa (43, 44), which expression is effectively increased by the immunotoxin. Saini et al. described that GRNLY delivered by cytotoxic T cells induces endoplasmic reticulum stress, but not recombinant GRNLY (26). However, we have demonstrated that recombinant GRNLY and also MFE23GRNLY are able to induce ER stress in the cells used in this study. The increased cytotoxic effect of the immunotoxin could be related with the higher amount of GRNLY brought to the cells surface by the scFv moiety in CEA-expressing cells. It has been described that ER stress promotes p47 and/or p53 activity (30), and we also demonstrate that p47 levels increase upon incubation with the immunotoxin, correlating with the induction of expression of its targets Puma and Noxa. In addition, Noxa can be also induced by CHOP activation. It was previously described that the main type of cell death produced by GRNLY was apoptosis through the mitochondrial apoptotic pathway (16, 17, 22, 25, 29)and in the present work we have confirmed those observations also for MFE23GRNLY-induced cell death in HeLa-CEA cells. However, we have also observed that A549 cells treated with GRNLY or with MFE23GRNLY suffer a caspase-independent cell death different from necroptosis, suggesting that other pathways could be involved. One of such pathways could be the caspase-independent cell death induced by AIF translocation from mitochondria to nucleus, which our group demonstrated that was also activated by GRNLY (22). In vitro data and also DAPI stainings performed in tumor sections from the in vivo experiments would support this hypothesis. We have also shown that the low efficiency in activating caspase-dependent cell death in A549 cells correlates with a high expression of the anti-apoptotic member of the Bcl-2 family Bcl-x L , and that this effect is completely reversed by the BH3 mimetic Bcl-x L inhibitor A1155463. The fact that MFE23GRNLY showed clear antitumor effects in athymic mice bearing A549 cancer xenografts when administered systemically broaden the possible clinical application of the immunotoxin not only to colon or gastric carcinomas, but also to CEA-expressing lung tumors. In addition, we showed that it exerts a remarkable synergic effect when combined in vitro with A1155463, a Bcl-x L specific inhibitor. This result suggested that the combination of this inhibitor with the immunotoxin could also increase the antitumor potential of this immunotoxin in vivo . However, although we confirmed A549 tumor targeting by the immunotoxin even at a lower dose, we did not observe the synergistic effect with the BH3 mimetic in vivo . BH3 mimetics, that inhibit specific anti-apoptotic proteins of the Bcl-2 family which are over-expressed in many cancer types, are a therapeutic option against cancer that has been extensively explored in the pre-clinical setting (45) and that has also reached the clinic. In 2018, the specific anti-Bcl-2 BH3 mimetic inhibitor venetoclax was approved for the treatment of chronic lymphocytic leukemia (CLL) of small cell lymphocytic leukemia (SLL); and in 2020 it was approved for the treatment of acute myeloid leukemia (AML) in combination with azacitidine, decitabine, or low-dose cytarabine (46, 47). A1155463 is another BH3 mimetic that specifically targets Bcl-x L . It has been observed that Bcl-x L inhibition induces platelet toxicity in experimentation animals, but that an on-off schedule of administration allows a good platelet function recovery (35, 37). A 10 mg/Kg dose has shown a partial retardation of tumor growth in an in vivo model of lung cancer (37), and good results in different types of leukemia, especially if combined with venetoclax (35). In our hands, we did not find any effect of A1155463 alone on the development of A549-derived tumors when used as a single agent. This could be due to the use of a lower dose compared with other works such as the one carried out by Tao et al., although in that study the effect as a single agent was only partial (37). However, we also lost the powerful synergistic effect observed in vitro on A549 cells, characterized by a high over-expression of Bcl-x L . The athymic mice used in our in vivo studies are almost completely devoid of T cells, and the humoral immune response mediated by B cells is also profoundly affected. However, these mice have more NK cells than wild-type mice, showing also a higher activation state. In our initial studies, we observed that intratumoral injection of granulysin induced a massive infiltration of NK cells in the xenografted tumors, indicating that the therapeutic effect of granulysin could be mediated, at least in part, by this intratumoral NK cell infiltration elicited by granulysin (18, 19). We confirmed this NK cell infiltration when systemically administering the MFE23GRNLY immunotoxin in athymic mice xenografted with HeLa-CEA cells (1, 48). NK cell development and survival is dependent on the anti-apoptotic proteins of the Bcl-2 family Mcl-1 and Bcl-2 itself, but to a lesser extent on Bcl-x L (49). However, NK cell activation is associated with a remarkable increased expression of Bcl-x L , that could be important for their survival and anti-tumoral function (50). If, at the same time that A1155463 is inducing A549 cell death in synergy with MFE23GRNLY, it is also negatively affecting NK cell infiltration and anti-tumoral function in vivo , then the global effect of the combination would be diminished. The NK cell infiltration data obtained indicates that this could be the case, although further studies will be needed to be certain about this possibility. In conclusion, MFE23GRNLY is an immunotoxin that causes ER stress and autophagosome accumulation in tumor cells, generally culminating in apoptotic cell death, except in cells overexpressing Bcl-x L , that seems to be executed by a caspase-independent mechanism. Overall, this ER stress-induced apoptosis and the possible synergy with Bcl-x L inhibitors could be an important strategy for future therapeutic approaches, also in lung tumors. Declarations Funding This work was supported by Gobierno de Aragón grant B31_23R and by the company Peaches Biotech. RIP and APT were contracted by the grant CPP2021-008506 financed by MCIN/AEI/10.13039/501100011033 and by the European Union-NextGenerationEU/PRTR Competing interests The authors have no relevant financial or non-financial interests to disclose Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Raquel Ibáñez-Pérez, Ana Pilar Tobajas, Patricia Guerrero-Ochoa, Ruth Soler-Agesta, Eduardo Romanos (animal experimentation), Eva Barrio (animal experimentation), Blanca Conde (animal experimentation), Isabel Marzo, Javier Naval, Laura Sanz and Alberto Anel. The first draft of the manuscript was written by Alberto Anel and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request Ethics approval Animal experimentation studies were carried out under Project Licence PI38/23 approved by the Ethic Committee for Animal Experiments from the University of Zaragoza. The care and use of animals were performed accordingly with the Spanish Policy for Animal Protection RD53/2013, which meets the European Union Directive 2010/63 on the protection of animals used for experimental and other scientific purposes. Acknowledgments The authors thank Cindy Giraldo and Alejandro Ibáñez from the microscopy and histopathology Core Unit, Institute for Health Sciences of Aragon (Zaragoza, Spain), for their advice and technical support. 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Supplementary Files SupplementalMaterial.doc Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 05 Sep, 2025 Reviews received at journal 17 Aug, 2025 Reviewers agreed at journal 31 Jul, 2025 Reviews received at journal 18 Apr, 2025 Reviewers agreed at journal 21 Mar, 2025 Reviewers agreed at journal 25 Feb, 2025 Reviewers agreed at journal 17 Jan, 2025 Reviewers invited by journal 29 Nov, 2024 Editor assigned by journal 13 Nov, 2024 Submission checks completed at journal 13 Nov, 2024 First submitted to journal 13 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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(A) HeLa, HeLa-CEA or A549 cells were incubated or not, as indicated, with MFE23 in PBS 5% FBS, followed by mouse anti-histidine tag antibody (1:500) and goat anti-mouse antibody bound to APC (1:500) or with the same reagents in the absence of MFE23, as indicated, and cells analysed by flow cytometry. (B) Cells were incubated with GRNLY or MFE23GRNLY (0,2 μM), as indicated, in PBS 5% FBS at 4°C, followed by mouse anti-histidine tag antibody (1:500) and goat anti-mouse antibody bound to FITC (1:500) or with the same reagents in the absence of GRNLY or MFE23GRNLY, as indicated, and cells analysed by flow cytometry.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5446761/v1/eab0ac2eeed269ada66a9592.png"},{"id":83943054,"identity":"5ec1cc2d-73e8-4168-879d-cd2435c46810","added_by":"auto","created_at":"2025-06-04 19:23:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":923649,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity dose-response of GRNLY and MFE23GRNLY on CEA-expressing tumor cells. HeLa-CEA (A) or A549 (B) tumor cells were incubated with increasing concentrations of recombinant GRNLY or MFE23GRNLY during 24h. Cell death was analysed by flow cytometry using Annexin-V-FITC and 7-AAD staining. Results are the mean ± SD of 3 independent experiments.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5446761/v1/1be5f38499380783f9e39a73.png"},{"id":83943075,"identity":"54322e55-8824-412a-827a-69d679dadd72","added_by":"auto","created_at":"2025-06-04 19:23:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1776760,"visible":true,"origin":"","legend":"\u003cp\u003eSystemic GRNLY and MFE23GRNLY treatment of athymic mice bearing A549 cancer xenografts. A) Treatment schedule: 2 × 10\u003csup\u003e6\u003c/sup\u003e A549 cells resuspended in Matrigel were injected s.c. in athymic mice. When tumor area reached 50 mm\u003csup\u003e2\u003c/sup\u003e, mice were randomized in three groups, control (black crosses), treated with recombinant GRNLY (purple diamonds) or treated with MFE23GRNLY (orange squares). Mice included in the treated groups received intraperitoneal injections of 10 nmol of the treatments in PBS every 2 days for 16 days, as indicated, and two days after the last injection, mice were euthanised. Mice in the control group received injections of PBS with the same time schedule. B) Tumor growth represented as tumor area in control and GRNLY groups. C) Tumor growwth represented as tumor area in control and MFE23GRNY groups. D) Tumor area after resection. E) Tumor weight after resection. Data are the mean ± SD of the tumor area or weight in each group of the study. In addition, individual values are shown in C and D. n ≥ 4. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5446761/v1/c63c0ed0c96642205431c234.png"},{"id":83943058,"identity":"326c56ac-cb23-4250-a828-ecf82016755c","added_by":"auto","created_at":"2025-06-04 19:23:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20880500,"visible":true,"origin":"","legend":"\u003cp\u003eHistochemistry and immunohistochemistry in tissue sections of A549-derived tumors from the experiment shown in Figure 3. H\u0026amp;E, representative images of hematoxylin/eosin staining on tumor sections of the indicated experimental groups. DAPI, representative images of tumor sections with nuclei stained using DAPI and photographed in a fluorescence microscope. Arrows indicate apoptotic, fragmented nuclei and circles indicate marginated chromatin nuclear phenotype. a-caspase-3, representative images of tumor sections incubated with an antibody against active caspase-3 and revealed by DAB staining.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5446761/v1/0e5c415975e66bc0399e0351.png"},{"id":83943056,"identity":"923575f5-44bd-475c-af45-26b6be35e6bc","added_by":"auto","created_at":"2025-06-04 19:23:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2183417,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of proteins involved in the PERK pathway. BIP, phosphorylated eIF2α and CHOP were determined by Western blot in A549 (A) or HeLa-CEA cells (B) treated with 10 µM GRNLY or MFE23GRNLY for 24h. Densitometric determination of band intensity was normalized to β-actin intensity for each lane.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5446761/v1/9b554c921fa2b5c214ee55ed.png"},{"id":83943064,"identity":"41e44f94-c406-4acd-9e54-517e70331cfa","added_by":"auto","created_at":"2025-06-04 19:23:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5021864,"visible":true,"origin":"","legend":"\u003cp\u003eAutophagy induction in HeLa-CEA and A549 cells treated with GRNLY or MFE23GRNLY. A) Histogram of autophagosome formation detected using the CYTO-ID® Autophagy Detection Kit by flow cytometry in HeLa-CEA or A549 cell lines treated with 5 µMGRNLY or MFE23GRNLY for 12h. B) Autophagosome structures detected by fluorescence microscopy using the CYTO-ID® Autophagy Detection Kit (green fluorescence) in A549 cells treated with 5 µM GRNLY or MFE23GRNLY for 24h. Nuclei were stained with Hoechst 33342 (blue fluorescence). C) Expression of LC3B-I, LC3B-II and p62 was determined by immunoblot in A549 or HeLa-CEA cells treated with 10 µM GRNLY or MFE23GRNLY for 24h. Densitometric determination of band intensity was normalized to β-actin levels in each lane.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5446761/v1/f727065eb0456d270ea62992.png"},{"id":83943060,"identity":"84173942-4ce7-42f6-a6b8-aed1d97d218f","added_by":"auto","created_at":"2025-06-04 19:23:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1025309,"visible":true,"origin":"","legend":"\u003cp\u003eStudy of the type of cell death induced by GRNLY or MFE23GRNLY in HeLa-CEA (A) or A549 (B) cells. Cells were preincubated for 1h in the presence or absence of 100 µM Z-VAD-fmk or 20 mM NEC-1 alone or in combination. Subsequently, cells were incubated for 24h with the EC50 concentrations of GRNLY or MFE23GRNLY obtained in Fig 2 and cell death analysed by flow cytometry using Annexin-V-FITC and 7-AAD staining. Results are the mean ± SD of 3 independent experiments. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 **, \u003cem\u003eph \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5446761/v1/bf741a9e20e88d97cafc0d6f.png"},{"id":83943072,"identity":"8afc082b-594e-417e-a911-27ee9a0f9e51","added_by":"auto","created_at":"2025-06-04 19:23:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5007508,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of proteins involved in apoptosis. A)\u003cstrong\u003e \u003c/strong\u003eThe expression of\u003cstrong\u003e \u003c/strong\u003epro-apoptotic proteins PUMA and NOXA and anti-apoptotic proteins MCL-1, BCL-2 and BCL-xL was determined by Western blot in A549 or HeLa-CEA cells treated with 10 µM GRNLY or MFE23GRNLY for 24h. Densitometric determination of band intensity was normalized to β-actin levles for each lane.\u003c/p\u003e\n\u003cp\u003eB) Basal anti-apoptotic proteins expression (MCL-1, BCL-xL and BCL-2) were determined by immunoblot in several tumor cell lines (HeLa, HeLa-CEA, HT-29 and A549). Densitometric determination of band intensity was normalized to β-actin for each lane.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5446761/v1/9b4bb7bdd7fff771920101e9.png"},{"id":83943074,"identity":"a4f04a82-6c7f-421d-838d-ad25dd3b6658","added_by":"auto","created_at":"2025-06-04 19:23:20","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1166801,"visible":true,"origin":"","legend":"\u003cp\u003eCombinatory effect of MFE23GRNLY and A1155463 in A549 cells. A) A549 cells were preincubated for 1h in the presence or absence of 100 µM Z-VAD-fmk or 2 µM A1155463 alone or in combination. Subsequently, cells were incubated with 2 µM GRNLY or MFE23GRNLY for 48h and cell death was evaluated by flow cytometry using Annexin-V-FITC and 7-AAD staining. Fresh Z-VAD-fmk was added 24h after protein addition. B) Comparison of specific observed death and expected additive death of the combination of A1155463 with GRNLY or MFE23GRNLY. C) Dose-response of A1155463 on A549 cells. Cells were incubated with increasing concentrations of A1155463 during 48h. Cell death was analysed by flow cytometry using with Annexin-V-FITC and 7-AAD staining. Results are the mean ± SD of 3 different experiments. *** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-5446761/v1/4ef3ecc3aa73901671824a90.png"},{"id":83943077,"identity":"c9fd0988-c18b-435f-a67e-752c17c325d3","added_by":"auto","created_at":"2025-06-04 19:23:20","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":3616783,"visible":true,"origin":"","legend":"\u003cp\u003eSystemic treatment with the combination of MFE23GRNLY and A1155463 of athymic mice bearing A549 cancer xenografts. A) Treatment schedule: 5 × 10\u003csup\u003e6\u003c/sup\u003e A549 cells resuspended in Matrigel were injected s.c. in athymic mice. When tumor volume reached 35 mm\u003csup\u003e2\u003c/sup\u003e, mice were randmized in four groups, as follows: control (black crosses), treated with A1155463 (blue diamonts), treated with MFE23GRNLY (orange squares) or treated with the combination of MFE23GRNLY with A1155463 (green circles). Mice included in the treament groups received intraperitoneal injections of 5 nmol MFE23GRNLY three times a week and/or intraperitoneal injections of 260 nmol A1155463 two times a week, as indicated in the scheme, and two days after the last injection mice were euthanised. B) Tumor growth represented as tumor area in control and A1155463-treated groups. Data acquisition was done following the schedule of MFE23GRNLY injection, as indicated in A. Data are the mean ± SD. C) Tumor growth represented as tumor area in control and MFE23GRNY-treated groups. Data are the mean ± SD. D) Individual tumor growth for each mice represented as tumor area in control and MFE23GRNY plus A1155463-treated groups. Mice euthanised before the end of the experiment are idicated with red circles.\u0026nbsp;E) Tumor volume of\u003cstrong\u003e \u003c/strong\u003ethe resected tumors obtained from the different experimental groups. F) Tumor weight of\u003cstrong\u003e \u003c/strong\u003ethe resected tumors obtained from the different experimental groups. Data are the mean ± SD, and individual values are also shown. Mice euthanised before the end of the experiment are indicated with red cicles. n ≥ 5, \u0026nbsp;**\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-5446761/v1/c998759653c7eefea64283be.png"},{"id":83943066,"identity":"244b5ab0-c0a8-4d31-8c41-634e03e7651a","added_by":"auto","created_at":"2025-06-04 19:23:19","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":42301003,"visible":true,"origin":"","legend":"\u003cp\u003eHistochemistry and immunohistochemistry in tissue sections of A549-derived tumors from the experiment shown in Figure 10. A) H\u0026amp;E, representative images of hematoxylin/eosin staining on tumor sections of the indicated experimental groups. DAPI, representative images of tumor sections with nuclei stained using DAPI and photographed in a fluorescence microscope. Arrows indicate apoptotic, fragmented nuclei and circles indicate marginated chromatin nuclear phenotype. a-caspase-3, representative images of tumor sections incubated with an antibody against active caspase-3 and revealed by DAB staining. B) Immunohistochemistry detection of NK infiltration in tissue sections of A549-derived tumors shown in Figure 10. Tumor sections from the indicated experimental groups were incubated with an antibody against NKp46 and revealed by DAB staining. In the combination group (lower panels), representative images from three individual mice are shown, two from tumors with a higher volume than the media of the control group (indicated as “high-volume tumors”), and one from the smallest tumor in the group (indicated as “small-volume tumor”)\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-5446761/v1/4c389e69d11570c914d9709b.png"},{"id":83943619,"identity":"51302e26-1b52-4405-a361-5d7f3ceeac91","added_by":"auto","created_at":"2025-06-04 19:39:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":80794998,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5446761/v1/f722698e-861d-48c0-9a0d-2a077085b528.pdf"},{"id":83943063,"identity":"0c112fc7-d851-4415-81b0-6a446cfc63cd","added_by":"auto","created_at":"2025-06-04 19:23:19","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1895424,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.doc","url":"https://assets-eu.researchsquare.com/files/rs-5446761/v1/0364e81e9d69f4e5132ec05d.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEfficient\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e anti-tumoral effect of the anti-CEA, granulysin-based immunotoxin MFE23GRNLY in a lung adenocarcinoma model. Combination with a Bcl-x\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eL\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-selective BH3 mimetic\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMFE232GRNLY is an immunotoxin resulting from the fusion of GRNLY, a cytolytic human protein, and the scFv of MFE23, an anti-carcinoembryonic antigen (CEA) antibody, described in a previous work if our group (1). The carcinoembryonic antigen (CEA), also known as CEACAM5 (2) was originally described in colorectal cancer (CRC) (3) as one of the first tumor-associated antigens, although it is also expressed at lower levels in normal tissues. Serum levels of CEA are useful in the prognosis, clinical control and monitoring the response to treatment of CRC (4). The expression of CEACAM5 in at least a fraction of primary lung tumors, especially in small-cell lung cancer, has also been described (5, 6). Although CEA is secreted from tumor cells, it has been shown that the antitumoral effects of bispecific anti-CEA x anti-CD3 antibodies (7) or of anti-CEA targeted CAR T cells (8) are not inhibited by soluble CEA. On the other hand, antibody-drug conjugates or immunotoxins based on CEA targeting are being explored in clinical and preclinical assays especially for CRC treatment\u003c/p\u003e\n\u003cp\u003eThe cytotoxic 9 kDa isoform of granulysin (GRNLY) is present in the granules of activated human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells (9). This protein shows cytolytic activity against a wide variety of microbes, such as bacteria, fungi and protozoan parasites and its physiological role is related with killing intracellular bacteria such as \u003cem\u003eM. Tuberculosis\u003c/em\u003e in concert with perforin (10-14). It also acts against protozoan parasites in concert with granzyme B (15). \u003c/p\u003e\n\u003cp\u003eOur group demonstrated that GRNLY was able to induce apoptotic cell death in several types of tumor cell lines \u003cem\u003ein vitro\u003c/em\u003e (16, 17) and also on \u003cem\u003eex vivo\u003c/em\u003e samples from B-CLL patients, without showing toxicity against peripheral mononuclear cells (PBMC) from healthy donors (16). Afterwards, we demonstrated its antitumor potential by intratumoral injection in \u003cem\u003ein vivo\u003c/em\u003e xenotransplantation models of human tumors in athymic mice (18, 19). \u003c/p\u003e\n\u003cp\u003eIn order to achieve tumor targeting after systemic injection, we developed the anti-CEA MFE23GRNLY immunotoxin and demonstrated its increased efficiency in eliminating CEA-expressing tumors \u003cem\u003ein vivo\u003c/em\u003e, specifically the colon carcinoma HT-29 and the cervix carcinoma HeLa transfected with CEA, HeLa-CEA cells (1). In line with this, we have also developed anti-MUC1-Tn granulysin-based immunotoxins and demonstrated their \u003cem\u003ein vivo\u003c/em\u003e anti-tumoral potential (20), although these immunotoxins had lower stability or yield of production than MFE23GRNLY.\u003c/p\u003e\n\u003cp\u003eIn the present work, we have first demonstrated the efficiency of MFE23GRNLY against the CEA-expressing human lung carcinoma A549 and analysed the mechanism of cell death induced by the immunotoxin compared with GRNLY alone in HeLa-CEA and A549 cells, finding interesting connections between the type and extent of cell death induced in the lung model and the expression of Bcl-x\u003csub\u003eL\u003c/sub\u003e, that have been also explored \u003cem\u003ein vivo\u003c/em\u003e. \u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA549 lung adenocarcinoma,\u0026nbsp;HT-29 colon adenocarcinomas, HeLa cervix carcinoma and\u0026nbsp;HeLa cervix carcinoma\u0026nbsp;stably transfected with CEA (HeLa-CEA cells) were cultured in DMEM medium (Gibco, Barcelona) supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin (all of them from Pan Biotech, Aidenbach, Germany) and GlutaMAX (Invitrogen, Barcelona), at 37ºC and 5% CO\u003csub\u003e2\u003c/sub\u003e using standard procedures. All cell lines were routinely tested for mycoplasma contamination by PCR.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFungi culture\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePichia pastoris\u003c/em\u003e was grown for 24h at 30°C with agitation at 250 rpm in BMGY medium (1% yeast extract, 2% peptone, 1.34% YNB, 1% glycerol, 400 μg/L biotin, and 0.1 M potassium phosphate, pH 6.0). Then, BMGY medium was changed to BMM medium (1.34% YNB, 0,5% methanol, 400 μg/L biotin, 0,03% Anti-foam 204, 1% casamino acids and 0.1 M potassium phosphate, pH 6.0) and incubated at 18°C and 250 rpm for 24h. Subsequently, 1% methanol was added and yeast cultured for 48h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression and purification of recombinant granulysin and of the granulysin-MFE23 immunotoxin in \u003cem\u003ePichia pastoris\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eP. pastoris\u003c/em\u003e strain SMD1168 transfected with GRNLY or with MFE23GRNLY was cultured in BMGY medium at 30 °C in a shaking incubator (250 rpm) overnight. The following day, the culture was transferred to BMMY medium at and incubated overnight at 18 °C in a shaking incubator (250 rpm) overnight for induction. For protein secretion induction, the culture was supplemented with 1.5 % methanol (v/v) every 24 h for 2 days. Subsequently, supernatants were collected and proteins were purified using by Ni\u003csup\u003e2+\u003c/sup\u003e affinity chromatography (Ni-NTA agarose, Qiagen). Finally, the elution buffer was replaced by PBS by\u0026nbsp;dialysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry analysis of CEA binding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of the binding of both MF23 or MF23GRNLY to the CEA antigen on the surface of living cells was performed as follows: 10\u003csup\u003e5\u003c/sup\u003e cells per well were placed in a 96-well roundbottom plate and incubated with MF23 or with MFE23GRNLY (0,2 μM) in PBS supplemented with 5% FBS for 30 min at 4°C followed by incubation with a mouse anti-histidine tag antibody (1:500; Genscript, Netherlands) and goat anti-mouse antibody bound to APC (1:500; Biolegend, San Diego) or FITC (1:500; Caltag, Barcelona). After each incubation step, cells were washed with in PBS supplemented with 5% FBS. CEA-binding specificity was determined on CEA-positive HeLa-CEA cells and compared to HeLa cells (negative for CEA expression), using a FACScalibur flow cytometer (BD Biosciences, Madrid).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter SDS-electrophoresis, the proteins were transferred to a nitrocelulose membrane. Then, membranes were blocked overnight at 4ºC with agitation with TBS-T buffer (10 mM Tris / HCl, pH 8.0, 0.12 M NaCl, 0.1% Tween-20, 0.05% thimerosal) containing 5% non-fat milk. Afterwards, membranes were washed with TBS-T buffer and incubated with primary antibodies against diferent proteins (see Table 1), diluted in TBS-T containing 5% BSA. After that, membranes were washed again with TBS-T and incubated with 0.2 µg/mL of the secondary antibody conjugated with peroxidase (Sigma). Finally, proteins levels were determined by chemiluminescence using the peroxidase substrate Pierce ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1. Antibodies used for western blot.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntibody\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompany\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eβ-Actin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBCL-xL/S (2H12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCHOP\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEIF2α-P (Ser51)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCell Signaling Technology\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLC3B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMCL1 (S-19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNoxa (114C307)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ep62 (D-3)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePUMA (G-3)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eα-Tubuline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSigma\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;cytotoxicity assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3x10\u003csup\u003e4\u003c/sup\u003e suspension cells or 1.5x10\u003csup\u003e4\u003c/sup\u003e adherent cells were seeded per well in 96-well plates. Adherent cells were seeded in the plates 24h before the experiment begins and medium was refreshed just before the incubation with the cytotoxic proteins. Subsequently, GRNLY or MFE23GRNLY were added at the indicated concentrations. In control wells, equal volumes of PBS were added. In some experiments,\u0026nbsp;cells were preincubated for 1h in the presence or absence of 100 µM Z-VAD-fmk or 20 µM NEC-1 alone or in combination followed by the incubation with GRNLY or MFE23GRNLY for 24 h. In other experiments cells were preincubated for 1h in the presence or absence of 100 µM Z-VAD-fmk or 2 µM A-1155463 alone or in combination, followed by the incubation with GRNLY or MFE23GRNLY for 48h, with Z-VAD-fmk refreshing evey 24h. Then, cells\u0026nbsp;were incubated at 37ºC with Annezin-V and t-AAD in annexin-binding buffer (140 mM NaCl, 2.5 mM CaCl\u003csub\u003e2\u003c/sub\u003e,10 mM Hepes/NaOH, pH 7.4) for 15 min at room temperature and cell death analysed by flow cytometry using a FACSCalibur (BD Bioscience, Madrid).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAutophagosome detection by CYTO-ID®\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAutophagy\u0026nbsp;Detection\u0026nbsp;Kit\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells (5x10\u003csup\u003e4\u003c/sup\u003e per well) were seeded in 48-well plates and were treated with GRNLY or MFE23GRNLY for 12h. Then, cells were stained with CYTO-ID®\u0026nbsp;Autophagy\u0026nbsp;Detection\u0026nbsp;Kit (Enzo Life Sciences, Farmingdale, USA)\u0026nbsp;as per manufacturer’s specifications and analysed by flow cytometry.\u003c/p\u003e\n\u003cp\u003eIn addition, fluorescence microscopy images were taken as follows: 5x10\u003csup\u003e4\u003c/sup\u003e cells were seeded per well in 24 well plates and were treated with GRNLY or MFE23GRNLY for 12h. Then, cells were simultaneously stained with CYTOID®\u0026nbsp;Autophagy\u0026nbsp;Detection\u0026nbsp;Kit\u0026nbsp;and Hoechst 33342 (Thermo Fisher, Barcelona) nuclear dye and photographed in a\u0026nbsp;E600/E400 Nikon fluorescence microscope equipped with a digital camera (DXM1200F, Nikon).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eI\u003c/em\u003e\u003cstrong\u003e\u003cem\u003en vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;experiments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmune-deficient athymic mice, Swiss nu/nu strain (Charles River, Barcelona), six-week-old males, were used in the present study. All procedures were carried out under Project Licence PI38/23 approved by the Ethic Committee for Animal Experiments from the University of Zaragoza. The care and use of animals were performed accordingly with the Spanish Policy for Animal Protection RD53/2013, which meets the European Union Directive 2010/63 on the protection of animals used for experimental and other scientific purposes. Mice were kept under specific standard pathogen-free conditions (average ambient temperature 24°C, 12/12-h light/dark cycle) with water and food provided \u003cem\u003ead libitum\u003c/em\u003e throughout the study.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGRNLY and MFE23GRNLY experiments\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e2 x 10\u003csup\u003e6\u003c/sup\u003e A549 tumor cells were subcutaneously injected in athymic mice. Treatments were administered when tumors reached approximatively 50 mm\u003csup\u003e2\u003c/sup\u003e in size. \u0026nbsp;GRNLY and MFE23GRNLY groups received in total 8 intraperitoneal injections every 2 days at the concentration of 10 nmol diluted in PBS before euthanasia. Control group received intraperitoneal injections of PBS following the same treatment schedule as GRNLY and MFE23GRNLY.\u003c/p\u003e\n\u003cp\u003eTumor growth\u0026nbsp;was\u0026nbsp;daily measured\u0026nbsp;using a precision caliper\u0026nbsp;and tumor area was calculated\u0026nbsp;by collecting the width and length of the tumor.\u003c/p\u003e\n\u003cp\u003eTumors from euthanized mice were surgically excised, measured, weighed and stored in a preservation solution (10% buffered formalin) to perform histological analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMFE23GRNLY and A-1155463\u0026nbsp;\u003c/em\u003e\u003cem\u003ecombination experiments\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e5 x 10\u003csup\u003e6\u003c/sup\u003e A549 tumor cells were subcutaneously injected in nude mice. Treatments were administered when tumors reached approximately 35 mm\u003csup\u003e2\u003c/sup\u003ein size.\u0026nbsp;MFE23GRNLY-treated groups received\u0026nbsp;10\u0026nbsp;intraperitoneal injections\u0026nbsp;3 times a week\u0026nbsp;(5 nmol/dose\u0026nbsp;diluted\u0026nbsp;in PBS).\u0026nbsp;A-1155463-treated groups received\u0026nbsp;6\u0026nbsp;intraperitoneal injections (260 nmol/dose in 5% DMSO, 10% ethanol, 20% Cremophor® RH-40 and 65% dextrose (5% in water)), twice a week.\u0026nbsp;Control group received intraperitoneal injections of PBS following the same schedule than the MFE23GRNLY-treated group.\u003c/p\u003e\n\u003cp\u003eTumor growth was monitored by measuring the tumor 3 times a week with a precision caliper by measuring the width and length of the tumor.\u003c/p\u003e\n\u003cp\u003eTumors from euthanized mice were surgically excised, measured, weighed and stored in a preservation solution (10% buffered formalin) to perform histological analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHematoxylin-eosin staining\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTissue for light microscopy was fixed in 4% formaldehyde and embedded in paraffin using routine procedure. Sections 5 μm thick were cut from the tissue blocks and deparaffinized by immersion in two changes of xylene for 5 min each. After that, tissue sections were rehydrated by immersion in decreasing concentrations of ethanol (immersion in separated baths of 100%, 95% and 70% of ethanol for 2 min/each bath). The nuclei of the cells in tissue sections were stained by immersing in GILL II Hematoxylin for 3 min. Sections were rinsed in HCl 0.1% for 5 seconds and then, in tap water for 3-5 min. Cell cytoplasm was stained by immersing in 0.5% of eosin containing 0.2% of glacial acetic acid, for 3 min and dipped once quickly in water for few seconds. After eosin staining, sections were dehydrated by immersion in ascending alcohol solutions (70%, 95% and absolute alcohol 100%) for 5 seconds/each. Finally, all sections were cleaned by xylene for 5 seconds, dried at room temperature and mounted with DPX using glass coverslips\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFluorescence study of apoptotic nuclei and immunohistochemistry for detection of activated caspase-3\u0026nbsp;and NK cell infiltration\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTissue for fluorescence microscopy was fixed in 4% formaldehyde and embedded in paraffin using routine procedure. Sections of tumor samples (4 μm thick) were deparaffinized in xylene, hydrated through a graded series of ethanol, and then immersed in 3% hydrogen peroxide in 100% methanol for 30 min to inhibit endogenous peroxidase activity. For antigen retrieval, the sections were boiled in 10 mM citrate buffer, pH 6.0 for 30 min. Staining of nuclei was performed using DAPI and detected using a fluorescence microscope (E600/E400, Nikon) equipped with a digital camera (DXM1200F, Nikon).\u003c/p\u003e\n\u003cp\u003eThe detection of activated caspase-3 or of NK cell infiltration was performed by immunohistochemistry using rabbit polyclonal antibodies either anti-human cleaved caspase-3 antibody (Cell Signalling, Barcelona) or anti-NKp46 (Biorbyt, Cambridge, UK). After rinsing the samples with PBS-Tween 20 for 2 times (2 min each), the sections were incubated with 200 μl blocking solution (5% rabbit or horse serum diluted in PBS) for 1hr at room temperature, and then incubated overnight at 4°C in humid chambers with the primary antibodies at 1/200 dilution. Labeling was revealed using DAB staining. Isotype control staining was also performed, giving no substantial signal in the experimental conditions described.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComputer-based statistical analysis was performed using GraphPad Prism 4.0 program (GrandPath Software Inc). Results are shown as mean±SD. Statistical significance was evaluated by using two-way ANOVA for \u003cem\u003ein vitro\u003c/em\u003e assays and multiple t-test analysis, using the Holm-Sidak method or the Welch’s t-test analysis for \u003cem\u003ein vivo\u003c/em\u003e assays. Differences were considered significant if \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt;0.05 (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eBinding of GRNLY and MFE23GRNLY to HeLa-CEA and A549 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn our previous study, we demonstrated the specific binding of MFE23GRNLY to the purified CEACAM5 antigen by ELISA, showing a similar titration pattern than the MFE23 scFv. We also showed that it bound to the antigen expressed on the surface of CEACAM5\u003csup\u003e+\u003c/sup\u003e cells, such as the colon adenocarcinoma HT-29 or HeLa cells transfected with CEA (HeLa-CEA cells) (1). \u003c/p\u003e\n\u003cp\u003eTo confirm CEACAM5 expression on the surface of lung adenocarcinoma A549 cells, we performed a flow cytometry assay using the MFE23 scFv moiety alone. As shown in Fig 1A, HeLa cells proved to be negative for CEACAM5 expression, while HeLa-CEA cells showed a high surface expression of the antigen, with A549 cells showing an intermediate, but significant level of expression. Then, we tested the binding of MFE23GRNLY to the lung adenocarcinoma A549, and compared with HeLa-CEA staining as a positive control and with HeLa cells as a negative control (Fig 1B). In these experiments, we also tested GRNLY binding to the surface of these cells, since it is a protein that interacts with membrane phospholipids (21). In HeLa cells, the binding of the immunotoxin was indistinguishable from that of GRNLY alone, indicating no contribution of the antibody moiety to this binding, due to the absence of CEA expression on the surface of these cells. On the contrary, a substantial shift of the labelling was observed in HeLa-CEA cells incubated with the immunotoxin, showing a 3-fold increase in MFI compared to GRNLY. A similar situation was observed in the case of A549 cells, further confirming the presence of the CEACAM5 antigen on their surface. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDose-response of GRNLY and MFE23GRNLY on CEA-expressing tumor cell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we performed complete dose-response cytotoxicity studies on HeLa-CEA and A549 cells, to calculate the corresponding IC\u003csub\u003e50\u003c/sub\u003e (Fig 2A). As previously shown (1), MFE23GRNLY was more cytotoxic than GRNLY alone on HeLa-CEA cells, with an IC\u003csub\u003e50\u003c/sub\u003e of 6 µM vs. 25 µM. A similar situation was observed for A549 cells, with an IC\u003csub\u003e50 \u003c/sub\u003eof 4 µM for MF23GRNLY vs. 10 µM for GRNLY alone (Fig 2B). \u003c/p\u003e\n\u003cp\u003eAltogether, these data would suggest the potential use of MFE23GRNLY not only for the treatment of colon or gastric tumors, but also for CEACAM5\u003csup\u003e+\u003c/sup\u003e lung tumors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e experiment on athymic mice xenografted with A549 tumor cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test this hypothesis, we performed \u003cem\u003ein vivo\u003c/em\u003e experiments using systemic administration of the treatments in a model of A549 cells xenotransplanted in athymic mice (Fig 3A). When recombinant granulysin was administered systemically (i.p.), it did not result in a significant reduction in tumor growth (Fig 3B). However, administration of the immunotoxin clearly reduced tumor growth, being this reduction statistically significant from the 1st injection onwards (Fig 3C). Relative to the control group, mean tumor growth was reduced around 50% by the immunotoxin at the end of the experiment. This reduction was also observed when the size (Fig 3D) or weight (Fig 3E) of the resected tumors were analysed.\u003c/p\u003e\n\u003cp\u003eNext, we performed histological studies on tissue sections obtained from resected tumors. Hematoxylin/eosin staining (H/E in Figure 4) showed that untreated tumors presented a compact tissue structure with high cellularity (upper left panel), while tumors from GRNLY-treated mice showed somewhat lower cellularity (middle left panel). By contrast, in the case of tumors from MFE23GRNLY-treated mice a dramatic loss of cellularity was observed, with most of the sections occupied by unstained stroma (lower left panel). Upon DAPI labeling, untreated tumors displayed nuclei with non-condensed chromatin (Fig 4, DAPI column, upper middle panel), a phenotype that was the predominant in tumors from mice treated by systemic GRNLY injection, although some cells with condensed chromatin were also observed (arrows, Fig 4, bottom middle panel). Resected tumors from MFE23GRNLY-treated mice showed some cell with condensed chromatin and fragmented nuclei (indicated with arrows in the lower panel). However, the major phenotype in this case corresponded to cells with perinuclear-condensed chromatin (indicated by circles in the same image). This nuclear morphology was also observed in our previous \u003cem\u003ein vivo\u003c/em\u003e study using HeLa-CEA cells (1), being characteristic of the caspase-independent apoptosis-inducing factor (AIF)-mediated cell death (22). Finally, active caspase-3 immunochemistry analysis provided some limited staining in tumors from GRNLY-treated mice (Fig 4, caspase-3 column, middle right panel), while a relative increase was observed in the case of tumors derived from MFE23GRNLY-treated mice (lower right panel).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMFE23GRNLY induces ER stress \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOnce demonstrated the \u003cem\u003ein vivo\u003c/em\u003e efficacy of MFE23GRNLY against A549-derived tumors, we sought to determine the cell death mechanism elicited by the immunotoxin on tumor cells. \u003c/p\u003e\n\u003cp\u003eAlterations in the endoplasmic reticulum (ER) folding environment are known to cause the accumulation of misfolded proteins, leading to ER stress. To deal with this and to restore normal ER functions, cells activate the unfolded protein response (UPR) via the induction of protein kinase RNA-like endoplasmic reticulum kinase (PERK). The interaction of the chaperone BiP (Binding immunoglobulin Protein) with misfolded proteins releases PERK, which phosphorylates eIF2α, inhibiting protein synthesis and inducing the selective translation of Activating Transcription Factor 4 (ATF4). ATF4 subsequently induces the upregulation of proapoptotic factor C/EBP homologous protein (CHOP), a biomarker of the ER stress response (23). \u003c/p\u003e\n\u003cp\u003eRecombinant GRNLY induces an alteration in plasma membrane structure resulting in intracelullar [Ca\u003csup\u003e2+\u003c/sup\u003e] increase (21, 22, 24-26), which in turn could induce ER stress. Hence, we analysed the expression of proteins involved in the PERK pathway in tumor cell lines treated with MFE23GRNLY or with GRNLY. We clearly observed in both A549 and HeLa-CEA cells a down-modulation of BIP chaperone expression and an increase in CHOP levels upon MFEGRNLY incubation. In the case of GRNLY, we observed a similar pattern for BIP expression, although we did not detect an increase in CHOP expression in A549 cells. Altogether, these data indicate that tumor cells treated with GRNLY, and especially with MFE23GRNLY, suffer activation of the PERK pathway and ER stress (Fig 5).\u003c/p\u003e\n\u003cp\u003eIn addition, an increase in eIF2α phosphorylation was observed in A549 cells treated with GRNLY or MFE23GRNLY (Fig 5A), but not in HeLa-CEA cells (Fig 5B). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMFE23GRNLY induces incomplete authophagy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAutophagy is a fundamental cell survival mechanism that allows cells to adapt to metabolic stress through the degradation and recycling of intracellular components to generate macromolecular precursors and produce energy.We first explored the formation of autophagosomes upon treatment with GRNLY or MFE23GRNLY using the Cyto-ID flow cytometry technique (27). As shown in Fig 6A\u003cstrong\u003e, \u003c/strong\u003eGRNLY and the immunotoxin clearly induced autophagosome formation on HeLa-CEA and A549 cells, albeit the immunotoxin was more active at the same concentration. Autophagy induction could be also clearly detected by fluorescence microscopy in A549 cells, showing the formation of multiple cytoplasmic punctate structures corresponding to autophagosomes (Fig 6B) .\u003c/p\u003e\n\u003cp\u003eWe also analysed the lipidation of the LC3 protein and the expression levels of p62 as other hallmarks of autophagy induction, that can be detected by immunoblot. The lipidation of LC3 was detected by immunoblot through the conversion of LC3-I to LC3-II. As shown in Fig 6C, MFE23GRNLY increased the level of LC3BII, while keeping constant the levels of p62 in both HeLa-CEA and A549 cells. These results suggests that autophagy was initiated as demonstrated by the formation of autophagosomes, but it was not completed, since p62 was not induced neither finally degraded. This observation is compatible with the phenotype of cells observed by phase-contrast microscopy after being treated with GRNLY or MFE23GRNLY, in which abundant cytoplasmic punctate structures, compatible with autophagosome accumulation, were clearly detected before the completion of cell death (Supplementary Fig 1). In addition, blocking the initiation of autophagy by 3-Methyladenine (3-MA), a widely used inhibitor of class III PI3K, in HeLa-CEA cells treated with GRNLY or MFE23GRNLY, resulted in an increase of the percentage of cell death (Supplementary Figure 2). Therefore, these data indicate that autophagy activation induced by GRNLY or MFE23GRNLY treatments seems to be a protective process put in place by cells to cope with stress and manage to survive.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMFE23GRNLY death mechanism is dependent on the cell line analysed\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt was previously described that GRNLY induced cell death mainly through the mitochondrial apoptotic pathway (22, 25, 28, 29), which results in the caspase-dependent phosphatidylserine (PS) exposure on the external membrane hemileaflet and final membrane permeabilization. PS exposure can be analysed by Annexin-V staining and membrane permeabilization by 7-AAD nuclear staining using flow cytometry. In the subsequent cytotoxicity experiments we have used the IC\u003csub\u003e50\u003c/sub\u003e values for GRNLY and for the immunotoxin calculated for each molecule in the second section of Results (see Fig 2). \u003c/p\u003e\n\u003cp\u003eTo study the mechanism of cell death induction by MFE23GRNLY as compared with GRNLY we used inhibitory molecules of known death pathways. The general caspase inhibitor Z-VAD-fmk significantly prevented death in HeLa-CEA cells induced by MFE23GRNLY, although only partially (from 50% to 30%), while the necroptosis inhibitor necrostatin-1 (NEC-1) had no effect either alone or in combination with Z-VAD-fmk (Fig 7A). Similar results were observed with GRNLY, although in this case NEC-1 also partially prevented cell death, suggesting that GRNLY is able to partially induce necroptotic death in these cells. In our previous work, a minor necroptotic component was also observed in GRNLY-induced cell death in other cell lines, especially if caspases were inhibited (16). \u003c/p\u003e\n\u003cp\u003eRemarkably, in A549 cells treated with MFE23GRNLY or with GRNLY (Fig 7B), no effect on cell death was oberved when combined with Z-VAD-fmk or NEC-1, suggesting the activation of a caspase-independent cell death pathway different to necroptosis.\u003c/p\u003e\n\u003cp\u003eWilling to understand the cell death mechanism in A549 cells, we analysed the expression of pro- and anti-apoptotic proteins of the Bcl-2 family before and after treatment with the same concentrations of the recombinant cytotoxic proteins. The expression of pro-apoptotic BH3-only proteins like PUMA and Noxa were increased in MFE23GRNLY-treated A549 cells. However, anti-apoptotic proteins like Mcl-1 and Bcl-x\u003csub\u003eL\u003c/sub\u003e were also substantially increased (Fig 8A). In the case of HeLa-CEA cells, an increase in Puma expression, but also in Mcl-1, was clearly observed (Fig 8A). \u003c/p\u003e\n\u003cp\u003eConnecting these results with the observed induction of ER stress, it has been described that ER stress promotes p47 and/or p53 activity, being Puma and Noxa targets of these transcription factors (30, 31). In Suppl. Fig 3, it can be observed that, especially after MFE23GRNLY treatment of A549 cells, an increase in the expression of p47 is clearly observed.\u003c/p\u003e\n\u003cp\u003eIt was previously described that GRNLY was able, in addition to activate the mitochondrial apoptotic pathway leading to caspase activation, to induce the translocation of AIF from mitochondria to the nucleus, a process that leads to caspase-independent cell death (22, 32). AIF is sytnthesized as a precursor of about 67 kDa and it is then imported to mitochondria, where it is processed to a 62/64 kDa mature form. Upon exposition to cell death-inducing agents, AIF is processed into a 57 kDa and lower molecular weight forms, which are translocated to the cytoplasm and finally to the nucleus (33, 34). In Suppl Fig 4, we show that, especially in the case of A549 cells treated with MFE23GRNLY, a processing of AIF to truncated isoforms with MW lower than 62 kDa took place, suggesting the efficient activation of this pathway.\u003c/p\u003e\n\u003cp\u003eThese results suggest that although A549 cells increased expression of pro-apoptotic proteins levels in the response to the treatments, they were protected from apoptosis leading to the execution of an alternative cell death pathway. \u003c/p\u003e\n\u003cp\u003eRemarkably, if analyzing the basal expression of anti-apoptotic members of the Bcl-2 family in several tumor cell lines, we found that basal Bcl-x\u003csub\u003eL\u003c/sub\u003e expression was very high in A549 cells, while the expression of this protein was moderate in HeLa-CEA cells, inversely correlating with the sensitivity to Z-VAD-fmk inhibition of cell death in these cell lines (Fig 8B). \u003c/p\u003e\n\u003cp\u003eWe reasoned that the protection from caspase-dependent cell death observed in A549 cells could be due to the high basal Bcl-x\u003csub\u003eL\u003c/sub\u003e expression detected in this particular cell line and that it could be reversed by the BH3 mimetic Bcl-x\u003csub\u003eL\u003c/sub\u003e inhibitor A1155463. As shown in Fig 9A, the combination of A1155463 with GRNLY or with MFE23GRNLY greatly increased cell death in this cell line, and, in addition, this increase was completely abrogated by the general caspase inhibitor Z-VAD-fmk. The effect observed was synergic, since there were statistically significant differences when comparing the expected additive cell death with the result obtained (Fig 9B). It should be noted that the synergy of A1155463 with GRNLY or MFE23GRNLY was achieved at a low 2 µM concentration, that is not toxic for A549 cells, with overt cytotoxicity obtained only at a 10-fold higher concentration, 20 µM (see Fig 9C). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e experiment combining MFE23GRNLY with the BH3 mimetic \u003c/strong\u003e\u003cstrong\u003eA1155463\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next explored this remarkable synergy in the same \u003cem\u003ein vivo\u003c/em\u003e model described in Figures 3 and 4. In order to achieve the same synergistic effect observed \u003cem\u003ein vitro\u003c/em\u003e, we reduced the dose of MFE23GRNLY used in the previous experiments from 10 to 5 nmol per injection (9.4 mg/Kg). In the case of A1155463, we also lowered the 10 mg/Kg dose reported in previous \u003cem\u003ein vivo\u003c/em\u003e studies (35-37) to a dose of 7 mg/Kg (260 nmol per injection). In addition, we followed a 72h injection schedule to give time to the animals to recover from its platelet toxicity, as reported in previous studies (35, 37). Hence, for the 10 MFE23GRNLY injections performed in the study, following the described 48h schedule, mice in the A1155463 groups received 6 injections of the agent, as indicated in the protocol scheme shown in Figure 10A. \u003c/p\u003e\n\u003cp\u003eAs shown in Figure 10B, the BH3 mimetic had no effect on the growth of tumors generated by the A549 lung adenocarcinoma. However, when the recombinant immunotoxin was administered systemically (i.p.), the effect was similar to that observed in the previous experiments, clearly reducing tumor growth, being this reduction statistically significant from the 5th injection onwards (Fig 10C). Relative to the control group, mean tumor growth was reduced more than 40% by the immunotoxin at the end of the experiment. When analyzing the combined effect of A1155463 and MFE23GRNLY (Fig 10D), the result was highly variable across the animals included in this experimental group. While in four mice the combination had a similar effect than MFE23GRNLY alone or even in two of these mice the combined effect was higher, in two other mice, the combination was much less effective than MFE23GRNLY alone. Remarkably, when we analysed the volume and weight of resected tumors after the end of the experiment, the variability described above was not reproduced. In this analysis, both the immunotoxin and its combination with the BH3 mimetic significantly reduced the volume and weight of the tumors by 50% (Figs 10E and 10F). In any case, the synergic effect observed \u003cem\u003ein vitro\u003c/em\u003e between both agents was not reproduced \u003cem\u003ein vivo\u003c/em\u003e, and the effect of the combination was equivalent to the effect obtained with the immunotoxin alone. \u003c/p\u003e\n\u003cp\u003eAdditionally, we performed histological studies on tissue sections obtained from resected tumors (Figure 11A). Hematoxylin/eosin staining showed that tumors from the control group or from the group treated with A1155463 presented a compact tissue structure with high cellularity, while tumors from MFE23GRNLY-treated mice, alone or in combination with A1155463 showed a great cellularity loss, with big sections occupied by unlabeled stroma. Upon DAPI labeling, untreated tumors or tumors treated by A1155463 displayed nuclei with non-condensed chromatin. In the case of resected tumors from MFE23GRNLY-treated mice, some cells with condensed chromatin and/or fragmented nuclei were observed (indicated with arrows), being in fact more abundant the cells with the AIF-related perinuclear-condensed chromatin (indicated with circles). When combined with A1155463, DAPI labeling showed a rather increased number of cells with condensed chromatin and fragmented nuclei (arrows) relative to cells with the mentioned AIF-related nuclear phenotype (circles). Finally, active caspase-3 immunochemistry showed no labelling in control or A1155463-treated mice (upper panels), a limited labeling in the case of tumors from MFE23GRNLY-treated mice (lower-middle panel), and a somewhat increased labeling in the case of tumors from mice treated with the combination of the immunotoxin with the BH3 mimetic (lower panel).\u003c/p\u003e\n\u003cp\u003eResults regarding NK cell infiltration, obtained by anti-NKp46 immunochemistry, are shown in Fig 11B. No NK cell infiltration was observed in control or A1155463-treated mice, while a clear NK infiltration was observed in all samples from the MFE23GRNLY experimental group. In the case of tumors treated with the combination of A1155463 with MFE23GRNLY, while in those mice in which tumor growth was no apparently affected by the treatment (see Figure 10D; indicated as “high-volume tumors” in the lower panels of Fig 11B), no NK infiltration was observed, the infiltration was present in the tumor which size was even smaller than that of the mean of the MFE23GRNLY treated group (see Fig 10D; indicated as “small-volume tumor” in the lower panels of Fig 11B). \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have previously demonstrated \u003cem\u003ein vitro \u003c/em\u003eand\u003cem\u003e in vivo\u003c/em\u003e that the immunotoxin MFE23GRNLY notably increases the cytotoxic activity of GRNLY against CEA-expressing human tumor cell lines. Now, we report molecular events that take place when tumor cells were treated with this immunotoxin and which could help to understand its death-inducing mechanism.\u003c/p\u003e\n\u003cp\u003eER is an essential site for the regulation of apoptotic pathways and it has also been recently recognized as an important component of autophagic signaling (38). Autophagy is a mechanism which enables the elimination of unfolded or misfolded proteins under ER stress conditions (39). Hence\u003cstrong\u003e, \u003c/strong\u003eER stress and autophagy are intimately connected under cell stress conditions and their cooperation can result in both survival or death induction (38).\u003c/p\u003e\n\u003cp\u003eIf the overload of unfolded or misfolded proteins in the ER is not resolved, the prolonged UPR will induce ER stress-associated apoptosis (40), probably through CHOP expression (41). In addition, CHOP is involved not only in apoptosis induction, but also in the suppression of autophagy interacting with LC3B (41, 42). ER stress is also connected with the mitochondrial apoptotic pathway through the increase in expression and activation of several BH3-only pro-apoptotic members of the Bcl-2 family such as Puma and Noxa (43, 44), which expression is effectively increased by the immunotoxin. \u003c/p\u003e\n\u003cp\u003eSaini et al. described that GRNLY delivered by cytotoxic T cells induces endoplasmic reticulum stress, but not recombinant GRNLY (26). However, we have demonstrated that recombinant GRNLY and also MFE23GRNLY are able to induce ER stress in the cells used in this study. The increased cytotoxic effect of the immunotoxin could be related with the higher amount of GRNLY brought to the cells surface by the scFv moiety in CEA-expressing cells. \u003c/p\u003e\n\u003cp\u003eIt has been described that ER stress promotes p47 and/or p53 activity (30), and we also demonstrate that p47 levels increase upon incubation with the immunotoxin, correlating with the induction of expression of its targets Puma and Noxa. In addition, Noxa can be also induced by CHOP activation. \u003c/p\u003e\n\u003cp\u003eIt was previously described that the main type of cell death produced by GRNLY was apoptosis through the mitochondrial apoptotic pathway (16, 17, 22, 25, 29)and in the present work we have confirmed those observations also for MFE23GRNLY-induced cell death in HeLa-CEA cells. However, we have also observed that A549 cells treated with GRNLY or with MFE23GRNLY suffer a caspase-independent cell death different from necroptosis, suggesting that other pathways could be involved. One of such pathways could be the caspase-independent cell death induced by AIF translocation from mitochondria to nucleus, which our group demonstrated that was also activated by GRNLY (22). \u003cem\u003eIn vitro \u003c/em\u003edata and also DAPI stainings performed in tumor sections from the \u003cem\u003ein vivo\u003c/em\u003e experiments would support this hypothesis. \u003c/p\u003e\n\u003cp\u003eWe have also shown that the low efficiency in activating caspase-dependent cell death in A549 cells correlates with a high expression of the anti-apoptotic member of the Bcl-2 family Bcl-x\u003csub\u003eL\u003c/sub\u003e, and that this effect is completely reversed by the BH3 mimetic Bcl-x\u003csub\u003eL\u003c/sub\u003e inhibitor A1155463. \u003c/p\u003e\n\u003cp\u003eThe fact that MFE23GRNLY showed clear antitumor effects in athymic mice bearing A549 cancer xenografts when administered systemically broaden the possible clinical application of the immunotoxin not only to colon or gastric carcinomas, but also to CEA-expressing lung tumors. In addition, we showed that it exerts a remarkable synergic effect when combined \u003cem\u003ein vitro\u003c/em\u003e with A1155463, a Bcl-x\u003csub\u003eL\u003c/sub\u003e specific inhibitor. This result suggested that the combination of this inhibitor with the immunotoxin could also increase the antitumor potential of this immunotoxin \u003cem\u003ein vivo\u003c/em\u003e. However, although we confirmed A549 tumor targeting by the immunotoxin even at a lower dose, we did not observe the synergistic effect with the BH3 mimetic \u003cem\u003ein vivo\u003c/em\u003e. \u003c/p\u003e\n\u003cp\u003eBH3 mimetics, that inhibit specific anti-apoptotic proteins of the Bcl-2 family which are over-expressed in many cancer types, are a therapeutic option against cancer that has been extensively explored in the pre-clinical setting (45) and that has also reached the clinic. In 2018, the specific anti-Bcl-2 BH3 mimetic inhibitor venetoclax was approved for the treatment of chronic lymphocytic leukemia (CLL) of small cell lymphocytic leukemia (SLL); and in 2020 it was approved for the treatment of acute myeloid leukemia (AML) in combination with azacitidine, decitabine, or low-dose cytarabine (46, 47). \u003c/p\u003e\n\u003cp\u003eA1155463 is another BH3 mimetic that specifically targets Bcl-x\u003csub\u003eL\u003c/sub\u003e. It has been observed that Bcl-x\u003csub\u003eL\u003c/sub\u003e inhibition induces platelet toxicity in experimentation animals, but that an on-off schedule of administration allows a good platelet function recovery (35, 37). A 10 mg/Kg dose has shown a partial retardation of tumor growth in an \u003cem\u003ein vivo\u003c/em\u003e model of lung cancer (37), and good results in different types of leukemia, especially if combined with venetoclax (35). \u003c/p\u003e\n\u003cp\u003eIn our hands, we did not find any effect of A1155463 alone on the development of A549-derived tumors when used as a single agent. This could be due to the use of a lower dose compared with other works such as the one carried out by Tao et al., although in that study the effect as a single agent was only partial (37). However, we also lost the powerful synergistic effect observed \u003cem\u003ein vitro\u003c/em\u003e on A549 cells, characterized by a high over-expression of Bcl-x\u003csub\u003eL\u003c/sub\u003e. \u003c/p\u003e\n\u003cp\u003eThe athymic mice used in our \u003cem\u003ein vivo\u003c/em\u003e studies are almost completely devoid of T cells, and the humoral immune response mediated by B cells is also profoundly affected. However, these mice have more NK cells than wild-type mice, showing also a higher activation state. In our initial studies, we observed that intratumoral injection of granulysin induced a massive infiltration of NK cells in the xenografted tumors, indicating that the therapeutic effect of granulysin could be mediated, at least in part, by this intratumoral NK cell infiltration elicited by granulysin (18, 19). We confirmed this NK cell infiltration when systemically administering the MFE23GRNLY immunotoxin in athymic mice xenografted with HeLa-CEA cells (1, 48). NK cell development and survival is dependent on the anti-apoptotic proteins of the Bcl-2 family Mcl-1 and Bcl-2 itself, but to a lesser extent on Bcl-x\u003csub\u003eL\u003c/sub\u003e (49). However, NK cell activation is associated with a remarkable increased expression of Bcl-x\u003csub\u003eL\u003c/sub\u003e, that could be important for their survival and anti-tumoral function (50). If, at the same time that A1155463 is inducing A549 cell death in synergy with MFE23GRNLY, it is also negatively affecting NK cell infiltration and anti-tumoral function \u003cem\u003ein vivo\u003c/em\u003e, then the global effect of the combination would be diminished. The NK cell infiltration data obtained indicates that this could be the case, although further studies will be needed to be certain about this possibility. \u003c/p\u003e\n\u003cp\u003eIn conclusion, MFE23GRNLY is an immunotoxin that causes ER stress and autophagosome accumulation in tumor cells, generally culminating in apoptotic cell death, except in cells overexpressing Bcl-x\u003csub\u003eL\u003c/sub\u003e, that seems to be executed by a caspase-independent mechanism. Overall, this ER stress-induced apoptosis and the possible synergy with Bcl-x\u003csub\u003eL\u003c/sub\u003e inhibitors could be an important strategy for future therapeutic approaches, also in lung tumors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Gobierno de Aragón grant B31_23R and by the company Peaches Biotech. RIP and APT were contracted by the grant CPP2021-008506 financed by MCIN/AEI/10.13039/501100011033 and by the European Union-NextGenerationEU/PRTR\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Raquel Ibáñez-Pérez, Ana Pilar Tobajas, Patricia Guerrero-Ochoa, Ruth Soler-Agesta, Eduardo Romanos (animal experimentation), Eva Barrio (animal experimentation), Blanca Conde (animal experimentation), Isabel Marzo, Javier Naval, Laura Sanz and Alberto Anel. The first draft of the manuscript was written by Alberto Anel and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experimentation studies were carried out under Project Licence PI38/23 approved by the Ethic Committee for Animal Experiments from the University of Zaragoza. The care and use of animals were performed accordingly with the Spanish Policy for Animal Protection RD53/2013, which meets the European Union Directive 2010/63 on the protection of animals used for experimental and other scientific purposes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Cindy Giraldo and Alejandro Ibáñez from the microscopy and histopathology Core Unit, Institute for Health Sciences of Aragon (Zaragoza, Spain), for their advice and technical support.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eGRNLY, granulysin;\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eCTL, cytotoxic T lymphocyte; NK, natural killer; PBS, phosphate buffered saline; FITC, fluorescein isothiocianate; 7-AAD, 7-amino-actinomycin D; CEA, carcinoembrionic antigen; scFv, single chain fraction variable; ER, endoplasmatic reticulum\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eIb\u0026aacute;\u0026ntilde;ez-P\u0026eacute;rez R, Guerrero-Ochoa P, Al-Wasaby S et al. 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The Adv Hematol. 10: 2040620719882822. \u003c/li\u003e\n\u003cli\u003eIb\u0026aacute;\u0026ntilde;ez-P\u0026eacute;rez R (2021) Inmunotoxina anti-CEA basada en la granulisina como una nueva terapia anti-tumoral. Tesis Doctoral Universidad de Zaragoza. \u003c/li\u003e\n\u003cli\u003eCarrington E, Tarlinton D, Gray D, Huntington N, Zhan Y, Lew A (2017) The life and death of immune cell types: the role of BCL-2 anti-apoptotic molecules. Immunol Cell Biol. 95: 870-7. \u003c/li\u003e\n\u003cli\u003eReina-Ortiz C, Mozas M, Ovelleiro D, Gao F, Villalba M, Anel A (2023) Dynamic Changes in miRNA Expression during the Generation of Expanded and Activated NK Cells. . Int J Mol Sci. 24: 13556. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"immunotoxin, scFv, CEA, granulysin, apoptosis, Bcl-xL","lastPublishedDoi":"10.21203/rs.3.rs-5446761/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5446761/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMFE23GRNLY is an immunotoxin formed by the recombinant fusion of granulysin and the single chain fraction variable (scFv) of the anti-CEA antibody MFE23. Granulysin is a protein stored in the granules of human cytotoxic T lymphocytes (CTL) and NK cells with antimicrobial and antitumoral activity. Previous work from our group demonstrated that recombinant MFE23GRNLY preserves the cytotoxic activity of GRNLY and the specific binding to CEA, targeting efficiently the antitumoral activity of GRNLY towards CEA-expressing tumors after systemic injection \u003cem\u003ein vivo\u003c/em\u003e. In the present work we observed that the lung adenocarcinoma A549 expressed CEA and was sensitive to the immunotoxin \u003cem\u003ein vitro\u003c/em\u003e. We have then demonstrated the efficacy of MFE23GRNLY against this tumor \u003cem\u003ein vivo,\u003c/em\u003e broadening the application of the immunotoxin to the treatment of lung tumors. Furthermore, we attempted tu unveil MFE23GRNLY mechanism of action compared to GRNLY on HeLa-CEA and on A549 cells. We demonstrated that MFE23GRNLY treated human tumor cells showed signs of ER stress and autophagosome accumulation. Remarkably, the general caspase inhibitor Z-VAD-fmk prevented MFE23GRNLY-induced death of HeLa-CEA cells but not of A549 cells, a feature that correlated with high levels of expression of the anti-apoptotic protein Bcl-x\u003csub\u003eL\u003c/sub\u003e in A549 cells. Accordingly, the combination of the BH3 mimetic A1155463, inhibitimg the activity of the anti-apoptotic member of the Bcl-2 family Bcl-x\u003csub\u003eL\u003c/sub\u003e, with MFE23GRNLY, showed a potent \u003cem\u003ein vitro\u003c/em\u003e synergistic effect with MFE23GRNLY in A549 cells. However, when we combined both agents \u003cem\u003ein vivo\u003c/em\u003e in athymic mice xenotransplanted with A549 cells, the synergistic effect was not observed, although the anti-tumoral potential of the systemic injection of the immunotoxin was again confirmed.\u003c/p\u003e","manuscriptTitle":"Efficient in vivo anti-tumoral effect of the anti-CEA, granulysin-based immunotoxin MFE23GRNLY in a lung adenocarcinoma model. Combination with a Bcl-xL-selective BH3 mimetic","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-04 19:23:12","doi":"10.21203/rs.3.rs-5446761/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-05T15:15:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-18T02:42:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"221662923534978095999738247035868543601","date":"2025-07-31T12:56:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-18T17:08:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3179175664254664063821292232112027214","date":"2025-03-21T10:17:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"260719723438896270026411590710158022121","date":"2025-02-25T10:06:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"139210100141871065422975263920244196253","date":"2025-01-17T16:10:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-29T17:00:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-13T12:21:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-13T12:19:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2024-11-13T11:50:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4384781c-5237-4e4a-b5ee-b58f3c53f340","owner":[],"postedDate":"June 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-03T21:23:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-04 19:23:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5446761","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5446761","identity":"rs-5446761","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}