Role of Intracellular and Extracellular Annexin A1 in MIA PaCa-2 Spheroids Formation and Drug Sensitivity

Simple Summary In order to improve the investigation of pancreatic cancer (PC), often supported through analyzes two-dimensional (2D) cell monolayers, we proposed to create a spheroid-based in vitro three-dimensional (3D) model using wild-type (WT) and ANXA1 knock-out (KO) MIA PaCa-2 PC cells. However, the production of spheroids still represents a technical challenge. Here, we have developed a protocol to obtain well-organized spheroids and have proved that Annexin A1 (ANXA1) affects the spheroid formation, because the WT cells have a greater ability to form this 3D model when compared to the ANXA1 KO examples. We also investigated how ANXA1 action could influence the PC pharmacological response both in basal conditions and by mimicking a tumor system through the addition of autocrine EVs. ANXA1, via EVs, significantly improves the formation, the stability and the drug resistance of this model, particularly compared to the ANXA1 KO one, which shows a structural instability and a greater drug sensitivity. Among solid tumors, pancreatic cancer (PC) remains a leading cause of death. In PC, the protein ANXA1 has been identified as an oncogenic factor acting in an autocrine/paracrine way, and also as a component of tumor-deriving extracellular vesicles. Here, we proposed the experimental protocol to obtain spheroids from the two cell lines, wild-type (WT) and Annexin A1 (ANXA1) knock-out (KO) MIA PaCa-2, this last previously obtained through CRISPR/Cas9 genome editing system. The use of three-dimensional (3D) models, like spheroids, can be useful to mimic tumor characteristics and for preclinical chemo-sensitivity studies. By using PC spheroids, we have assessed the activity of intracellular and extracellular ANXA1. Indeed, we have proved that the intracellular protein influences in vitro tumor development and growth by spheroids analysis, in addition to defining the modification about cell protein pattern in ANXA1 KO model compared to the WT one. Moreover, we have tested the response to FOLFIRINOX chemotherapy regimen whose cytostatic effect appeared notably increased in ANXA1 KO spheroids. Additionally, this study has highlighted that the extracellular ANXA1 action is strengthened through the EVs supporting spheroids growth and resistance to drug treatment, mainly affecting tumor progression. Thus, our data interestingly suggest the relevance of ANXA1 as a potential therapeutic PC marker

The combination of mesoglycan and VEGF promotes skin wound repair by enhancing the activation of endothelial cells and fibroblasts and their cross-talk

Skin wound healing requires accurate therapeutic topical managements to accelerate tissue regeneration. Here, for the first time, we found that the association mesoglycan/VEGF has a strong pro-healing activity. In detail, this combination induces angiogenesis in human endothelial cells promoting in turn fibroblasts recruitment. These ones acquire a notable ability to invade the matrigel coating and to secrete an active form of metalloproteinase 2 in presence of endothelial cells treated with mesoglycan/VEGF. Next, by creating intrascapular lesions on the back of C57Bl6 mice, we observed that the topical treatments with the mesoglycan/VEGF promotes the closure of wounds more than the single substances beside the control represented by a saline solution. As revealed by eosin/hematoxylin staining of mice skin biopsies, treatment with the combination mesoglycan/VEGF allows the formation of a well-structured matrix with a significant number of new vessels. Immunofluorescence analyses have revealed the presence of endothelial cells at the closed region of wounds, as evaluated by CD31, VE-cadherin and fibronectin staining and of activated fibroblasts assessed by vimentin, col1A and FAP1α. These results encourage defining the association mesoglycan/VEGF to activate endothelial and fibroblast cell components in skin wound healing promoting the creation of new vessels and the deposition of granulation tissue

The pro-healing effects of heparan sulfate and growth factors are enhanced by the heparinase enzyme: New association for skin wound healing treatment

: Effective treatment strategies for skin wound repair are the focus of numerous studies. New pharmacological approaches appear necessary to guarantee a correct and healthy tissue regeneration. For these reasons, we purposed to investigate the effects of the combination between heparan sulfate and growth factors further adding the heparinase enzyme. Interestingly, for the first time, we have found that this whole association retains a marked pro-healing activity when topically administered to the wound. In detail, this combination significantly enhances the motility and activation of the main cell populations involved in tissue regeneration (keratinocytes, fibroblasts and endothelial cells), compared with single agents administered without heparinase. Notably, using an experimental C57BL/6 mouse model of skin wounding, we observed that the topical treatment of skin lesions with heparan sulfate + growth factors + heparinase promotes the highest closure of wounds compared to each substance mixed with the other ones in all the possible combinations. Eosin/hematoxylin staining of skin biopsies revealed that treatment with the whole combination allows the formation of a well-structured matrix with numerous new vessels. Confocal analyses for vimentin, FAP1α, CK10 and CD31 have highlighted the presence of activated fibroblasts, differentiated keratinocytes and endothelial cells at the closed region of wounds. Our results encourage defining this combined treatment as a new and appealing therapy expedient in skin wound healing, as it is able to activate cell components and promote a dynamic lesions closure

The Pyrazolyl-Urea Gege3 Inhibits the Activity of ANXA1 in the Angiogenesis Induced by the Pancreatic Cancer Derived EVs

The pyrazolyl-urea Gege3 molecule has shown interesting antiangiogenic effects in the tumor contest. Here, we have studied the role of this compound as interfering with endothelial cells activation in response to the paracrine effects of annexin A1 (ANXA1), known to be involved in promoting tumor progression. ANXA1 has been analyzed in the extracellular environment once secreted through microvesicles (EVs) by pancreatic cancer (PC) cells. Particularly, Gege3 has been able to notably prevent the effects of Ac2-26, the ANXA1 mimetic peptide, and of PC-derived EVs on endothelial cells motility, angiogenesis, and calcium release. Furthermore, this compound also inhibited the translocation of ANXA1 to the plasma membrane, otherwise induced by the same ANXA1-dependent extracellular stimuli. Moreover, these effects have been mediated by the indirect inhibition of protein kinase C\u3b1 (PKC\u3b1), which generally promotes the phosphorylation of ANXA1 on serine 27. Indeed, by the subtraction of intracellular calcium levels, the pathway triggered by PKC\u3b1 underwent a strong inhibition leading to the following impediment to the ANXA1 localization at the plasma membrane, as revealed by confocal and cytofluorimetry analysis. Thus, Gege3 appeared an attractive molecule able to prevent the paracrine effects of PC cells deriving ANXA1 in the tumor microenvironment

The Procoagulant Activity of Emoxilane®: A New Appealing Therapeutic Use in Epistaxis of the Combination of Sodium Hyaluronate, Silver Salt, α-tocopherol and D-panthenol

Epistaxis is one of the most frequent hemorrhages resulting from local or systemic factors. Its management without hospitalization has prompted an interest in locally applied hemostatic agents. Generally, the therapy approaches involve sprays or creams acting as a physical barrier, even used as tampons or gauze. In this study, we have investigated the activity of Emoxilane®, a combination of sodium hyaluronate, silver salt, α-tocopherol acetate and D-panthenol, which is known to be able to separately act in a different biological manner. Our in vitro results, obtained on endothelial and nasal epithelial cells, have shown that the association of these molecules presented a notable antioxidant activity mainly due to the α-tocopherol and D-panthenol and a significant antimicrobial role thanks to the silver compound. Moreover, remarkable hemostatic activity was found by evaluating plasmin inhibition attributable to the sodium hyaluronate. Interestingly, on human plasma, we have confirmed that Emoxilane® strongly induced the increase of thrombin levels. These data suggest that the use of this association could represent an appealing pharmacological approach to actively induce hemostasis during epistaxis. Our future perspective will aim to the creation of a formulation for an easy topical application in the nose which is able to contrast the bleeding

Manipulation of the immune system for cancer defeat: a focus on the T cell inhibitory checkpoint molecules

The immune system actively counteracts the tumorigenesis process; a breakout of the immune system function, or its ability to recognize transformed cells, can favor cancer development. Cancer becomes able to escape from immune system control by using multiple mechanisms, which are only in part known at a cellular and molecular level. Among these mechanisms, in the last decade, the role played by the so-called "inhibitory immune checkpoints" is emerging as pivotal in preventing the tumor attack by the immune system. Physiologically, the inhibitory immune checkpoints work to maintain the self-tolerance and attenuate the tissue injury caused by pathogenic infections. Cancer cell exploits such immune-inhibitory molecules to contrast the immune intervention and induce tumor tolerance. Molecular agents that target these checkpoints represent the new frontier for cancer treatment. Despite the heterogeneity and multiplicity of molecular alterations among the tumors, the immune checkpoint targeted therapy has been shown to be helpful in selected and even histologically different types of cancer, and are currently being adopted against an increasing variety of tumors. The most frequently used is the moAb-based immunotherapy that targets the Programmed Cell Death 1 protein (PD-1) or the PD-1 Ligand (PD-L1) or the cytotoxic T lymphocyte antigen-4 (CTLA4). However, new therapeutic approaches are currently in development, along with the discovery of new immune checkpoints exploited by the cancer cell. This article aims to review the inhibitory checkpoints, which are known up to now, along with the mechanisms of cancer immunoediting. An outline of the immune checkpoint targeting approaches, also including combined immunotherapies and the existing trials, is also provided. Notwithstanding the great efforts devoted by researchers in the field of biomarkers of response, to date, no validated FDA-approved immunological biomarkers exist for cancer patients. We highlight relevant studies on predictive biomarkers and attempt to discuss the challenges in this field, due to the complex and largely unknown dynamic mechanisms that drive the tumor immune tolerance

Identification of a highly suppressive Treg subset associated to immunotherapy response

Background Cancer immunotherapy has shown surprising efficacy in several types of advanced and incurable tumors, particularly, malignant melanoma. There are several immunotherapeutic strategies aimed at enhancing immunological defenses against tumor. Among these, monoclonal antibodies against the so-called “immune checkpoint inhibitors”, that counteract tumor-induced immune-disarming pathways, have shown the best outcomes. Regulatory T lymphocytes or Tregs are a subset of lymphocytes involved in immune-surveillance and maintenance of self-tolerance. Tumor often exploits Tregs to allow tolerance to its own antigens and avoid immune system attack. Tregs are usually increased in melanoma patients. It is noticeable that Tregs is a heterogeneous population with respect to their immunosuppressive capability. Lymphocytes are particularly rich in FKBP51 (FKBP5 gene), an immunophilin better known as the intracellular receptor for FK506 and rapamycin. Melanoma aberrantly expresses this immunophilin, which supports cancer resistance and invasion. Recently, our group has shown that melanoma interaction with immune cells, through PD-L1/PD1, bidirectionally generated the splicing of FKBP5 gene inducing a lower molecular weight form of FKBP51, termed FKBP51s, in both melanoma and lymphocyte. A study performed on PBMC of 64 patients with advanced melanoma (stage III/IV) showed that FKBP51s marks a Treg subset which was correlated, as an independent variable, to anti-CTLA4 (ipilimumab) response. More precisely, a low frequency of Treg FKBP51spos (1.2 and 0.04 and < 0.8%. After a transient increase registered following the first administration, the count decreased to 0.3+0.2% in responder patients. Interestingly, a patient with count = 0.72% developed autoimmune side effects that led to therapy discontinuation. Resolution of side effects was accompanied by an increase in Treg FKBP51s+ value to 9.9%; thenafter, anti-PD1 re-administration produced a successful and objective response. In vitro iTreg generation suggested that FKBP51s was induced in Treg CD25high, Ki67high and p70S6khigh , corresponding to a highly metabolically active profile associated with strong suppressive capability. Conclusion Our data reinforce the hypothesis that melanoma patients that benefit from immune checkpoint targeted therapy are recognizable by an expansion of a Treg subset which plays a central role in de-activation of stimulatory co-signalling pathways, in support of tumor immune evasion. Such a Treg subset is marked by FKBP51s, a splicing protein isoform generated by triggering of surface antigens (PD-L1, PD1) that are abundantly expressed on highly suppressive Tregs