cancers Article Potent Anticancer Activity of CXCR4-Targeted Nanostructured Toxins in Aggressive Endometrial Cancer Models Esperanza Medina-Gutiérrez 1,2 , Annabel García-León 1,2 , Alberto Gallardo 1,3 , Patricia Álamo 1,2,4 , Lorena Alba-Castellón 1,2 , Ugutz Unzueta 1,2,4,5 , Antonio Villaverde 4,5,6 , Esther Vázquez 4,5,6 , Isolda Casanova 1,2,4, * and Ramon Mangues 1,2,4, * 1 Oncogenesis and Antitumor Drugs Group, Institut d’Investigació Biomèdica Sant Pau (IIB Sant Pau), 08041 Barcelona, Spain 2 Oncogenesis and Antitumor Drugs Group, Institut de Recerca Contra la Leucèmia Josep Carreras, 08025 Barcelona, Spain 3 Department of Pathology, Hospital de la Santa Creu i Sant Pau, 08041 Barcelona, Spain 4 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain 5 Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 6 Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain * Correspondence: icasanova@santpau.cat (I.C.); rmangues@santpau.cat (R.M.) Simple Summary: The fact that most endometrial cancer (EC) patients overexpress the CXCR4 receptor in tumor tissue, especially at incurable advanced stages, opens an avenue for developing novel therapies targeting CXCR4+ EC cells to add new armamentarium against this malignancy. We have generated novel CXCR4-targeted nanotoxins, T22-DITOX-H6 and T22-PE24-H6, which we here evaluate in EC mouse models. We observed a selective killing of CXCR4+ EC cells by apoptosis induction in cultured cells as well as in tumor models, which inhibit tumor growth and increase mouse survival after repeated intravenous doses. Moreover, T22-DITOX-H6 induced a significant Citation: Medina-Gutiérrez, E.; block of metastasis dissemination without toxicity in non-tumor tissues. Therefore, both nanotoxins García-León, A.; Gallardo, A.; Álamo, may become alternative therapeutics for CXCR4+ high-risk EC patients, who nowadays lack effective P.; Alba-Castellón, L.; Unzueta, U.; therapies. Villaverde, A.; Vázquez, E.; Casanova, I.; Mangues, R. Potent Abstract: Patients with advanced endometrial cancer (EC) show poor outcomes. Thus, the develop- Anticancer Activity of ment of new therapeutic approaches to prevent metastasis development in high-risk patients is an CXCR4-Targeted Nanostructured unmet need. CXCR4 is overexpressed in EC tumor tissue, epitomizing an unexploited therapeutic Toxins in Aggressive Endometrial target for this malignancy. The in vitro antitumor activity of two CXCR4-targeted nanoparticles, in- Cancer Models. Cancers 2023, 15, 85. cluding either the C. diphtheriae (T22-DITOX-H6) or P. aeruginosa (T22-PE24-H6) toxin, was evaluated https://doi.org/10.3390/ using viability assays. Apoptotic activation was assessed by DAPI and caspase-3 and PARP cleavage cancers15010085 in cell blocks. Both nanotoxins were repeatedly administrated to a subcutaneous EC mouse model, Academic Editor: Magali whereas T22-DITOX-H6 was also used in a highly metastatic EC orthotopic model. Tumor burden was Gary-Bobo assessed through bioluminescence, while metastatic foci and toxicity were studied using histological Received: 5 December 2022 or immunohistochemical analysis. We found that both nanotoxins exerted a potent antitumor effect Revised: 20 December 2022 both in vitro and in vivo via apoptosis and extended the survival of nanotoxin-treated mice without Accepted: 20 December 2022 inducing any off-target toxicity. Repeated T22-DITOX-H6 administration in the metastatic model Published: 23 December 2022 induced a dramatic reduction in tumor burden while significantly blocking peritoneal, lung and liver metastasis without systemic toxicity. Both nanotoxins, but especially T22-DITOX-H6, represent a promising therapeutic alternative for EC patients that have a dismal prognosis and lack effective therapies. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article Keywords: advanced endometrial cancer; metastasis; protein nanoparticles; bacterial toxins; targeted distributed under the terms and drug delivery; CXCR4 conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cancers 2023, 15, 85. https://doi.org/10.3390/cancers15010085 https://www.mdpi.com/journal/cancers Cancers 2023, 15, 85 2 of 20 1. Introduction Endometrial cancer (EC) is the most common gynecological cancer in western coun- tries. It involves 90% of all uterine cancers. It accounts for 90,000 deaths worldwide per year [1], whereas the five-year survival of patients diagnosed at advanced stages is only 20% [2,3]. Despite the recent increase in molecularly targeted therapies approved by the FDA, including immune checkpoint inhibitors (pembralizumab, dostarlimab), angiogenesis inhibitors (lenvatinib, bevacizumab) or mTOR inhibitors (everolimus, temsirolimus), the first line therapy remains still surgery, chemotherapy and radiotherapy [4]. The modest response to conventional treatments, along with their high rates of resistance to chemother- apy, explains why current therapies are not successful in treating high-risk, recurrent or metastatic EC patients [5]. Thus, there is an unmet medical need calling to develop new therapeutic approaches that improve current treatment, both by preventing metastatic development in high-risk EC patients and achieving a higher inhibition of metastatic dissemination, along with a significant reduction in its associated systemic toxicity and severe adverse effects [6,7]. Moreover, while selective therapeutic alternatives such as antibody–drug conjugates (ADC), consisting of a targeted monoclonal antibody chemically attached to a cytotoxic agent, have been approved for other cancer types [8]; however there are none currently available to treat EC [9]. The development of toxin-based therapies, and particularly the immunotoxins dis- playing antitumor activity, is a highly dynamic research field, which is being evaluated in clinical trials despite their low therapeutic window. Immunotoxins are similar to ADC since they pursue targeted drug delivery; thus, they consist of a targeting monoclonal antibody, or cytokine, chemically linked to a cytotoxic domain of bacterial, plant or animal toxins [10]. Among the clinical trials that evaluated toxin-based therapies against cancer, however, the FDA approved only those to treat hematological neoplasias. In contrast, immunotoxins did not show sufficient activity against solid tumors because of poor specificity and severe adverse effects [11]. To prevent these handicaps, we are developing novel nanoparticles displaying multivalent-CXCR4 targeting ligands. These nanoparticles are capable of selectively de- livering conjugated drugs or cytotoxic protein domains to cancer cells that overexpress the CXCR4 receptor (CXCR4+ ) in cancer tissues, avoiding their delivery in healthy organs because of the low or negligible CXCR4 expression level in normal cells [12,13]. Thus, our goal is to tackle the urgent need to develop therapeutic agents that could be exploited to achieve targeted delivery of bacterial exotoxins to CXCR4+ tumor cells and thus reach personalized therapy that enhances the antineoplastic effect while reducing adverse effects in EC patients [14]. CXCR4 is a chemokine receptor overexpressed in EC tumor cells both at mRNA and protein levels [15], showing mostly membrane staining in cancer tissues of EC patients, which has been reported to promote metastases in our previous work [16]. We also recently proved the role of CXCR4 in enhancing metastasis in the EC mouse model. Moreover, we demonstrated a CXCR4-dependent biodistribution for the T22-GFP-H6 nanocarrier, and its enhanced uptake in tumor tissue in a subcutaneous EC model. In this work, we selected the CXCR4+ Luciferase+ AN3CA cell line, among the tested HEC1A, ARK-2 and AN3CA cells, to generate the subcutaneous and metastatic EC models to be used in the evaluation of these novel therapeutic nanoparticles because of their higher internalization of our CXCR4- targeted nanocarrier in this cell line. Moreover, the orthotopic model derived from CXCR4+ Luciferase+ AN3CA yielded the highest metastatic and aggressive malignancy [16]. In these models, two nanotoxins were tested: T22-DITOX-H6, which incorporates the deimmunized catalytic domain of the exotoxin from Corynebacterium diphtheriae (DITOX) and T22-PE24-H6, which incorporates the Pseudomonas aeruginosa (PE24) exotoxin domain as previously described [17] (Figure S1). Both nanotoxins maintained the same nanostruc- ture as that described for the T22-GFP-H6 nanocarrier and, similarly, formed self-assembled protein-only nanoparticles that achieve a multivalent display of CXCR4-targeted T22 lig- Cancers 2023, 15, 85 3 of 20 ands. These nanotoxins had previously shown high preclinical therapeutic potential in head and neck and colorectal cancer models and in hematological cancer models [18–20]. Additional therapeutic nanoparticles derived from the same carrier that include human proapoptotic domains, specifically BAK, PUMA or BAX pore-forming domains [13], are here compared with the nanotoxins regarding their cytotoxic effect in vitro. Thus, in this work, we performed a screening of nanotoxins and proapoptotic nanopar- ticles in terms of their anticancer effect in vitro, selecting only those with a higher effect, namely T22-DITOX-H6 and T22-PE24-H6. Then, we evaluated the antitumor and an- timetastatic effect of the nanotoxins in aggressive subcutaneous and orthotopic EC models. Our results support the nanotoxins as alternative therapies that could improve the current treatment of high-risk EC because of their capacity to prevent metastasis development at the clinically relevant dissemination sites for EC using our recently developed orthotopic model of metastatic CXCR4+ EC. 2. Materials and Methods 2.1. Cell Culture AN3CA cell line (ATCC) and transduced CXCR4+ Luciferase+ AN3CA were cultured in DMEM and F12 medium 1:1, while HEC1A (ATCC) and transduced CXCR4+ Luciferase+ HEC1A were cultured in high glucose DMEM. All cell lines were maintained in their medium supplemented with 10% fetal bovine serum and penicillin/streptomycin, at 37 ◦ C and 5% CO2 . The CXCR4+ Luciferase+ AN3CA cell line was obtained previously by lentivi- ral transduction in a previous work by our group. CXCR4 cell expression was assessed before all in vitro and in vivo experiments by flow cytometry as previously described [16]. 2.1.1. Cytotoxic Effect In vitro cytotoxicity was evaluated using XTT assay (Roche, Basel, Switzerland) fol- lowing the manufacturer’s instructions; 25,000 cells per well were seeded in 96 well plates and exposed to therapeutic nanoparticles at different concentrations ranging from 1 nM to 1000 nM for 48 h. A competition assay was performed by preincubating the cells with 1 µM CXCR4 antagonist AMD3100 for 1 h, followed by nanoparticle exposure. Absorbance at 490 nm was read (FLUOstar OPTIMA, BMG Labtech, Ortenberg, Germany) and cell viability was quantified and expressed in relation to the viability of buffer-treated cells. 2.1.2. Apoptosis Assessment by Flow Cytometry The percentage of cells undergoing early or late apoptosis was quantified using an Annexin V-FITC/propidium iodide (PI) detection kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s instructions. Data were analyzed using MACSQuant cytome- ter and MACS Quantify v2.3 software (Miltenyi Biotec, Bergisch Gladbach, Germany). 2.1.3. Paraffin-Embedded Cell Blocks In total, 3.5 × 106 CXCR4+ Luciferase+ AN3CA cells were seeded in 150 cm2 cell culture flasks. Then, 24 h later, they were treated with either buffer or 4 nM of T22-DITOX- H6 or T22-PE24-H6 nanotoxins for 6, 12, 24 or 48 h. Supernatants were collected and attached cells were washed, trypsinized, collected in the supernatant and washed in PBS. Cell blocks were obtained by gently mixing 2 drops of human plasma and 2 drops of human thrombin (Roche). Cell blocks were fixed in paraformaldehyde 4% and embedded in paraffin for further immunocytochemistry or DAPI staining analysis. All slides were examined under an optic or fluorescence microscope (Olympus BX53, Olympus, Tokyo, Japan) and representative pictures were taken using an Olympus DP73 camera. 2.1.4. DAPI Fluorescent Staining ProLong Gold Antifade Mountant with DAPI (Invitrogen, Waltham, MA, USA) was used to assess DNA condensation and apoptotic cell bodies in paraffin slides of cell blocks. Cancers 2023, 15, 85 4 of 20 4 µM slides were incubated at 60 ◦ C for 1 h and rehydrated with 3 × 5 min incubations in xylene and decreasing concentrations of alcohol to 76◦ to be stained with DAPI. 2.1.5. Immunocytochemistry Anti-active caspase-3 (1:300; BD Pharmingen, 559565) and anti-cleaved poly (ADP- ribose) polymerase PARP p85 (1:300; Promega, G73411) were used to determine apoptosis induction in cell blocks. Staining was performed in a DAKO Autostainer Link48 (Agilent, Santa Clara, CA, USA) following the manufacturer’s instructions, using 4 µm paraffin sections of cell pellets. All slides were examined by a blind observer under an optic micro- scope and representative pictures were taken using an Olympus DP73 camera. Positively stained cells were identified using QuPath software v.0.2.3 (Queen’s University, Belfast, Northern Ireland, UK) [21] at 400× magnification and compared to control samples. 2.2. Animals Five-week-old female Swiss nude (Crl:NU(Ico)-Foxn1nu ) and NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ) mice (Charles River, Écully, France) used for the experiments were main- tained under specific pathogen-free conditions, being fed with irradiated food (14% protein, 4% fat, Teklad Global diet, ENVIGO) and reverse osmosis autoclaved water ad libitum. Their light/dark cycle was 12 h and they were housed in individually ventilated cage units (Sealsafe Plus GM500, Techniplast, Buguggiate, Italy) in groups of five. All procedures were performed using sterile material in a type II biosafety cabinet (BIO-II-A/P, Telstar, Terrassa, Spain). During surgery, eye dryness was avoided using a saline drip and body temperature was maintained using a heating blanket under a drape. The correct implantation in vivo of EC cells, either in the subcutaneous or the ortho- topic model, was assessed using firefly D-luciferin (Perkin Elmer, Waltham, MA, USA), a substrate for luciferase that is expressed in transduced AN3CA cells. Mice were injected intraperitoneally with 2.25 mg/mouse, and anesthetized with 3% isoflurane in oxygen to capture the intensity of the emitted bioluminescence (BLI) 5 min after injection. Images were analyzed using Living Image v.4.7.3. software (Perkin Elmer, Waltham, MA, USA). The endpoint criteria included 10–20% body weight loss, signs of pain or distress, such as abnormal postures or development of ulcers, alopecia, ruffled fur, abnormal breathing, abnormal activity, coma, ataxia or tremors. All mice were euthanized by cervical dislocation at the experimental endpoint. All the in vivo procedures were approved by the Hospital de la Santa Creu i de Sant Pau Animal Ethics Committee and Generalitat de Catalunya (FUE-2018-00819447, 24 April 2019) and performed according to European Council directives (CEA-OH/9721/2). 2.2.1. Antitumor Effect of Nanotoxins in a Subcutaneous CXCR4+ EC Model In total, 40 Swiss nude mice were anesthetized with 2% isofluorane, and the back was swabbed with povidone-iodine. Then, 107 cells were inoculated subcutaneously (SC) in both flanks. Tumor growth was monitored using a caliper and the formula V = width2 × length/2, twice a week. To assess the effect of each nanotoxin on tumor growth and mice survival, animals were recruited when tumors reached 30–70 mm3 . They were randomized into three groups (n = 10/group), receiving either 10 µg T22-DITOX-H6, 10 µg T22-PE24-H6 or carbonate buffer as a control, three times a week for a maximum of 15 doses (Figure S2). The tumor size endpoint criterium was set at a volume of 900 mm3 . In case mice did not reach this criterium after the administration of the 15th dose, they were left untreated until the endpoint. To determine the activation of protein markers of apoptotic cell death, animals were recruited when tumors reached 150–200 mm3 to receive a single bolus of either T22-DITOX- H6 (30 µg), T22-PE24-H6 (100 µg) or carbonate buffer (n = 2 per group). Mice were euthanized at 24 or 48 h after treatment. Cancers 2023, 15, 85 5 of 20 Finally, tumor and non-tumor organs (lung, kidneys, liver, spleen) were collected, fixed in 4% formaldehyde and paraffin-embedded for histopathological or IHC evaluation of metastasis, apoptotic markers and toxicity. 2.2.2. Antimetastatic Effect of T22-DITOX-H6 Using a Highly Metastatic Orthotopic CXCR4+ EC Model In total, 22 NSG mice were anesthetized with 100 mg/kg ketamine (Ketolar, Pfizer, New York, NY, USA) and 10 mg/kg xylacyn (Rompun, Bayer, Barmen, Germany). Then, 106 CXCR4+ Luciferase+ AN3CA cells resuspended in 25 uL of culture medium were inoculated through the myometrium into the endometrial cavity using a 29G Hamilton syringe (Microliter Serie 800, Hamilton, Reno, NV, USA), according to the procedure described in [16]. Three days after implantation, cell engraftment and the bioluminescence emission were assessed using IVIS Spectrum 200. Mice were then separated into two groups (n = 10/group) to receive either 5 µg T22-DITOX-H6 or carbonate buffer as control, three times a week. Whole body BLI was determined twice a week (Figure S3). All mice were euthanized when at least one of them reached endpoint criteria, such as a high primary tumor or peritoneal carcinomatosis growth or abdominal distension, which happened two days after the 14th dose. All clinically relevant organs (uterus and ovaries, peritoneal carcinomatosis, lung, kidneys, liver, spleen, abdominal lymph nodes) were collected, fixed in 4% formaldehyde and paraffin-embedded for histopathological analysis, IHC evaluation of metastatic foci and possible toxicity of T22-DITOX-H6 on normal organs. 2.2.3. Histological Examination Paraffin sections of all organs were stained with hematoxylin-eosin (H&E) and ana- lyzed by two independent blind observers to assess the possible toxicity on non-tumor organs. 2.2.4. Immunohistochemistry Immunohistochemical staining was performed in a DAKO Autostainer Link48 (Agi- lent) following the manufacturer’s instructions, using 4 µm paraffin sections of cell pellets or organs. Anti-human vimentin (Dako IGA63061-) was used to determine the presence of tumor cells in clinically relevant organs. Anti-active caspase-3 (1:300; BD Pharmingen, 559565) and anti-cleaved poly (ADP-ribose) polymerase PARP p85 (1:300; Promega, G73411) were used to determine apoptosis induction in vivo. All slides were examined under an optic microscope and representative pictures were taken using an Olympus DP73 camera. Liver sections were processed with cellSens software (Olympus), while lung sections were analyzed with QuPath, both at 200× magnifications. 2.3. Statistical Analysis All in vitro experiments were performed in biological triplicates. Mice number for in vivo experiments was defined in preliminary experiments to measure the interindividual variability among mice in terms of tumor growth and metastatic load. Randomizations of animals in experimental and control groups were performed using a dice. Two mice with undetectable orthotopic cell engraftment assessed by bioluminescence were excluded from the experiment. The investigator was not blinded to group allocation during in vivo manipulation of animals; nevertheless, she was blinded for the histological analysis. All results were expressed as mean ± standard error (SE). Differences between groups were analyzed using either the Kruskal–Wallis test, Student T-test or Mann–Whitney test, after the determination of normality by the Shapiro–Wilk test. Differences were considered statistically significant at p ≤ 0.05. Statistical calculations were performed using SPSS software v.23 (IBM, Armonk, NY, USA). Cancers 2023, 15, 85 6 of 20 3. Results 3.1. Potent CXCR4-Dependent Cytotoxic Effect Induced by T22-DITOX-H6 and T22-PE24-H6 in CE Cells In Vitro CXCR4 expression was validated in three human EC cell lines in previous work. As previously explained, we selected the transduced AN3CA cell line among ARK-2 and HEC- 1A to perform the in vitro work because it internalized our CXCR4-targeted nanocarrier selectively, but also because it showed higher aggressiveness in vivo, obtaining models that could be used afterward in the in vivo evaluation of selected nanoparticles screened in the in vitro studies [16]. Nevertheless, we assessed the cytotoxic activity of two nanotoxins and four distinct proapoptotic nanoparticles in both CXCR4+ Luciferase+ AN3CA and CXCR4+ Luciferase+ HEC1A cell lines. Interestingly, T22-PUMA-GFP-H6, T22-BAXPORO-GFP-H6 and T22-BAK-GFP-H6 proapoptotic nanoparticles did not show cytotoxic effect in any of the tested cell lines after cell exposure to 1000 nM nanoparticles for 48 h. Interestingly, T22-PE24-H6 and T22-DITOX-H6 showed a dramatically higher cytotoxic effect on the CXCR4+ AN3CA cell line, compared to the CXCR4+ HEC1A cell line (Figure 1A). Thus, we continued the in vitro characterization of the nanotoxins effect using the AN3CA cell line. After decreasing the concentration range of nanotoxins, both T22-DITOX-H6 and T22-PE24-H6 were found to exert a potent anticancer effect in the low nanomolar rank, with IC50 of 1.6 nM and 2.5 nM, respectively. Moreover, none of the tested nanoparticles in the CXCR4− parental AN3CA cell line showed a cytotoxic effect; therefore, their anticancer effect was CXCR4-dependent (Figure 1B,C). In addition, a competition assay was also performed, by blocking the CXCR4 receptor with CXCR4 antagonist AMD3100 in CXCR4+ Luciferase+ AN3CA cells, by their pretreatment for 1 h with 1000 nM AMD3100 before nanotoxin exposure. We observed that the cytotoxic effect induced by the T22-DITOX-H6 or T22-PE24-H6 was inhibited using cytotoxic concentrations of the nanotoxins (Figure 1D,E). Therefore, we observed that both T22-DITOX-H6 and T22-PE24-H6 induce a high cytotoxic effect in vitro and also that this activity is mediated by the CXCR4 receptor. 3.2. T22-DITOX-H6 and T22-PE24-H6-Induced EC Cell Death In Vitro Is Mediated by Apoptosis Induction Translocation of phosphatidyl serine was assessed by annexin V/PI assay in CXCR4+ Luciferase+ AN3CA cells exposed to 4 nM T22-DITOX-H6 or T22-PE24-H6. While early apoptosis only could be assessed in T22-DITOX-H6 treated cells at 6 h, late apoptosis can be found at 24 h in cells treated with any of the two nanotoxins (Figure 2A–C). Cancers 2023, 15, x FOR PEER REVIEW 7 of 21 Cancers 2023, 15, 85 7 of 20 Figure 1. The CXCR4-dependent cytotoxic effect induced by T22-DITOX-H6 and T22-PE24-H6, at a low nanomolar range, in human endometrial cancer cells in vitro. (A) Screening of therapeutic Figure 1. The CXCR4-dependent cytotoxic effect induced by T22-DITOX-H6 and T22-PE24-H6, at nanoparticles targeted to the CXCR4endometrial receptor, at 1cancer µM protein concentration, in the CXCR4 + AN3CA a low nanomolar range, in human cells in vitro. (A) Screening of therapeutic and + CXCR4 HEC1A nanoparticles targetedcell lines. to the T22-DITOX-H6 CXCR4 receptor, at 1 and T22-PE24-H6 µ M protein show ain concentration, higher cytotoxic the CXCR4 + effect AN3CA on CXCR4 + +AN3CA cells than the rest of pro-apoptotic nanoparticles. (B,C) Dose–response curve and CXCR4 HEC1A cell lines. T22-DITOX-H6 and T22-PE24-H6 show a higher cytotoxic effect on CXCR4+ AN3CA expressed cellsofthan as percent the restviable remaining CXCR4+ and of pro-apoptotic CXCR4- AN3CA nanoparticles. (B,C)cells Dose–response curve after exposure to ex- T22- pressed as (B) DITOX-H6 percent of remaining or T22-PE24-H6 viable (C) at theCXCR4 + 1–50 nM and CXCR4 range. - AN3CA cells after exposure to T22- (D,E) Competition assays, measuring the DITOX-H6 percent (B) orCXCR4 of viable T22-PE24-H6 + AN3CA (C) cells at theafter 1–50exposure nM range. to (D,E) Competition T22-DITOX-H6 (D)assays, measuring the or T22-DITOX-H6 (E) percent of viable CXCR4+ AN3CA cells after exposure to T22-DITOX-H6 (D) or T22-DITOX-H6 (E) without or with a prior exposure to the CXCR4 antagonist AMD3100. * p < 0.005; ** p = 0.001 (mean without or with a prior exposure to the CXCR4 antagonist AMD3100. * p < 0.005; ** p = 0.001 (mean ±± SEM; SEM; Mann–Whitney). Mann–Whitney). After decreasing the concentration range of nanotoxins, both T22-DITOX-H6 and T22-PE24-H6 were found to exert a potent anticancer effect in the low nanomolar rank, with IC50 of 1.6 nM and 2.5 nM, respectively. Moreover, none of the tested nanoparticles Cancers 2023, 15, 85 8 of 20 Moreover, apoptosis markers such as nuclear condensation, according to the results of DAPI staining, and expression of apoptotic markers (cleaved caspase-3 and its cleavage product, and proteolyzed PARP), increase at 6 h after exposure to both nanoparticles, being this effect higher in cells treated with T22-DITOX-H6 (Figure 2D–F). Positive cells for all Cancers 2023, 15, x FOR PEER REVIEWthese markers were determined using QuPath software and statistically significant 8 ofdiffer- 21 ences were found when comparing all treated samples at different timepoints, compared to control cells (Figure 2E,F). Overall, these results show a high cytotoxic effect in vitro for both nanotoxins through inthe theactivation CXCR4- parental AN3CA cell of the apoptotic line showed pathway, a cytotoxic as supported by effect; therefore,oftheir the activation anti- of markers cancer effect was CXCR4-dependent apoptotic induction markers. (Figures 1B,C). In addition, a competition assay was also performed, by blocking the CXCR4 receptor with CXCR4 antagonist AMD3100 in CXCR4 + Luciferase+ AN3CA cells, by their pretreatment for 1 h with 1000 nM AMD3100 3.3. Repeated T22-DITOX-H6 or T22-PE24-H6 Treatment Inhibit Tumor Growth and Increases before nanotoxin Survival exposure.Toxicity without Systemic We observed that +the in a CXCR4 cytotoxic AN3CA effect induced Subcutaneous by the T22-DI- CE Model TOX-H6 Since both nanotoxins showed a high cytotoxic effect in the CXCR4nanotoxins or T22-PE24-H6 was inhibited using cytotoxic concentrations of the + Luciferase+ (Figure 1D,E). Therefore, we observed that both T22-DITOX-H6 and T22-PE24-H6 AN3CA cell line, we used the subcutaneous EC model derived from this cell line induce to test atheir highanticancer cytotoxic effect in vitro and also that this activity is mediated by the CXCR4 activity in vivo. Thus, mice bearing subcutaneous tumors derived from thisrecep- tor. cell line were recruited once the tumors had a volume of 30–70 mm3 , and were randomized to three groups, which received repeated intravenous doses of either buffer, 10 µg T22- 3.2. T22-DITOX-H6 DITOX-H6 or 10 µgand T22-PE24-H6-Induced T22-PE24-H6. Mice wereEC Cell Death Inup administered Vitro is Mediated to 15 by all of them doses, and Apoptosis Induction were left to survival, being euthanized once the tumors reached 900 mm . 3 Translocation of phosphatidyl Nanotoxin-treated serine awas groups showed assessedinby reduction annexin tumor V/PIrate growth assay asin CXCR4+ to compared Luciferase the controlAN3CA + cells group, as wellexposed to 4 mice as a longer nM T22-DITOX-H6 survival, whichorwasT22-PE24-H6. statisticallyWhile earlyfor significant apoptosis only could be assessed in T22-DITOX-H6 treated cells at 6 h, late both nanotoxins, and these effects were more pronounced in the T22-DITOX-H6-treated apoptosis can be found at 24 h group (Figure 3).in cells treated with any of the two nanotoxins (Figure 2A–C). Figure 2. Cont. Cancers 2023, 15, x FOR PEER REVIEW 9 of 21 Cancers 2023, 15, 85 9 of 20 Figure 2. T22-DITOX-H6 and T22-PE24-H6-induced cell death in vitro in CXCR4+ AN3CA endome- Figure 2. T22-DITOX-H6 and T22-PE24-H6-induced cell death in vitro in CXCR4+ AN3CA endome- trialcancer trial cancercells cellsisismediated mediated byby apoptosis. apoptosis. (A)(A) Representative Representative dot-plot dot-plot andand quantitation quantitation of viable of viable cells (green), at early (light blue) or late (navy blue) apoptosis using Annexin V-FITC/PI cells (green), at early (light blue) or late (navy blue) apoptosis using Annexin V-FITC/PI assay, after assay, after their exposure for 2, 6, 24 and 48 h to 4 nM of T22-DITOX-H6 (B) or T22-PE24-H6 (C), expressed as as their exposure for 2, 6, 24 and 48 h to 4 nM of T22-DITOX-H6 (B) or T22-PE24-H6 (C), expressed mean mean± ± SEM. (D) SEM. (D)Representative Representative images images ofofDAPI DAPI oror immunohistochemistry immunohistochemistry staining ofof staining the theapopto- apoptosis sis mediators, active cleaved caspase-3 and cleaved PARP. Experiments were performed usingusing mediators, active cleaved caspase-3 and cleaved PARP. Experiments were performed CXCR4+ CXCR4 + AN3CA cell pellets after 6, 12, 24 or 48 h exposure to 4 nM of T22-DITOX-H6 or T22-PE24- AN3CA cell pellets after 6, 12, 24 or 48 h exposure to 4 nM of T22-DITOX-H6 or T22-PE24-H6. Bar: H6. Bar: 50 µ m. (E) Quantification of positive cell percentage, compared to control, regarding the 50 µm. (E,F) Quantification of positive cell percentage, compared to control, regarding the induction induction of apoptotic bodies (DAPI staining) and cleavage of caspase-3 (act. Casp-3) and PARP of apoptotic (pPARP) bodies (DAPI (Mann–Whitney; p