TGF-ß signalling limits effector function capacity of NK cell anti-tumour immunity in human bladder cancer.

BACKGROUND: Natural killer (NK) cells are important innate immunity players and have unique abilities to recognize and eliminate cancer cells, particularly in settings of antibody-opsonization and antibody-dependant cellular cytotoxicity (ADCC). However, NK cell-based responses in bladder cancers to therapeutic antibodies are typically immunosuppressed, and these immunosuppressive mechanisms are largely unknown. METHODS: Single cell RNA sequencing (scRNA-seq) and high-dimensional flow cytometry were used to investigate the phenotype of tumour-infiltrating NK cells in patients with bladder cancer. Further, in vitro, and in vivo models of this disease were used to validate these findings. FINDINGS: NK cells within bladder tumours displayed reduced expression of FcγRIIIa/CD16, the critical Fc receptor involved in ADCC-mediated cytotoxicity, on both transcriptional and protein levels. Transcriptional signatures of transforming growth factor (TGF)-ß-signalling, a pleiotropic cytokine known for its immunosuppressive and tissue residency-inducing effects, were upregulated in tumour-infiltrating NK cells. TGF-ß mediated CD16 downregulation on NK cells, was further validated in vitro, which was accompanied by a transition into a tissue residency phenotype. This CD16 downregulation was also abrogated by TGF-ßR signalling inhibition, which could also restore the ADCC ability of NK cells subject to TGF-ß effects. In a humanized mouse model of bladder cancer, mice treated with a TGF-ß inhibitor exhibited increased ADCC activity compared to mice treated only with antibodies. INTERPRETATION: This study highlights how TGF-ß-rich bladder cancers inhibit NK cell-mediated ADCC by downregulating CD16. TGF-ß inhibition represents new avenues to reverse immunosuppression and enhance the tumoricidal capacity of NK cells in bladder cancer. FUNDING: The Guimaraes Laboratory is funded by a US Department of Defense-Breast Cancer Research Program-Breakthrough Award Level 1 (#BC200025), a grant (#2019485) awarded through the Medical Research Future Fund (MRFF, with the support of the Queensland Children's Hospital Foundation, Microba Life Sciences, Richie's Rainbow Foundation, Translational Research Institute (TRI) and UQ), and a grant (#RSS_2023_085) funded by a Metro South Health Research Support Scheme. J.K.M.W. is funded by a UQ Research Training Program PhD Scholarship and N.O. is funded by a NHMRC Postgraduate Scholarship (#2021932).

Exploring NK cell receptor dynamics in paediatric leukaemias: implications for immunotherapy and prognosis.

Objectives: Immunotherapies targeting natural killer (NK) cell receptors have shown promise against leukaemia. Unfortunately, cancer immunosuppressive mechanisms that alter NK cell phenotype prevent such approaches from being successful. The study utilises advanced cytometry to examine how cancer immunosuppressive pathways affect NK cell phenotypic changes in clinical samples. Methods: In this study, we conducted a high-dimensional examination of the cell surface expression of 16 NK cell receptors in paediatric patients with acute myeloid leukaemia and acute lymphoblastic leukaemia, as well as in samples of non-age matched adult peripheral blood (APB) and umbilical cord blood (UCB). An unsupervised analysis was carried out in order to identify NK cell populations present in paediatric leukaemias. Results: We observed that leukaemia NK cells clustered together with UCB NK cells and expressed relatively higher levels of the NKG2A receptor compared to APB NK cells. In addition, CD56dimCD16+CD57- NK cells lacking NKG2A expression were mainly absent in paediatric leukaemia patients. However, CD56br NK cell populations expressing high levels of NKG2A were highly represented in paediatric leukaemia patients. NKG2A expression on leukaemia NK cells was found to be positively correlated with the expression of its ligand, suggesting that the NKG2A-HLA-E interaction may play a role in modifying NK cell responses to leukaemia cells. Conclusion: We provide an in-depth analysis of NK cell populations in paediatric leukaemia patients. These results support the development of immunotherapies targeting immunosuppressive receptors, such as NKG2A, to enhance innate immunity against paediatric leukaemia.

Nanobiomaterials to modulate natural killer cell responses for effective cancer immunotherapy.

Natural killer (NK) cells have emerged as a major target for cancer immunotherapies, particularly as cellular therapy modalities because they have relatively less toxicity than T lymphocytes. However, NK cell-based therapy suffers from many challenges, including problems with its activation, resistance to genetic engineering, and large-scale expansion needed for therapeutic purposes. Recently, nanobiomaterials have emerged as a promising solution to control the challenges associated with NK cells. This focused review summarises the recent advances in the field and highlights current and future perspectives of using nanobiomaterials to maximise anticancer responses of NK cells for safe and effective immunotherapy. Finally, we provide our opinion on the role of smart materials in activating NK cells as a potential cellular therapy of the future.

A simple method to evaluate the toxic effects of Prorocentrum lima extracts to fish (sea bass) kidney cells.

The diarrhetic shellfish toxins (DSTs) okadaic acid (OA) and its analogues - the dinophysistoxins (DTXs) - are produced by dinoflagellates such as Prorocentrum lima and can bioaccumulate in filter-feeding organisms as they are transferred through the food web. Although there is no assessment of the harmful effects of these toxins on the fish's immune system, this study developed a primary culture protocol for kidney cells from marine fish Centropomus parallelus and evaluated the immunotoxic effects to P. lima extracts containing DSTs. The cells were obtained by mechanical dissociation, segregated with Percoll gradient, and incubated for 24 h at 28 °C in a Leibovitz culture medium supplemented with 2% fetal bovine serum and antibiotics. The exposed cells were evaluated in flow cytometry using the CD54 PE antibody. We obtained >5.0 × 106 viable cells per 1.0 g of tissue that exhibited no cell differentiation. Exposure to 1.2 or 12 ng DST mL-1 stimulated the immune system activation and increased the proportion of activated macrophages and monocytes in 48 to 52% and in 127 to 146%, respectively. The protocol proved to be an alternative tool to assess the immunotoxic effects of DST exposure on fish's anterior kidney cells.

Profiling of lung SARS-CoV-2 and influenza virus infection dissects virus-specific host responses and gene signatures.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in late 2019 has spread globally, causing a pandemic of respiratory illness designated coronavirus disease 2019 (COVID-19). A better definition of the pulmonary host response to SARS-CoV-2 infection is required to understand viral pathogenesis and to validate putative COVID-19 biomarkers that have been proposed in clinical studies. METHODS: Here, we use targeted transcriptomics of formalin-fixed paraffin-embedded tissue using the NanoString GeoMX platform to generate an in-depth picture of the pulmonary transcriptional landscape of COVID-19, pandemic H1N1 influenza and uninfected control patients. RESULTS: Host transcriptomics showed a significant upregulation of genes associated with inflammation, type I interferon production, coagulation and angiogenesis in the lungs of COVID-19 patients compared to non-infected controls. SARS-CoV-2 was non-uniformly distributed in lungs (emphasising the advantages of spatial transcriptomics) with the areas of high viral load associated with an increased type I interferon response. Once the dominant cell type present in the sample, within patient correlations and patient-patient variation, had been controlled for, only a very limited number of genes were differentially expressed between the lungs of fatal influenza and COVID-19 patients. Strikingly, the interferon-associated gene IFI27, previously identified as a useful blood biomarker to differentiate bacterial and viral lung infections, was significantly upregulated in the lungs of COVID-19 patients compared to patients with influenza. CONCLUSION: Collectively, these data demonstrate that spatial transcriptomics is a powerful tool to identify novel gene signatures within tissues, offering new insights into the pathogenesis of SARS-COV-2 to aid in patient triage and treatment.

Enhancing Natural Killer Cell Targeting of Pediatric Sarcoma.

Osteosarcoma, Ewing sarcoma (EWS), and rhabdomyosarcoma (RMS) are the most common pediatric sarcomas. Conventional therapy for these sarcomas comprises neoadjuvant and adjuvant chemotherapy, surgical resection of the primary tumor and/or radiation therapy. Patients with metastatic, relapsed, or refractory tumors have a dismal prognosis due to resistance to these conventional therapies. Therefore, innovative therapeutic interventions, such as immunotherapy, are urgently needed. Recently, cancer research has focused attention on natural killer (NK) cells due their innate ability to recognize and kill tumor cells. Osteosarcoma, EWS and RMS, are known to be sensitive to NK cell cytotoxicity in vitro. In the clinical setting however, NK cell cytotoxicity against sarcoma cells has been mainly studied in the context of allogeneic stem cell transplantation, where a rapid immune reconstitution of NK cells plays a key role in the control of the disease, known as graft-versus-tumor effect. In this review, we discuss the evidence for the current and future strategies to enhance the NK cell-versus-pediatric sarcoma effect, with a clinical focus. The different approaches encompass enhancing antibody-dependent NK cell cytotoxicity, counteracting the NK cell mechanisms of self-tolerance, and developing adoptive NK cell therapy including chimeric antigen receptor-expressing NK cells.

Electrospun polyvinyl-alcohol/gum arabic nanofibers: Biomimetic platform for in vitro cell growth and cancer nanomedicine delivery.

The design of powerful in vitro cell culture platforms to support precision medicine can contribute to predict therapeutic success of cancer patients. Electrospun nanofibers applied to cell culture can mimic extracellular matrix and improve in vitro cell behavior. Here, we describe biocompatible blended polyvinyl-alcohol (PVA)/gum arabic (GA) extracellular matrix (ECM)-like nanofibers for in vitro cell cultures capable of delivering nanocomposite for desired biomedical application. Therefore, PVA/GA ECM-like electrospun nanofibers were developed and characterized. Heat treatment was used to crosslink the nanofibers and biocompatibility was evaluated, which demonstrated the ability of developed platform to provide a cell culture-friendly environment. Previous work demonstrated that GA-gold nanoparticles (GA-AuNPs) in non-cytotoxic concentrations can reduce key metastatic cellular events such as invasion and colony formation of metastatic melanoma cells. Thus, crosslinked nanofibers were functionalized with GA-AuNPs and its cellular delivery was evaluated. GA-AuNPs were efficiently adsorbed onto the PVA/GA nanofibers surface and the system effectively delivered the nanocomposites to metastatic melanoma cells. In conclusion, the described biocompatible system could be prospected as a valuable in vitro tool for precision medicine.

Production of a novel recombinant brown spider hyaluronidase in baculovirus-infected insect cells.

Hyaluronidases are low expressed toxins of brown spider venoms, but, as highly active molecules, they present an important role as spreading factors. By degrading extracellular matrix components, these enzymes favor the diffusion of toxins in the affected tissue and at systemic level. Here, a novel isoform of hyaluronidase of Loxosceles intermedia Mello-Leitão (1934) venom was cloned, expressed in a baculovirus-insect cell expression system and fully active purified. This recombinant enzyme, named LiHyal2 (Loxosceles intermedia Hyaluronidase isoform 2), shares high identity with hyaluronidases of other spiders and scorpions. The catalytic and sugar binding amino acid residues are conserved in LiHyal2, human, and honeybee venom hyaluronidases and the molecular model of LiHyal2 shares major similarities with their crystal structures, including the active site. LiHyal2 was expressed as a 45 kDa protein and degraded hyaluronic acid (HA) and chondroitin sulphate as demonstrated by HA zymography and agarose gel electrophoresis. Lectin blot analysis revealed that LiHyal2 is post-translationally modified by the addition of high mannose N-linked carbohydrates. In vivo experiments showed that LiHyal2 potentialize dermonecrosis and edema induced by a recombinant phospholipase-D (PLD) of L. intermedia venom, as well as enhance the increase in capillary permeability triggered by this PLD, indicating that these toxins act synergistically during envenomation. Altogether, these results introduce a novel approach to express spider recombinant toxins, contribute to the elucidation of brown spider venom mechanisms and add to the development of a more specific treatment of envenomation victims.

Tumor Microenvironment-Associated Extracellular Matrix Components Regulate NK Cell Function.

The tumor microenvironment (TME) is composed of multiple infiltrating host cells (e.g., endothelial cells, fibroblasts, lymphocytes, and myeloid cells), extracellular matrix, and various secreted or cell membrane-presented molecules. Group 1 innate lymphoid cells (ILCs), which includes natural killer (NK) cells and ILC1, contribute to protecting the host against cancer and infection. Both subsets are able to quickly produce cytokines such as interferon gamma (IFN-γ), chemokines, and other growth factors in response to activating signals. However, the TME provides many molecules that can prevent the potential effector function of these cells, thereby protecting the tumor. For example, TME-derived tumor growth factor (TGF)-ß and associated members of the superfamily downregulate NK cell cytotoxicity, cytokine secretion, metabolism, proliferation, and induce effector NK cells to upregulate ILC1-like characteristics. In concert, a family of carbohydrate-binding proteins called galectins, which can be produced by different cells composing the TME, can downregulate NK cell function. Matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase (ADAM) are also enzymes that can remodel the extracellular matrix and shred receptors from the tumor cell surface, impairing the activation of NK cells and leading to less effective effector functions. Gaining a better understanding of the characteristics of the TME and its associated factors, such as infiltrating cells and extracellular matrix, could lead to tailoring of new personalized immunotherapy approaches. This review provides an overview of our current knowledge on the impact of the TME and extracellular matrix-associated components on differentiation, impairment, and function of NK cells.

Microscopic analysis of colonization of Colletotrichum abscissum in citrus tissues.

Postbloom fruit drop (PFD), caused mainly by Colletotrichum abscissum, is one of the most severe citrus diseases and can causes up to 80% fruit loss in favorable climatic conditions. According to the literature, other Colletotrichum species colonize hosts using distinct strategies: intracellular hemibiotrophic or subcuticular intramural necrotrophic colonization. However, so far, for C. abscissum only the necrotrophic stage has been described and some aspects remain unclear in PFD disease cycle. To better understand the disease cycle, microscopy studies could be applied. However, even using eGFP strains (expressing green fluorescent protein), the results are unclear due to the autofluorescence of citrus leaves. To eliminate this problem and to study the interaction between C. abscissum-citrus we used a destaining and staining methodologies, and we observed that in leaves, even applying injury before inoculation, C. abscissum does not colonize adjacent tissues. Apparently, in the leaves the fungus only uses the nutrients exposed in the artificial lesions for growth, and then produces large amount of spores. However, in flowers, C. abscissum penetrated and colonized the tissues of the petals 12 h after inoculation. In the early stages of infection, we observed the development of primary biotrophic hyphae, suggesting this species as a hemibiotrophic fungus, with a short biotrophic phase during flower colonization followed by dominant necrotrophic colonization. In conclusion, the use of an eGFP strain of C. abscissum and a different methodology of destaining and staining allowed a better understanding of the morphology and mechanisms used by this citrus pathogen to colonize the host.

A low-molecular-weight galactofucan from the seaweed, Spatoglossum schröederi, binds fibronectin and inhibits capillary-like tube formation in vitro.

A low-molecular-weight (LMW) heterofucan (designated fucan B) was obtained from the brown seaweed, Spatoglossum schröederi, and its activity as an inhibitor of capillary-like tube formation by endothelial cells (ECs) was analyzed. Chemical, infrared and electrophoretic analyses confirmed the identity of fucan B. In contrast to other LMW fucans, fucan B (0.012-0.1 mg/mL) inhibited ECs capillary-like tube formation in a concentration-dependent manner. In addition, fucan B (0.01-0.05 mg/mL) did not affect ECs proliferation. Fucan B also inhibited ECs migration on a fibronectin-coated surface, but not on laminin- or collagen-coated surfaces. Biotinylated fucan B was used as a probe to identify its localization. Confocal microscopy experiments revealed that biotinylated fucan did not bind to the cell surface, but rather only to fibronectin. Our findings suggest that fucan B inhibits ECs capillary-like tube formation and migration by binding directly to fibronectin and blocking fibronectin sites recognized by cell surface ligands. However, further studies are needed to evaluate the in vivo effects of fucan B.

Differential effects of Zincum metallicum on cell models.

INTRODUCTION: Zinc is an essential trace element necessary for life. Traditional and complementary medicines use zinc-based formulations to treat different classes of diseases. Basic research on homeopathic preparations of zinc are rare and there are a few published clinical cases describing its effects on patients. The use of cell-based models in drug screening is a reliable source of evidence. METHODS: We sought to investigate experimental end-points using cell-based models to determine the effects of dilutions of Zincum metallicum prepared according to the Brazilian Homeopathic Pharmacopoeia. Murine RAW 264.7 macrophages and melanoma B16-F10 cell lines were cultured according to standard procedures. Cells were treated with either 5c, 6c or 30c Zincum metallicum and control cells with its respective vehicle (5c, 6c, or 30c Lactose). Macrophage activation by CD54 immunolabeling and intracellular reactive oxygen species (ROS) using DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) were detected by flow cytometry. Phagocytic capacity (endocytic index) was quantified by light microscopy. Features of melanoma cells were analyzed by colorimetric assays to determine melanin content and cell proliferation rate. All obtained data were submitted to normality test followed by statistical analysis. RESULTS: Zincum metallicum 6c shifted high ROS-producing macrophages to a low ROS-producing phenotype. Macrophage CD54 expression was increased by Zincum metallicum 5c. No changes in endocytic index were observed. Melanoma cells were not affected by any treatment we tested. CONCLUSIONS: Differing responses and non-linearity were found on macrophages challenged with Zincum metallicum at high dilutions. No changes in melanoma cells were observed. Customised assays using target cells can be useful to investigate high-dilution effects. Other cell types and conditions should be explored.

Increased cellular uptake of lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles due to surface modification with folic acid.

Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid were synthesized by miniemulsion polymerization in just one step. In vitro biocompatibility and cytotoxicity assays on L929 (murine fibroblast), human red blood, and HeLa (uterine colon cancer) cells were performed. The effect of folic acid at the nanoparticles surface was evaluated through cellular uptake assays in HeLa cells. Results showed that the presence of folic acid did not affect substantially the polymer particle size (~120 nm), the superparamagnetic behavior, the encapsulation efficiency of lauryl gallate (~87 %), the Zeta potential (~38 mV) of the polymeric nanoparticles or the release profile of lauryl gallate. The release profile of lauryl gallate from superparamagnetic poly(methyl methacrylate) nanoparticles presented an initial burst effect (0-1 h) followed by a slow and sustained release, indicating a biphasic release system. Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles with folic acid did not present cytotoxicity effects on L929 and human red blood cells. However, free lauryl gallate presented significant cytotoxic effects on L929 and human red blood cells at all tested concentrations. The presence of folic acid increased the cytotoxicity of lauryl gallate loaded in nanoparticles on HeLa cells due to a higher cellular uptake when HeLa cells were incubated at 37 °C. On the other hand, when the nanoparticles were incubated at low temperature (4 °C) cellular uptake was not observed, suggesting that the uptake occurred by folate receptor mediated energy-dependent endocytosis. Based on presented results our work suggests that this carrier system can be an excellent alternative in targeted drug delivery by folate receptor.