Human amniotic membrane inhibits migration and invasion of muscle-invasive bladder cancer urothelial cells by downregulating the FAK/PI3K/Akt/mTOR signalling pathway.

Bladder cancer is the 10th most commonly diagnosed cancer with the highest lifetime treatment costs. The human amniotic membrane (hAM) is the innermost foetal membrane that possesses a wide range of biological properties, including anti-inflammatory, antimicrobial and anticancer properties. Despite the growing number of studies, the mechanisms associated with the anticancer effects of human amniotic membrane (hAM) are poorly understood. Here, we reported that hAM preparations (homogenate and extract) inhibited the expression of the epithelial-mesenchymal transition markers N-cadherin and MMP-2 in bladder cancer urothelial cells in a dose-dependent manner, while increasing the secretion of TIMP-2. Moreover, hAM homogenate exerted its antimigratory effect by downregulating the expression of FAK and proteins involved in actin cytoskeleton reorganisation, such as cortactin and small RhoGTPases. In muscle-invasive cancer urothelial cells, hAM homogenate downregulated the PI3K/Akt/mTOR signalling pathway, the key cascade involved in promoting bladder cancer. By using normal, non-invasive papilloma and muscle-invasive cancer urothelial models, new perspectives on the anticancer effects of hAM have emerged. The results identify new sites for therapeutic intervention and are prompt encouragement for ongoing anticancer drug development studies.

Recommendations from the COST action CA17116 (SPRINT) for the standardization of perinatal derivative preparation and in vitro testing.

Many preclinical studies have shown that birth-associated tissues, cells and their secreted factors, otherwise known as perinatal derivatives (PnD), possess various biological properties that make them suitable therapeutic candidates for the treatment of numerous pathological conditions. Nevertheless, in the field of PnD research, there is a lack of critical evaluation of the PnD standardization process: from preparation to in vitro testing, an issue that may ultimately delay clinical translation. In this paper, we present the PnD e-questionnaire developed to assess the current state of the art of methods used in the published literature for the procurement, isolation, culturing preservation and characterization of PnD in vitro. Furthermore, we also propose a consensus for the scientific community on the minimal criteria that should be reported to facilitate standardization, reproducibility and transparency of data in PnD research. Lastly, based on the data from the PnD e-questionnaire, we recommend to provide adequate information on the characterization of the PnD. The PnD e-questionnaire is now freely available to the scientific community in order to guide researchers on the minimal criteria that should be clearly reported in their manuscripts. This review is a collaborative effort from the COST SPRINT action (CA17116), which aims to guide future research to facilitate the translation of basic research findings on PnD into clinical practice.

Methods and criteria for validating the multimodal functions of perinatal derivatives when used in oncological and antimicrobial applications.

Perinatal derivatives or PnDs refer to tissues, cells and secretomes from perinatal, or birth-associated tissues. In the past 2 decades PnDs have been highly investigated for their multimodal mechanisms of action that have been exploited in various disease settings, including in different cancers and infections. Indeed, there is growing evidence that PnDs possess anticancer and antimicrobial activities, but an urgent issue that needs to be addressed is the reproducible evaluation of efficacy, both in vitro and in vivo. Herein we present the most commonly used functional assays for the assessment of antitumor and antimicrobial properties of PnDs, and we discuss their advantages and disadvantages in assessing the functionality. This review is part of a quadrinomial series on functional assays for the validation of PnDs spanning biological functions such as immunomodulation, anticancer and antimicrobial, wound healing, and regeneration.

Mesenchymal Stem/Stromal Cells May Decrease Success of Cancer Treatment by Inducing Resistance to Chemotherapy in Cancer Cells.

The tumour microenvironment, which is comprised of various cell types and the extracellular matrix, substantially impacts tumour initiation, progression, and metastasis. Mesenchymal stem/stromal cells (MSCs) are one of the key stromal cells in the tumour microenvironment, and their interaction with cancer cells results in the transformation of naïve MSCs to tumour-associated MSCs. The latter has an important impact on tumour growth and progression. Recently, it has been shown that they can also contribute to the development of chemoresistance in cancer cells. This review provides an overview of 42 studies published between 1 January 2001 and 1 January 2022 that examined the effect of MSCs on the susceptibility of cancer cells to chemotherapeutics. The studies showed that MSCs affect various signalling pathways in cancer cells, leading to protection against chemotherapy-induced damage. Promising results emerged from the use of inhibitors of various signalling pathways that are affected in cancer cells due to interactions with MSCs in the tumour microenvironment. These studies present a good starting point for the investigation of novel treatment approaches and demonstrate the importance of targeting the stroma in the tumour microenvironment to improve treatment outcomes.

Supramolecular organizing centers at the interface of inflammation and neurodegeneration.

The pathogenesis of neurodegenerative diseases involves the accumulation of misfolded protein aggregates. These deposits are both directly toxic to neurons, invoking loss of cell connectivity and cell death, and recognized by innate sensors that upon activation release neurotoxic cytokines, chemokines, and various reactive species. This neuroinflammation is propagated through signaling cascades where activated sensors/receptors, adaptors, and effectors associate into multiprotein complexes known as supramolecular organizing centers (SMOCs). This review provides a comprehensive overview of the SMOCs, involved in neuroinflammation and neurotoxicity, such as myddosomes, inflammasomes, and necrosomes, their assembly, and evidence for their involvement in common neurodegenerative diseases. We discuss the multifaceted role of neuroinflammation in the progression of neurodegeneration. Recent progress in the understanding of particular SMOC participation in common neurodegenerative diseases such as Alzheimer's disease offers novel therapeutic strategies for currently absent disease-modifying treatments.

The Role of Innate Immune System in the Human Amniotic Membrane and Human Amniotic Fluid in Protection Against Intra-Amniotic Infections and Inflammation.

Intra-amniotic infection and inflammation (IAI) affect fetal development and are highly associated with preterm labor and premature rupture of membranes, which often lead to adverse neonatal outcomes. Human amniotic membrane (hAM), the inner part of the amnio-chorionic membrane, protects the embryo/fetus from environmental dangers, including microbial infection. However, weakened amnio-chorionic membrane may be breached or pathogens may enter through a different route, leading to IAI. The hAM and human amniotic fluid (hAF) respond by activation of all components of the innate immune system. This includes changes in 1) hAM structure, 2) presence of immune cells, 3) pattern recognition receptors, 4) cytokines, 5) antimicrobial peptides, 6) lipid derivatives, and 7) complement system. Herein we provide a comprehensive and integrative review of the current understanding of the innate immune response in the hAM and hAF, which will aid in design of novel studies that may lead to breakthroughs in how we perceive the IAI.

Detrimental Effect of Various Preparations of the Human Amniotic Membrane Homogenate on the 2D and 3D Bladder Cancer In vitro Models.

Despite being among the ten most common cancers with high recurrence rates worldwide, there have been no major breakthroughs in the standard treatment options for bladder cancer in recent years. The use of a human amniotic membrane (hAM) to treat cancer is one of the promising ideas that have emerged in recent years. This study aimed to investigate the anticancer activity of hAM homogenate on 2D and 3D cancer models. We evaluated the effects of hAM homogenates on the human muscle invasive bladder cancer urothelial (T24) cells, papillary cancer urothelial (RT4) cells and normal porcine urothelial (NPU) cells as well as on human mammary gland non-tumorigenic (MCF10a) cells and low-metastatic breast cancer (MCF7) cells. After 24 h, we observed a gradual detachment of cancerous cells from the culture surface, while the hAM homogenate did not affect the normal cells. The most pronounced effect hAM homogenate had on bladder cancer cells; however, the potency of their detachment was dependent on the treatment protocol and the preparation of hAM homogenate. We demonstrated that hAM homogenate significantly decreased the adhesion, growth, and proliferation of human bladder invasive and papillary cancer urothelial cells and did not affect normal urothelial cells even in 7-day treatment. By using light and electron microscopy we showed that hAM homogenate disrupted the architecture of 2D and 3D bladder cancer models. The information provided by our study highlights the detrimental effect of hAM homogenate on bladder cancer cells and strengthens the idea of the potential clinical application of hAM for bladder cancer treatment.

Antimicrobial Activity of Human Fetal Membranes: From Biological Function to Clinical Use.

The fetal membranes provide a supportive environment for the growing embryo and later fetus. Due to their versatile properties, the use of fetal membranes in tissue engineering and regenerative medicine is increasing in recent years. Moreover, as microbial infections present a crucial complication in various treatments, their antimicrobial properties are gaining more attention. The antimicrobial peptides (AMPs) are secreted by cells from various perinatal derivatives, including human amnio-chorionic membrane (hACM), human amniotic membrane (hAM), and human chorionic membrane (hCM). By exhibiting antibacterial, antifungal, antiviral, and antiprotozoal activities and immunomodulatory activities, they contribute to ensuring a healthy pregnancy and preventing complications. Several research groups investigated the antimicrobial properties of hACM, hAM, and hCM and their derivatives. These studies advanced basic knowledge of antimicrobial properties of perinatal derivatives and also provided an important insight into the potential of utilizing their antimicrobial properties in a clinical setting. After surveying the studies presenting assays on antimicrobial activity of hACM, hAM, and hCM, we identified several considerations to be taken into account when planning future studies and eventual translation of fetal membranes and their derivatives as antimicrobial agents from bench to bedside. Namely, (1) the standardization of hACM, hAM, and hCM preparation to guarantee rigorous antimicrobial activity, (2) standardization of the antimicrobial susceptibility testing methods to enable comparison of results between various studies, (3) investigation of the antimicrobial properties of fetal membranes and their derivatives in the in vivo setting, and (4) designation of donor criteria that enable the optimal donor selection. By taking these considerations into account, future studies will provide crucial information that will enable reaching the optimal treatment outcomes using the fetal membranes and their derivatives as antimicrobial agents.

The Antibacterial Activity of Human Amniotic Membrane against Multidrug-Resistant Bacteria Associated with Urinary Tract Infections: New Insights from Normal and Cancerous Urothelial Models.

Urinary tract infections (UTIs) represent a serious global health issue, especially due to emerging multidrug-resistant UTI-causing bacteria. Recently, we showed that the human amniotic membrane (hAM) could be a candidate for treatments and prevention of UPEC and Staphylococcus aureus infections. However, its role against multidrug-resistant bacteria, namely methicillin-resistant S. aureus (MRSA), extended-spectrum beta-lactamases (ESBL) producing Escherichia coli and Klebsiella pneumoniae, vancomycin-resistant Enterococci (VRE), carbapenem-resistant Acinetobacter baumannii, and Pseudomonas aeruginosa has not yet been thoroughly explored. Here, we demonstrate for the first time that the hAM homogenate had antibacterial activity against 7 out of 11 tested multidrug-resistant strains, the greatest effect was on MRSA. Using novel approaches, its activity against MRSA was further evaluated in a complex microenvironment of normal and cancerous urinary bladder urothelia. Even short-term incubation in hAM homogenate significantly decreased the number of bacteria in MRSA-infected urothelial models, while it did not affect the viability, number, and ultrastructure of urothelial cells. The hAM patches had no antibacterial activity against any of the tested strains, which further exposes the importance of the hAM preparation. Our study substantially contributes to basic knowledge on the antibacterial activity of hAM and reveals its potential to be used as an antibacterial agent against multidrug-resistant bacteria.

The Cells and Extracellular Matrix of Human Amniotic Membrane Hinder the Growth and Invasive Potential of Bladder Urothelial Cancer Cells.

Bladder cancer is one of the most common cancers among men in industrialized countries and on the global level incidence and mortality rates are increasing. In spite of progress in surgical treatment and chemotherapy, the prognosis remains poor for patients with muscle-invasive bladder cancer. Therefore, there is a great need for the development of novel therapeutic approaches. The human amniotic membrane (hAM) is a multi-layered membrane that comprises the innermost part of the placenta. It has unique properties that make it suitable for clinical use, such as the ability to promote wound healing and decrease scarring, low immunogenicity, and immunomodulatory, antimicrobial and anticancer properties. This study aimed to investigate the effect of (i) hAM-derived cells and (ii) hAM scaffolds on the growth dynamics, proliferation rate, and invasive potential of muscle-invasive bladder cancer T24 cells. Our results show that 24 and 48 h of co-culturing T24 cells with hAM-derived cells (at 1:1 and 1:4 ratios) diminished the proliferation rate of T24 cells. Furthermore, when seeded on hAM scaffolds, namely (1) epithelium of hAM (e-hAM), (2) basal lamina of hAM (denuded; d-hAM), and (3) stroma of hAM (s-hAM), the growth dynamic of T24 cells was altered and proliferation was reduced, even more so by the e-hAM scaffolds. Importantly, despite their muscle-invasive potential, the T24 cells did not disrupt the basal lamina of hAM scaffolds. Furthermore, we observed a decrease in the expression of epithelial-mesenchymal transition (EMT) markers N-cadherin, Snail and Slug in T24 cells grown on hAM scaffolds and individual T24 cells even expressed epithelial markers E-cadherin and occludin. Our study brings new knowledge on basic mechanisms of hAM affecting bladder carcinogenesis and the results serve as a good foundation for further research into the potential of hAM-derived cells and the hAM extracellular matrix to serve as a novel bladder cancer treatment.

Amniotic Membrane Preparation Crucially Affects Its Broad-Spectrum Activity Against Uropathogenic Bacteria.

Urinary tract infections are among the most common bacterial infections in humans. Moreover, they are highly recurrent and increasingly often resistant to antibiotics. The antimicrobial properties of the amniotic membrane (AM), the innermost layer of fetal membranes, have been briefly reported in the literature, however, the results of published studies are often inconsistent and unclear; moreover, its effect on uropathogenic bacteria has not yet been investigated. Further, there is no data in the literature about the effect of AM preparation and storage on its antimicrobial properties. To examine the impact of several preparation procedures on the antimicrobial properties of AM, we prepared patches and homogenates of fresh (fAM) and cryopreserved (cAM) human AM and tested them on 14 selected Gram-positive and Gram-negative uropathogenic bacteria. By employing novel antimicrobial efficiency assays we showed that fAM and cAM homogenates have broad-spectrum antimicrobial activity against all here tested uropathogenic bacteria, except for Serratia marcescens. Moreover, they had a potent effect also on the multiple-resistant clinical strains of uropathogenic Escherichia coli. Interestingly, the patches of fAM and cAM had no antimicrobial effect on any of the tested strains. We therefore prepared and stored AM patches according to the standard procedure for clinical use in ophthalmology, which includes the cryopreservation of antibiotic-treated AM, and performed antimicrobial efficiency assays. Our findings suggest that the ultrastructure of AM patches could enable the retention of added antibiotics. In addition, we also prepared gentamicin-resistant uropathogenic E. coli strains, which confirmed that the antimicrobial effect of antibiotic-treated AM patches can be attributed to the antibiotic alone. To summarize, here we describe novel protocols for preparation and storage of AM to ensure the preservation of its antimicrobial factors. Moreover, we describe the mechanism of AM retention of antibiotics, based on which the AM could potentially be used as a drug delivery vehicle in future clinically applicable approaches.

Different Effects Of Amniotic Membrane Homogenate On The Growth Of Uropathogenic Escherichia coli, Staphylococcus aureus And Serratia marcescens.

PURPOSE: Due to the emergence and spread of bacterial strains resistant to antibiotics, the development of new antimicrobials is imperative. The antimicrobial effect of the amniotic membrane (AM) has been explored to a limited extent so far. MATERIALS AND METHODS: We collected 12 biological samples of AM homogenates and tested their antimicrobial effect on 4 pathogens, including the clinical strain of uropathogenic Escherichia coli (UPEC), the wild-type strain of Staphylococcus aureus, and the wild-type strain and a clinical strain of Serratia marcescens. To quantify the antibacterial effect of AM, we monitored the effect of AM homogenate on bacterial growth using plate count method and agar diffusion method. Additionally, minimal inhibitory concentrations (MICs) for AM homogenate dilutions were determined and S. marcescens growth in AM homogenate alone was evaluated. RESULTS: Our results demonstrated that AM homogenate had a bacteriostatic effect on studied UPEC and S. aureus. Interestingly, when used in lower concentrations, the AM homogenate had a bactericidal effect on both strains. In contrast, S. marcescens was completely resistant to the growth-inhibitory substances of AM homogenate. Its growth was slightly accelerated in liquid culture medium in the presence of AM homogenate and the strain was able to grow in undiluted, 2-fold and 4-fold diluted AM homogenate. CONCLUSION: Obtained results illustrated that AM homogenate could be a candidate for treatments and prevention of UPEC and S. aureus infections, but not that of S. marcescens, whose growth is enhanced by AM homogenate. Moreover, the established liquid culture medium assay can be used as a time- and cost-effective method for a personalized evaluation of drug effect on the growth of chosen bacterial strains with parallel testing of resistance or susceptibility to multiple drugs. The susceptibility of bacteria to AM homogenate in solid and liquid culture media is encouraging for its use in biomedical applications.

Human Amniotic Membrane and Amniotic Membrane-Derived Cells: How Far Are We from Their Use in Regenerative and Reconstructive Urology?

Human amniotic membrane (hAM) is the innermost layer of fetal membranes, which surrounds the developing fetus and forms the amniotic cavity. hAM and hAM-derived cells possess many properties that make them suitable for use in regenerative medicine, such as low immunogenicity, promotion of epithelization, anti-inflammatory properties, angiogenic and antiangiogenic properties, antifibrotic properties, antimicrobial properties, and anticancer properties. Many pathological conditions of the urinary tract lead to organ damage or complete loss of function. Consequently, the reconstruction or replacement of damaged organs is needed, which makes searching for new approaches in regenerative and reconstructive urology a necessity. The use of hAM for treating defects in kidneys, ureters, urinary bladder, and urethra was tested in vitro in cell cultures and in vivo in mice, rats, rabbits, cats, dogs, and also in humans. These studies confirmed the advantages and the potential of hAM for use in regenerative and reconstructive urology as stated above. However, they also pointed out a few concerns we have to take into consideration. These are (1) the lack of a standardized protocol in hAM preparation and storage, (2) the heterogeneity of hAM, and especially (3) low mechanical strength of hAM. Before any wider use of hAM for treating urological defects, the protocols for preparation and storage will need to be standardized, followed by more studies on larger animals and clinical trials, which will altogether extensively assess the potential of hAM use in urological patients.