Clinical Relevance of Tumour-Infiltrating Immune Cells in HER2-Negative Breast Cancer Treated with Neoadjuvant Therapy.

Currently, therapy response cannot be accurately predicted in HER2-negative breast cancer (BC). Measuring stromal tumour-infiltrating lymphocytes (sTILs) and mediators of the tumour microenvironment and characterizing tumour-infiltrating immune cells (TIICs) may improve treatment response in the neoadjuvant setting. Tumour tissue and peripheral blood samples were retrospectively collected from 118 patients, and sTILs were evaluated. Circulating exosomes and myeloid-derived suppressor cells were determined by flow cytometry. TIICs markers (CD4, CD8, CD20, CD1a, and CD68) were assessed immunohistochemically. High sTILs were significantly associated with pathological complete response (pCR; p = 0.048) and event-free survival (EFS; p = 0.027). High-CD68 cells were significantly associated with pCR in triple-negative (TN, p = 0.027) and high-CD1a cells with EFS in luminal-B (p = 0.012) BC. Cluster analyses of TIICs revealed two groups of tumours (C1 and C2) that had different immune patterns and clinical outcomes. An immunoscore based on clinicopathological variables was developed to identify high risk (C1) or low-risk (C2) patients. Additionally, cluster analyses revealed two groups of tumours for both luminal-B and TNBC. Our findings support the association of sTILs with pCR and show an immunological component in a subset of patients with HER2-negative BC. Our immunoscore may be useful for future escalation or de-escalation treatments.

Hydrogels with dual sensitivity to temperature and pH in physiologically relevant ranges as supports for versatile controlled cell detachment.

Thermosensitive hydrogels based on the N-vinyl caprolactam (VCL), capable of allowing for cell adhesion and proliferation, as well as non-aggressive detachment by controlled temperature drop, were functionalized with 23 % or lower molar percentages of the cationizable hydrophobic unit 2-(diisopropylamino) ethyl methacrylate (DPAEMA), to obtain networks with dual sensitivity to temperature and pH. The swelling analysis of the systems has shown a transition pK (pKb) close to physiological values, dependent on the temperature of the medium (pKb of 6.6 and 6.9 when the temperature of the medium is above and below the transition temperature VPTT, respectively) and little dependence on the degree of functionalization of DPAEMA. In addition, at temperatures below the transition temperature (VPTT), the systems have shown large swelling variations as a function of the pH (i.e. below and above the pKb), exhibiting greater absorption capacity at pHs below pKb, where the DPAEMA units are cationized. Cytocompatibility and transplant capacity have been evaluated using the C166-GFP endothelial cell line. None of the thermosensitive hydrogels with variable DPAEMA content showed a delay with respect to the control without DPAEMA neither in terms of adhesion nor in proliferation. However, by increasing the percentage of DPAEMA functionalization -and decreasing thermosensitivity-, a correlative decrease in mitochondrial activity was obtained in the transplant, with significant differences for the hydrogels with DPAEMA molar percentage of 3 % or higher. Taking advantage of the proximity of the pKb to the physiological value, we have evaluated the cellular response and the capacity for transplantation after lowering the pH to 6.5, below pKb. A direct relationship of the DPAEMA functionalization degree on the detachment efficiency was observed, since the hydrogels with the highest molar load of DPAEMA showed higher mitochondrial metabolic activity after cell detachment.

Secreted Protein Acidic and Rich in Cysteine (SPARC) Polymorphisms in Response to Neoadjuvant Chemotherapy in HER2-Negative Breast Cancer Patients.

Secreted protein acidic and rich in cysteine (SPARC) expression has been proposed as a prognostic and predictive biomarker for some cancer types, but knowledge about the predictive value of SPARC polymorphisms in the context of neoadjuvant therapy for breast cancer (BC) is lacking. In 132 HER2-negative BC patients treated with neoadjuvant chemotherapy, we determined polymorphisms in the SPARC gene and analyzed their association with outcome. We also determined SPARC protein expression in tumor tissue. SPARC rs19789707 was significantly associated with response to treatment according to the Miller and Payne system in the breast (multivariate: odds ratio (OR), 3.81; p = 0.028). This association was significant in the subgroup of patients with luminal tumors (univariate: p = 0.047). Regarding survival, two SPARC variants showed significant associations with event-free survival: the rs19789707 variant in the subgroup of luminal A tumors (univariate: p = 0.006), and the rs4958487 variant in the subgroup of luminal B tumors (univariate: p = 0.022). In addition, SPARC rs4958487, rs10065756, and rs12153644 were significantly correlated with SPARC protein expression. Our findings suggest that SPARC polymorphisms could be good predictors of treatment response and survival in BC patients treated with neoadjuvant chemotherapy, especially those with luminal tumors.

High-precision targeting and destruction of cancer-associated PDGFR-ß+ stromal fibroblasts through self-assembling, protein-only nanoparticles.

The need for more effective and precision medicines for cancer has pushed the exploration of new materials appropriate for drug delivery and imaging, and alternative receptors for targeting. Among the most promising strategies, finding suitable cell surface receptors and targeting agents for cancer-associated platelet derived growth factor receptor ß (PDGFR-ß)+ stromal fibroblasts is highly appealing. As a neglected target, this cell type mechanically and biologically supports the growth, progression, and infiltration of solid tumors in non-small cell lung, breast, pancreatic, and colorectal cancers. We have developed a family of PDGFR-ß-targeted nanoparticles based on biofabricated, self-assembling proteins, upon hierarchical and iterative selective processes starting from four initial candidates. The modular protein PDGFD-GFP-H6 is well produced in recombinant bacteria, resulting in structurally robust oligomeric particles that selectively penetrates into PDGFR-ß+ stromal fibroblasts in a dose-dependent manner, by means of the PDGFR-ß ligand PDGFD. Upon in vivo administration, these GFP-carrying protein nanoparticles precisely accumulate in tumor tissues and enlighten them for IVIS observation. When GFP is replaced by a microbial toxin, selective tumor tissue destruction is observed associated with a significant reduction in tumor volume growth. The presented data validate the PDGFR-ß/PDGFD pair as a promising toolbox for targeted drug delivery in the tumor microenvironment and oligomeric protein nanoparticles as a powerful instrument to mediate highly selective biosafe targeting in cancer through non-cancer cells. STATEMENT OF SIGNIFICANCE: We have developed a transversal platform for nanoparticle-based drug delivery into cancer-associated fibroblasts. This is based on the engineered modular protein PDGFD-GFP-H6 that spontaneously self-assemble and selectively penetrates into PDGFR-ß+ stromal fibroblasts in a dose-dependent manner, by means of the PDGFR-ß ligand PDGFD. In vivo, these protein nanoparticles accumulate in tumor and when incorporating a microbial toxin, they destroy tumor tissues with a significant reduction in tumor volume, in absence of side toxicities. The data presented here validate the PDGFR-ß/PDGFD pair as a fully versatile toolbox for targeted drug delivery in the tumor microenvironment intended as a synergistic treatment.

Vat Photopolymerization 3D Printing of Hydrogels with Re-Adjustable Swelling.

Vat photopolymerization typically prints highly crosslinked networks. Printing hydrogels, which are also networks but with a high swelling capacity in water and therefore with low crosslinking density, is a challenge for this technique. However, it may be of interest in medicine and in other areas, since it would allow for the preparation of this type of 3D-shaped material. In this work, an approach for printing hydrogels via vat photopolymerization that uses a mixture of stable and hydrolysable crosslinkers has been evaluated so that an initial highly crosslinked network can be printed, although after hydrolysis it becomes a network with low crosslinking. This approach has been studied with PEO/PEG-related formulations, that is, with a PEG-dimethacrylate as a stable crosslinker, a PEO-related derivative carrying ß-aminoesters as a degradable crosslinker, and PEG-methyl ether acrylate and hydroxyethyl acrylate as monofunctional monomers. A wide family of formulations has been studied, maintaining the weight percentage of the crosslinkers at 15%. Resins have been studied in terms of viscosity, and the printing process has been evaluated through the generation of Jacobs working curves. It has been shown that this approach allows for the printing of pieces of different shapes and sizes via vat photopolymerization, and that these pieces can re-ajust their water content in a tailored fashion through treatments in different media (PBS or pH 10 buffer).

Technological advances in fibrin for tissue engineering.

Fibrin is a promising natural polymer that is widely used for diverse applications, such as hemostatic glue, carrier for drug and cell delivery, and matrix for tissue engineering. Despite the significant advances in the use of fibrin for bioengineering and biomedical applications, some of its characteristics must be improved for suitability for general use. For example, fibrin hydrogels tend to shrink and degrade quickly after polymerization, particularly when they contain embedded cells. In addition, their poor mechanical properties and batch-to-batch variability affect their handling, long-term stability, standardization, and reliability. One of the most widely used approaches to improve their properties has been modification of the structure and composition of fibrin hydrogels. In this review, recent advances in composite fibrin scaffolds, chemically modified fibrin hydrogels, interpenetrated polymer network (IPN) hydrogels composed of fibrin and other synthetic or natural polymers are critically reviewed, focusing on their use for tissue engineering.

Probing the Biosafety of Implantable Artificial Secretory Granules for the Sustained Release of Bioactive Proteins.

Among bio-inspired protein materials, secretory protein microparticles are of clinical interest as self-contained, slow protein delivery platforms that mimic secretory granules of the human endocrine system, in which the protein is both the drug and the scaffold. Upon subcutaneous injection, their progressive disintegration results in the sustained release of the building block polypeptides, which reach the bloodstream for systemic distribution and subsequent biological effects. Such entities are easily fabricated in vitro by Zn-assisted cross-molecular coordination of histidine residues. Using cationic Zn for the assembly of selected pure protein species and in the absence of any heterologous holding material, these granules are expected to be nontoxic and therefore adequate for different clinical uses. However, such presumed biosafety has not been so far confirmed and the potential protein dosage threshold not probed yet. By selecting the receptor binding domain (RBD) from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein as a model protein and using a mouse lab model, we have explored the toxicity of RBD-made secretory granules at increasing doses up to ∼100 mg/kg of animal weight. By monitoring body weight and biochemical blood markers and through the histological scrutiny of main tissues and organs, we have not observed systemic toxicity. Otherwise, the bioavailability of the material was demonstrated by the induction of specific antibody responses. The presented data confirm the intrinsic biosafety of artificial secretory granules made by recombinant proteins and prompt their further clinical development as self-contained and dynamic protein reservoirs.

Dopamine receptors D1 and D2 show prognostic significance and potential therapeutic applications for endometrial cancer patients.

OBJECTIVE: Catecholaminergic signaling has been a target for therapy in different type of cancers. In this work, we characterized the ADRß2, DRD1 and DRD2 expression in healthy tissue and endometrial tumors to evaluate their prognostic significance in endometrial cancer (EC), unraveling their possible application as an antitumor therapy. METHODS: 109 EC patients were included. The expression of the ADRß2, DRD1 and DRD2 proteins was evaluated by immunohistochemistry and univariate and multivariate analysis to assess their association with clinic-pathological and outcome variables. Finally, HEC1A and AN3CA EC cell lines were exposed to different concentrations of selective dopaminergic agents alone or in combination to study their effects on cellular viability. RESULTS: ADRß2 protein expression was not associated with clinico-pathological parameters or prognosis. DRD1 protein expression was reduced in tumors samples but showed a significant inverse association with tumor size and stage. DRD2 protein expression was significantly associated with non-endometrioid EC, high grade tumors, tumor size, worse disease-free survival (HR = 3.47 (95%CI:1.35-8.88)) and overall survival (HR = 2.98 (95%CI:1.40-6.34)). The DRD1 agonist fenoldopam showed a reduction of cellular viability in HEC1A and AN3CA cells. The exposure to domperidone, a DRD2 antagonist, significantly reduced cell viability compared to the control. Finally, DRD1 agonism and DRD2 antagonism combination induced a significant reduction in cell viability of the AN3CA cells compared to monotherapy, close to being an additive response than a synergistic effect (CI of 1.1 at 0.5% Fa). CONCLUSION: DRD1 and DRD2 expression levels showed a significant association with clinico-pathological parameters. Both the combined activation of DRD1 and blockage of DRD2 may form an innovative strategy to inhibit tumor growth in EC.

Lymphocyte infiltration and antitumoral effect promoted by cytotoxic inflammatory proteins formulated as self-assembling, protein-only nanoparticles.

Two human proteins involved in the inflammatory cell death, namely Gasdermin D (GSDMD) and the Mixed Lineage Kinase Domain-Like (MLKL) protein have been engineered to accommodate an efficient ligand of the tumoral cell marker CXCR4, and a set of additional peptide agents that allow their spontaneous self-assembling. Upon production in bacterial cells and further purification, both proteins organized as stable nanoparticles of 46 and 54 nm respectively, that show, in this form, a moderate but dose-dependent cytotoxicity in cell culture. In vivo, and when administered in mouse models of colorectal cancer through repeated doses, the nanoscale forms of tumor-targeted GSDMD and, at a lesser extent, of MLKL promoted CD8+ and CD20+ lymphocyte infiltration in the tumor and an important reduction of tumor size, in absence of systemic toxicity. The potential of these novel pharmacological agents as anticancer drugs is discussed in the context of synergistic approaches to more effective cancer treatments.

Evaluation of Poly(N-Ethyl Pyrrolidine Methacrylamide) (EPA) and Derivatives as Polymeric Vehicles for miRNA Delivery to Neural Cells.

MicroRNAs (miRNAs) are endogenous, short RNA oligonucleotides that regulate the expression of hundreds of proteins to control cells' function in physiological and pathological conditions. miRNA therapeutics are highly specific, reducing the toxicity associated with off-target effects, and require low doses to achieve therapeutic effects. Despite their potential, applying miRNA-based therapies is limited by difficulties in delivery due to their poor stability, fast clearance, poor efficiency, and off-target effects. To overcome these challenges, polymeric vehicles have attracted a lot of attention due to their ease of production with low costs, large payload, safety profiles, and minimal induction of the immune response. Poly(N-ethyl pyrrolidine methacrylamide) (EPA) copolymers have shown optimal DNA transfection efficiencies in fibroblasts. The present study aims to evaluate the potential of EPA polymers as miRNA carriers for neural cell lines and primary neuron cultures when they are copolymerized with different compounds. To achieve this aim, we synthesized and characterized different copolymers and evaluated their miRNA condensation ability, size, charge, cytotoxicity, cell binding and internalization ability, and endosomal escape capacity. Finally, we evaluated their miRNA transfection capability and efficacy in Neuro-2a cells and rat primary hippocampal neurons. The results indicate that EPA and its copolymers, incorporating ß-cyclodextrins with or without polyethylene glycol acrylate derivatives, can be promising vehicles for miRNA administration to neural cells when all experiments on Neuro-2a cells and primary hippocampal neurons are considered together.

The Environmental Impacts of Disposable Nonwoven Fabrics during the COVID-19 Pandemic: Case Study on the Francesc de Borja Hospital.

Hospitals generate huge amounts of nonwoven residues daily. This paper focused on studying the evolution of nonwoven waste generated in the Francesc de Borja Hospital, Spain, over the last few years and its relation to the COVID-19 pandemic. The main objective was to identify the most impacting pieces of nonwoven equipment in the hospital and to analyze possible solutions. The carbon footprint of the nonwoven equipment was studied through a life-cycle assessment. The results showed an apparent increase in the carbon footprint in the hospital from 2020. Additionally, due to the higher annual volume, the simple nonwoven gown used primarily for patients had a higher carbon footprint over a year than the more sophisticated surgical gowns. It can be concluded that developing a local circular economy strategy for medical equipment could be the solution to avoid the enormous waste generation and the carbon footprint of nonwoven production.

T22-PE24-H6 Nanotoxin Selectively Kills CXCR4-High Expressing AML Patient Cells In Vitro and Potently Blocks Dissemination In Vivo.

Despite advances in the development of targeted therapies for acute myeloid leukemia (AML), most patients relapse. For that reason, it is still necessary to develop novel therapies that improve treatment effectiveness and overcome drug resistance. We developed T22-PE24-H6, a protein nanoparticle that contains the exotoxin A from the bacterium Pseudomonas aeruginosa and is able to specifically deliver this cytotoxic domain to CXCR4+ leukemic cells. Next, we evaluated the selective delivery and antitumor activity of T22-PE24-H6 in CXCR4+ AML cell lines and BM samples from AML patients. Moreover, we assessed the in vivo antitumor effect of this nanotoxin in a disseminated mouse model generated from CXCR4+ AML cells. T22-PE24-H6 showed a potent, CXCR4-dependent antineoplastic effect in vitro in the MONO-MAC-6 AML cell line. In addition, mice treated with nanotoxins in daily doses reduced the dissemination of CXCR4+ AML cells compared to buffer-treated mice, as shown by the significant decrease in BLI signaling. Furthermore, we did not observe any sign of toxicity or changes in mouse body weight, biochemical parameters, or histopathology in normal tissues. Finally, T22-PE24-H6 exhibited a significant inhibition of cell viability in CXCR4high AML patient samples but showed no activity in CXCR4low samples. These data strongly support the use of T22-PE24-H6 therapy to benefit high-CXCR4-expressing AML patients.

Fabrication of 3D cylindrical thermosensitive hydrogels as supports for cell culture and detachment of tubular cell sheets.

Pseudo interpenetrating vinyl-caprolactam (VCL) based thermosensitive tubular hydrogels with a volume phase transition temperature, VPTT, around 35 °C, have been prepared by combining two different crosslinkers, a di-methacrylate (C1) and a di-vinyl urea (C2). The molar ratio between the two crosslinkers (for a global crosslinker molar percentage of 1.9) has shown to play a key role on the properties of the hydrogel. Increasing the amount of di-vinyl urea, leads to transparent but rather fragile materials and to a lower extent of thermosensitivity, that is, to a lower variation in the hydrogel swelling upon temperature change. However, tubes prepared with a selected crosslinker molar ratio C1/C2 of 65/35 provided a compromise between transparency, thermosensitivity and maneuverability and were, thus, evaluated as supports for cell culture using premyoblastic cells. These hydrogels, used as supports, allow for surface adhesion and cell proliferation until confluence, and eventually an efficient monolayer detachment (and transplant to a 3D-printed polylactic acid (PLA) support) through a controlled drop in temperature. As a result, this method permits to obtain tubular tissue constructs with potential applications in tissue engineering such as in the elaboration of vascular grafts.

Novel Endometrial Cancer Models Using Sensitive Metastasis Tracing for CXCR4-Targeted Therapy in Advanced Disease.

Advanced endometrial cancer (EC) lacks therapy, thus, there is a need for novel treatment targets. CXCR4 overexpression is associated with a poor prognosis in several cancers, whereas its inhibition prevents metastases. We assessed CXCR4 expression in EC in women by using IHC. Orthotopic models were generated with transendometrial implantation of CXCR4-transduced EC cells. After in vitro evaluation of the CXCR4-targeted T22-GFP-H6 nanocarrier, subcutaneous EC models were used to study its uptake in tumor and normal organs. Of the women, 91% overexpressed CXCR4, making them candidates for CXCR4-targeted therapies. Thus, we developed CXCR4+ EC mouse models to improve metastagenesis compared to current models and to use them to develop novel CXCR4-targeted therapies for unresponsive EC. It showed enhanced dissemination, especially in the lungs and liver, and displayed 100% metastasis penetrance at all clinically relevant sites with anti-hVimentin IHC, improving detection sensitivity. Regarding the CXCR4-targeted nanocarrier, 60% accumulated in the SC tumor; therefore, selectively targeting CXCR4+ cancer cells, without toxicity in non-tumor organs. Our CXCR4+ EC models will allow testing of novel CXCR4-targeted drugs and development of nanomedicines derived from T22-GFP-H6 to deliver drugs to CXCR4+ cells in advanced EC. This novel approach provides a therapeutic option for women with metastatic, high risk or recurrent EC that have a dismal prognosis and lack effective therapies.

Self-assembling protein nanocarrier for selective delivery of cytotoxic polypeptides to CXCR4+ head and neck squamous cell carcinoma tumors.

Loco-regional recurrences and distant metastases represent the main cause of head and neck squamous cell carcinoma (HNSCC) mortality. The overexpression of chemokine receptor 4 (CXCR4) in HNSCC primary tumors associates with higher risk of developing loco-regional recurrences and distant metastases, thus making CXCR4 an ideal entry pathway for targeted drug delivery. In this context, our group has generated the self-assembling protein nanocarrier T22-GFP-H6, displaying multiple T22 peptidic ligands that specifically target CXCR4. This study aimed to validate T22-GFP-H6 as a suitable nanocarrier to selectively deliver cytotoxic agents to CXCR4+ tumors in a HNSCC model. Here we demonstrate that T22-GFP-H6 selectively internalizes in CXCR4+ HNSCC cells, achieving a high accumulation in CXCR4+ tumors in vivo, while showing negligible nanocarrier distribution in non-tumor bearing organs. Moreover, this T22-empowered nanocarrier can incorporate bacterial toxin domains to generate therapeutic nanotoxins that induce cell death in CXCR4-overexpressing tumors in the absence of histological alterations in normal organs. Altogether, these results show the potential use of this T22-empowered nanocarrier platform to incorporate polypeptidic domains of choice to selectively eliminate CXCR4+ cells in HNSCC. Remarkably, to our knowledge, this is the first study testing targeted protein-only nanoparticles in this cancer type, which may represent a novel treatment approach for HNSCC patients.

Preparation and Characterization of Plasma-Derived Fibrin Hydrogels Modified by Alginate di-Aldehyde.

Fibrin hydrogels are one of the most popular scaffolds used in tissue engineering due to their excellent biological properties. Special attention should be paid to the use of human plasma-derived fibrin hydrogels as a 3D scaffold in the production of autologous skin grafts, skeletal muscle regeneration and bone tissue repair. However, mechanical weakness and rapid degradation, which causes plasma-derived fibrin matrices to shrink significantly, prompted us to improve their stability. In our study, plasma-derived fibrin was chemically bonded to oxidized alginate (alginate di-aldehyde, ADA) at 10%, 20%, 50% and 80% oxidation, by Schiff base formation, to produce natural hydrogels for tissue engineering applications. First, gelling time studies showed that the degree of ADA oxidation inhibits fibrin polymerization, which we associate with fiber increment and decreased fiber density; moreover, the storage modulus increased when increasing the final volume of CaCl2 (1% w/v) from 80 µL to 200 µL per milliliter of hydrogel. The contraction was similar in matrices with and without human primary fibroblasts (hFBs). In addition, proliferation studies with encapsulated hFBs showed an increment in cell viability in hydrogels with ADA at 10% oxidation at days 1 and 3 with 80 µL of CaCl2; by increasing this compound (CaCl2), the proliferation does not significantly increase until day 7. In the presence of 10% alginate oxidation, the proliferation results are similar to the control, in contrast to the sample with 20% oxidation whose proliferation decreases. Finally, the viability studies showed that the hFB morphology was maintained regardless of the degree of oxidation used; however, the quantity of CaCl2 influences the spread of the hFBs.

A Novel CXCR4-Targeted Diphtheria Toxin Nanoparticle Inhibits Invasion and Metastatic Dissemination in a Head and Neck Squamous Cell Carcinoma Mouse Model.

Loco-regional recurrences and metastasis represent the leading causes of death in head and neck squamous cell carcinoma (HNSCC) patients, highlighting the need for novel therapies. Chemokine receptor 4 (CXCR4) has been related to loco-regional and distant recurrence and worse patient prognosis. In this regard, we developed a novel protein nanoparticle, T22-DITOX-H6, aiming to selectively deliver the diphtheria toxin cytotoxic domain to CXCR4+ HNSCC cells. The antimetastatic effect of T22-DITOX-H6 was evaluated in vivo in an orthotopic mouse model. IVIS imaging system was utilized to assess the metastatic dissemination in the mouse model. Immunohistochemistry and histopathological analyses were used to study the CXCR4 expression in the cancer cells, to evaluate the effect of the nanotoxin treatment, and its potential off-target toxicity. In this study, we report that CXCR4+ cancer cells were present in the invasive tumor front in an orthotopic mouse model. Upon repeated T22-DITOX-H6 administration, the number of CXCR4+ cancer cells was significantly reduced. Similarly, nanotoxin treatment effectively blocked regional and distant metastatic dissemination in the absence of systemic toxicity in the metastatic HNSCC mouse model. The repeated administration of T22-DITOX-H6 clearly abrogates tumor invasiveness and metastatic dissemination without inducing any off-target toxicity. Thus, T22-DITOX-H6 holds great promise for the treatment of CXCR4+ HNSCC patients presenting worse prognosis.

A diphtheria toxin-based nanoparticle achieves specific cytotoxic effect on CXCR4+ lymphoma cells without toxicity in immunocompromised and immunocompetent mice.

High rates of relapsed and refractory diffuse large B-cell lymphoma (DLBCL) patients and life-threatening side effects associated with immunochemotherapy make an urgent need to develop new therapies for DLBCL patients. Immunotoxins seem very potent anticancer therapies but their use is limited because of their high toxicity. Accordingly, the self-assembling polypeptidic nanoparticle, T22-DITOX-H6, incorporating the diphtheria toxin and targeted to CXCR4 receptor, which is overexpressed in DLBCL cells, could offer a new strategy to selectively eliminate CXCR4+ DLBCL cells without adverse effects. In these terms, our work demonstrated that T22-DITOX-H6 showed high specific cytotoxicity towards CXCR4+ DLBCL cells at the low nanomolar range, which was dependent on caspase-3 cleavage, PARP activation and an increase of cells in early/late apoptosis. Repeated nanoparticle administration induced antineoplastic effect, in vivo and ex vivo, in a disseminated immunocompromised mouse model generated by intravenous injection of human luminescent CXCR4+ DLBCL cells. Moreover, T22-DITOX-H6 inhibited tumor growth in a subcutaneous immunocompetent mouse model bearing mouse CXCR4+ lymphoma cells in the absence of alterations in the hemogram, liver or kidney injury markers or on-target or off-target organ histology. Thus, T22-DITOX-H6 demonstrates a selective cytotoxicity towards CXCR4+ DLBCL cells without the induction of toxicity in non-lymphoma infiltrated organs nor hematologic toxicity.

CXCR4-targeted nanotoxins induce GSDME-dependent pyroptosis in head and neck squamous cell carcinoma.

BACKGROUND: Therapy resistance, which leads to the development of loco-regional relapses and distant metastases after treatment, constitutes one of the major problems that head and neck squamous cell carcinoma (HNSCC) patients currently face. Thus, novel therapeutic strategies are urgently needed. Targeted drug delivery to the chemokine receptor 4 (CXCR4) represents a promising approach for HNSCC management. In this context, we have developed the self-assembling protein nanotoxins T22-PE24-H6 and T22-DITOX-H6, which incorporate the de-immunized catalytic domain of Pseudomonas aeruginosa (PE24) exotoxin A and the diphtheria exotoxin (DITOX) domain, respectively. Both nanotoxins contain the T22 peptide ligand to specifically target CXCR4-overexpressing HNSCC cells. In this study, we evaluate the potential use of T22-PE24-H6 and T22-DITOX-H6 nanotoxins for the treatment of HNSCC. METHODS: T22-PE24-H6 and T22-DITOX-H6 CXCR4-dependent cytotoxic effect was evaluated in vitro in two different HNSCC cell lines. Both nanotoxins cell death mechanisms were assessed in HNSCC cell lines by phase-contrast microscopy, AnnexinV/ propidium iodide (PI) staining, lactate dehydrogenase (LDH) release assays, and western blotting. Nanotoxins antitumor effect in vivo was studied in a CXCR4+ HNSCC subcutaneous mouse model. Immunohistochemistry, histopathology, and toxicity analyses were used to evaluate both nanotoxins antitumor effect and possible treatment toxicity. GSMDE and CXCR4 expression in HNSCC patient tumor samples was also assessed by immunohistochemical staining. RESULTS: First, we found that both nanotoxins exhibit a potent CXCR4-dependent cytotoxic effect in vitro. Importantly, nanotoxin treatment triggered caspase-3/Gasdermin E (GSDME)-mediated pyroptosis. The activation of this alternative cell death pathway that differs from traditional apoptosis, becomes a promising strategy to bypass therapy resistance. In addition, T22-PE24-H6 and T22-DITOX-H6 displayed a potent antitumor effect in the absence of systemic toxicity in a CXCR4+ subcutaneous HNSCC mouse model. Lastly, GSDME was found to be overexpressed in tumor tissue from HNSCC patients, highlighting the relevance of this strategy. CONCLUSIONS: Altogether, our results show that T22-PE24-H6 and T22-DITOX-H6 represent a promising therapy for HNSCC patients. Remarkably, this is the first study showing that both nanotoxins are capable of activating caspase-3/GSDME-dependent pyroptosis, opening a novel avenue for HNSCC treatment.

A multivalent Ara-C-prodrug nanoconjugate achieves selective ablation of leukemic cells in an acute myeloid leukemia mouse model.

Current therapy in acute myeloid leukemia (AML) is based on chemotherapeutic drugs administered at high doses, lacking targeting selectivity and displaying poor therapeutic index because of severe adverse effects. Here, we develop a novel nanoconjugate that combines a self-assembled, multivalent protein nanoparticle, targeting the CXCR4 receptor, with an Oligo-Ara-C prodrug, a pentameric form of Ara-C, to highly increase the delivered payload to target cells. This 13.4 nm T22-GFP-H6-Ara-C nanoconjugate selectively eliminates CXCR4+ AML cells, which are protected by its anchoring to the bone marrow (BM) niche, being involved in AML progression and chemotherapy resistance. This nanoconjugate shows CXCR4-dependent internalization and antineoplastic activity in CXCR4+ AML cells in vitro. Moreover, repeated T22-GFP-H6-Ara-C administration selectively eliminates CXCR4+ leukemic cells in BM, spleen and liver. The leukemic dissemination blockage induced by T22-GFP-H6-Ara-C is significantly more potent than buffer or Oligo-Ara-C-treated mice, showing no associated on-target or off-target toxicity and, therefore, reaching a highly therapeutic window. In conclusion, T22-GFP-H6-Ara-C exploits its 11 ligands-multivalency to enhance target selectivity, while the Oligo-Ara-C prodrug multimeric form increases 5-fold its payload. This feature combination offers an alternative nanomedicine with higher activity and greater tolerability than current intensive or non-intensive chemotherapy for AML patients.