An integrated framework for quantifying immune-tumour interactions in a 3D co-culture model

AbstractInvestigational in vitro models that reflect the complexity of the interaction between the immune system and tumours are limited and difficult to establish. Herein, we present a platform to study the tumour-immune interaction using a co-culture between cancer spheroids and activated immune cells. An algorithm was developed for analysis of confocal images of the co-culture to evaluate the following quantitatively; immune cell infiltration, spheroid roundness and spheroid growth. As a proof of concept, the effect of the glucocorticoid stress hormone, cortisol was tested on 66CL4 co-culture model. Results were comparable to 66CL4 syngeneic in vivo mouse model undergoing psychological stress. Furthermore, administration of glucocorticoid receptor antagonists demonstrated the use of this model to determine the effect of treatments on the immune-tumour interplay. In conclusion, we provide a method of quantifying the interaction between the immune system and cancer, which can become a screening tool in immunotherapy design.

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Resolvins as Regulators of the Immune System

The Scientific World JOURNAL ◽

10.1100/tsw.2010.72 ◽

2010 ◽

Vol 10 ◽

pp. 818-831 ◽

Cited By ~ 20

Author(s):

Hiroyuki Seki ◽

Takaharu Sasaki ◽

Tomomi Ueda ◽

Makoto Arita

Keyword(s):

Immune System ◽

Acute Inflammation ◽

Immune Cell ◽

Inflammatory Responses ◽

Bioactive Metabolites ◽

Cell Functions ◽

First Response ◽

Beneficial Impact

Inflammation is the first response of the immune system to infection or injury, but excessive or inappropriate inflammatory responses contribute to a range of acute and chronic human diseases. Clinical assessment of dietary supplementation of ω-3 polyunsaturated fatty acids (i.e., eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) indicate that they have beneficial impact on these diseases, although the mechanisms are poorly understood at the molecular level. In this decade, it has been revealed that EPA and DHA are enzymatically converted to bioactive metabolites in the course of acute inflammation and resolution. These metabolites were shown to regulate immune cell functions and to display potent anti-inflammatory actions bothin vitroandin vivo. Because of their ability to resolve an acute inflammatory response, they are referred to as proresolving mediators, or resolvins. In this review, we provide an overview of the formation and actions of these lipid mediators.

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Immune System Dysfunction and Autoimmune Disease in Mice Lacking Emk (Par-1) Protein Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.21.9.3206-3219.2001 ◽

2001 ◽

Vol 21 (9) ◽

pp. 3206-3219 ◽

Cited By ~ 58

Author(s):

Jonathan B. Hurov ◽

Thaddeus S. Stappenbeck ◽

Christian M. Zmasek ◽

Lynn S. White ◽

Sheila H. Ranganath ◽

...

Keyword(s):

Immune System ◽

T Cell ◽

Protein Kinase ◽

Autoimmune Disease ◽

Immune Cell ◽

Interleukin 4 ◽

Parotid Glands ◽

Immune System Dysfunction

ABSTRACT Emk is a serine/threonine protein kinase implicated in regulating polarity, cell cycle progression, and microtubule dynamics. To delineate the role of Emk in development and adult tissues, mice lacking Emk were generated by targeted gene disruption.Emk −/− mice displayed growth retardation and immune cell dysfunction. Although B- and T-cell development were normal, CD4+T cells lacking Emk exhibited a marked upregulation of the memory marker CD44/pgp-1 and produced more gamma interferon and interleukin-4 on stimulation through the T-cell receptor in vitro. In addition, B-cell responses to T-cell-dependent and -independent antigen challenge were altered in vivo. AsEmk −/− animals aged, they developed splenomegaly, lymphadenopathy, membranoproliferative glomerulonephritis, and lymphocytic infiltrates in the lungs, parotid glands and kidneys. Taken together, these results demonstrate that the Emk protein kinase is essential for maintaining immune system homeostasis and that loss of Emk may contribute to autoimmune disease in mammals.

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Immune Landscape of Thyroid Cancers: New Insights

Frontiers in Endocrinology ◽

10.3389/fendo.2020.637826 ◽

2021 ◽

Vol 11 ◽

Author(s):

Elisa Menicali ◽

Martina Guzzetti ◽

Silvia Morelli ◽

Sonia Moretti ◽

Efisio Puxeddu

Keyword(s):

Thyroid Cancer ◽

Immune System ◽

Immune Cell ◽

The Cancer Genome Atlas ◽

Immune Checkpoints ◽

Thyroid Tumors ◽

Thyroid Cancers ◽

Immune Destruction

Immune system plays a key role in cancer prevention as well as in its initiation and progression. During multistep development of tumors, cells must acquire the capability to evade immune destruction. Both in vitro and in vivo studies showed that thyroid tumor cells can avoid immune response by promoting an immunosuppressive microenvironment. The recruitment of immunosuppressive cells such as TAMs (tumor-associated macrophages), TAMCs (tumor-associated mast cells), MDSC (myeloid-derived suppressor cells), TANs (tumor-associated neutrophils) and Tregs (regulatory T cells) and/or the expression of negative immune checkpoints, like PD-L1 (programmed death-ligand 1), CTLA-4 (cytotoxic T-lymphocyte associated protein 4), and/or immunosuppressive enzymes, as IDO1 (indoleamine 2,3-dioxygenase 1), are just some of the mechanisms that thyroid cancer cells exploit to escape immune destruction. Some authors systematically characterized immune cell populations and soluble mediators (chemokines, cytokines, and angiogenic factors) that constitute thyroid cancer microenvironment. Their purpose was to verify immune system involvement in cancer growth and progression, highlighting the differences in immune infiltrate among tumor histotypes. More recently, some authors have provided a more comprehensive view of the relationships between tumor and immune system involved in thyroid carcinogenesis. The Cancer Genome Atlas (TCGA) delivered a large amount of data that allowed to combine information on the inflammatory microenvironment with gene expression data, genetic and clinical-pathological characteristics, and differentiation degree of papillary thyroid carcinoma (PTC). Moreover, using a new sensitive and highly multiplex analysis, the NanoString Technology, it was possible to divide thyroid tumors in two main clusters based on expression of immune-related genes. Starting from these results, the authors performed an immune phenotype analysis that allowed to classify thyroid cancers in hot, cold, or intermediate depending on immune infiltration patterns of the tumor microenvironment. The aim of this review is to provide a comprehensive and updated view of the knowledge on immune landscape of thyroid tumors. Understanding interactions between tumor and microenvironment is crucial to effectively direct immunotherapeutic approaches in the treatment of thyroid cancer, particularly for those not responsive to conventional therapies.

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3D co-cultures of epithelial cells with immune cells as a model of HSV and oncolytic HSV infections

Access Microbiology ◽

10.1099/acmi.ac2020.po0501 ◽

2020 ◽

Vol 2 (7A) ◽

Author(s):

Ilaria Epifano ◽

Joe Conner ◽

Iain A. McNeish ◽

Sheila V. Graham

Keyword(s):

Animal Models ◽

Epithelial Cells ◽

Immune Cells ◽

Immune Cell ◽

Cell Killing ◽

Tissue Microenvironment ◽

Culture Model ◽

Human Immune

Virus infectivity is commonly investigated with in vitro monolayer cell cultures or in vivo animal models. Ease of growth and manipulation and low cost characterise standard cell culture. Animal models allow investigation of infectivity in the context of tissue structure and environment but are costly and can be limited by species variations. Neither approach can recapitulate the human context, the tissue microenvironment and human immune components. Three-dimensional organotypic raft tissue models can provide most of the advantages of in vitro and in vivo models. We successfully established HSV and oncolytic HSV (HSV1716) infection in 3D raft cultures of epithelial non-tumour (HaCaT) and tumour (SiHa, OVCAR3 and TOV21G) cell lines. Our 3D models allowed the evaluation and quantification of virus replication and the recovery of the virus both in culture media and tissues. We developed a complex 3D co-culture of epithelial cells with human immune cells in order to mimic the tissue microenvironment. This innovation allowed us to study the effect of immune cells in cell killing by HSV1716 in the in vitro tissues. In HSV1716-infected co-culture tissues, immune cells were identified throughout the tissue and some migrated to the areas of infection. The immune activity was identified through increased IL-8 release. Moreover, combining infection with immune cell infiltration increased tumour cell killing in the 3D co-culture model. This new co-culture model could be further developed to identify the role of immune cells in oncolytic viroimmunotherapy and to dissect the involvement of specific single immune cell subpopulations.

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Modulation of Matrix Metalloproteinases by Plant-Derived Products

Current Cancer Drug Targets ◽

10.2174/1568009620666201120144838 ◽

2020 ◽

Vol 20 ◽

Author(s):

Nur Najmi Mohamad Anuar ◽

Nurul Iman Natasya Zulkafali ◽

Azizah Ugusman

Keyword(s):

Clinical Trials ◽

Natural Products ◽

Matrix Metalloproteinases ◽

Animal Studies ◽

Current Knowledge ◽

In Vitro Models ◽

In Vivo Studies ◽

Extracellular Matrix Components

: Matrix metalloproteinases (MMPs) are a group of zinc-dependent metallo-endopeptidase that are responsible towards the degradation, repair and remodelling of extracellular matrix components. MMPs play an important role in maintaining a normal physiological function and preventing diseases such as cancer and cardiovascular diseases. Natural products derived from plants have been used as traditional medicine for centuries. Its active compounds, such as catechin, resveratrol and quercetin, are suggested to play an important role as MMPs inhibitors, thereby opening new insights into their applications in many fields, such as pharmaceutical, cosmetic and food industries. This review summarises the current knowledge on plant-derived natural products with MMP-modulating activities. Most of the reviewed plant-derived products exhibit an inhibitory activity on MMPs. Amongst MMPs, MMP-2 and MMP-9 are the most studied. The expression of MMPs is inhibited through respective signalling pathways, such as MAPK, NF-κB and PI3 kinase pathways, which contribute to the reduction in cancer cell behaviours, such as proliferation and migration. Most studies have employed in vitro models, but a limited number of animal studies and clinical trials have been conducted. Even though plant-derived products show promising results in modulating MMPs, more in vivo studies and clinical trials are needed to support their therapeutic applications in the future.

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Glucose Oligosaccharide and Long-Chain Glucomannan Feed Additives Induce Enhanced Activation of Intraepithelial NK Cells and Relative Abundance of Commensal Lactic Acid Bacteria in Broiler Chickens

Veterinary Sciences ◽

10.3390/vetsci8060110 ◽

2021 ◽

Vol 8 (6) ◽

pp. 110

Author(s):

Nathalie Meijerink ◽

Jean E. de Oliveira ◽

Daphne A. van Haarlem ◽

Guilherme Hosotani ◽

David M. Lamot ◽

...

Keyword(s):

Immune System ◽

Nk Cells ◽

Relative Abundance ◽

Nk Cell ◽

Feed Additives ◽

Microbiota Composition ◽

In Ovo ◽

Long Chain

Restrictions on the use of antibiotics in the poultry industry stimulate the development of alternative nutritional solutions to maintain or improve poultry health. This requires more insight in the modulatory effects of feed additives on the immune system and microbiota composition. Compounds known to influence the innate immune system and microbiota composition were selected and screened in vitro, in ovo, and in vivo. Among all compounds, 57 enhanced NK cell activation, 56 increased phagocytosis, and 22 increased NO production of the macrophage cell line HD11 in vitro. Based on these results, availability and regulatory status, six compounds were selected for further analysis. None of these compounds showed negative effects on growth, hatchability, and feed conversion in in ovo and in vivo studies. Based on the most interesting numerical results and highest future potential feasibility, two compounds were analyzed further. Administration of glucose oligosaccharide and long-chain glucomannan in vivo both enhanced activation of intraepithelial NK cells and led to increased relative abundance of lactic acid bacteria (LAB) amongst ileum and ceca microbiota after seven days of supplementation. Positive correlations between NK cell subsets and activation, and relative abundance of LAB suggest the involvement of microbiota in the modulation of the function of intraepithelial NK cells. This study identifies glucose oligosaccharide and long-chain glucomannan supplementation as effective nutritional strategies to modulate the intestinal microbiota composition and strengthen the intraepithelial innate immune system.

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Human Saliva-Mediated Hydrolysis of Eugenyl-β-D-Glucoside and Fluorescein-di-β-D-Glucoside in In Vivo and In Vitro Models

Biomolecules ◽

10.3390/biom11020172 ◽

2021 ◽

Vol 11 (2) ◽

pp. 172

Author(s):

Mariusz Dziadas ◽

Adam Junka ◽

Henryk Jeleń

Keyword(s):

Oral Cavity ◽

Control Sample ◽

Rapid Test ◽

Human Saliva ◽

In Vitro Models ◽

Microbial Hydrolysis ◽

Amoxicillin Trihydrate ◽

Potassium Clavulanate

Eugenyl-β-D-glucopyranoside, also referred to as Citrusin C, is a natural glucoside found among others in cloves, basil and cinnamon plants. Eugenol in a form of free aglycone is used in perfumeries, flavourings, essential oils and in medicinal products. Synthetic Citrusin C was incubated with human saliva in several in vitro models together with substrate-specific enzyme and antibiotics (clindamycin, ciprofloxacin, amoxicillin trihydrate and potassium clavulanate). Citrusin C was detected using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Citrusin C was completely degraded only when incubated with substrate-specific A. niger glucosidase E.C 3.2.1.21 (control sample) and when incubated with human saliva (tested sample). The addition of antibiotics to the above-described experimental setting, stopped Citrusin C degradation, indicating microbiologic origin of hydrolysis observed. Our results demonstrate that Citrusin C is subjected to complete degradation by salivary/oral cavity microorganisms. Extrapolation of our results allows to state that in the human oral cavity, virtually all β-D-glucosides would follow this type of hydrolysis. Additionally, a new method was developed for an in vivo rapid test of glucosidase activity in the human mouth on the tongue using fluorescein-di-β-D-glucoside as substrate. The results presented in this study serve as a proof of concept for the hypothesis that microbial hydrolysis path of β-D-glucosides begins immediately in the human mouth and releases the aglycone directly into the gastrointestinal tract.

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Biofilm formation in the lung contributes to virulence and drug tolerance of Mycobacterium tuberculosis

Nature Communications ◽

10.1038/s41467-021-21748-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Poushali Chakraborty ◽

Sapna Bajeli ◽

Deepak Kaushal ◽

Bishan Dass Radotra ◽

Ashwani Kumar

Keyword(s):

Mycobacterium Tuberculosis ◽

Immune System ◽

Biofilm Formation ◽

Antimycobacterial Activity ◽

Drug Tolerance ◽

Host Immune System ◽

Tissue Sections ◽

Slow Growing

AbstractTuberculosis is a chronic disease that displays several features commonly associated with biofilm-associated infections: immune system evasion, antibiotic treatment failures, and recurrence of infection. However, although Mycobacterium tuberculosis (Mtb) can form cellulose-containing biofilms in vitro, it remains unclear whether biofilms are formed during infection in vivo. Here, we demonstrate the formation of Mtb biofilms in animal models of infection and in patients, and that biofilm formation can contribute to drug tolerance. First, we show that cellulose is also a structural component of the extracellular matrix of in vitro biofilms of fast and slow-growing nontuberculous mycobacteria. Then, we use cellulose as a biomarker to detect Mtb biofilms in the lungs of experimentally infected mice and non-human primates, as well as in lung tissue sections obtained from patients with tuberculosis. Mtb strains defective in biofilm formation are attenuated for survival in mice, suggesting that biofilms protect bacilli from the host immune system. Furthermore, the administration of nebulized cellulase enhances the antimycobacterial activity of isoniazid and rifampicin in infected mice, supporting a role for biofilms in phenotypic drug tolerance. Our findings thus indicate that Mtb biofilms are relevant to human tuberculosis.

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O23: CHARACTERISING PATIENT-DERIVED COLORECTAL CANCER TISSUE-ORIGINATED ORGANOIDAL SPHEROIDS FOR HIGH-THROUGHPUT MICROFLUIDIC APPLICATIONS

British Journal of Surgery ◽

10.1093/bjs/znab117.023 ◽

2021 ◽

Vol 108 (Supplement_1) ◽

Author(s):

MI Khot ◽

M Levenstein ◽

R Coppo ◽

J Kondo ◽

M Inoue ◽

...

Keyword(s):

Colorectal Cancer ◽

Fluid Flow ◽

High Throughput ◽

Microfluidic Devices ◽

In Vitro Models ◽

Fluorescent Imaging ◽

Cancer Tissue ◽

Cell Models

Abstract Introduction Three-dimensional (3D) cell models have gained reputation as better representations of in vivo cancers as compared to monolayered cultures. Recently, patient tumour tissue-derived organoids have advanced the scope of complex in vitro models, by allowing patient-specific tumour cultures to be generated for developing new medicines and patient-tailored treatments. Integrating 3D cell and organoid culturing into microfluidics, can streamline traditional protocols and allow complex and precise high-throughput experiments to be performed with ease. Method Patient-derived colorectal cancer tissue-originated organoidal spheroids (CTOS) cultures were acquired from Kyoto University, Japan. CTOS were cultured in Matrigel and stem-cell media. CTOS were treated with 5-fluorouracil and cytotoxicity evaluated via fluorescent imaging and ATP assay. CTOS were embedded, sectioned and subjected to H&E staining and immunofluorescence for ABCG2 and Ki67 proteins. HT29 colorectal cancer spheroids were produced on microfluidic devices using cell suspensions and subjected to 5-fluorouracil treatment via fluid flow. Cytotoxicity was evaluated through fluorescent imaging and LDH assay. Result 5-fluorouracil dose-dependent reduction in cell viability was observed in CTOS cultures (p<0.01). Colorectal CTOS cultures retained the histology, tissue architecture and protein expression of the colonic epithelial structure. Uniform 3D HT29 spheroids were generated in the microfluidic devices. 5-fluorouracil treatment of spheroids and cytotoxic analysis was achieved conveniently through fluid flow. Conclusion Patient-derived CTOS are better complex models of in vivo cancers than 3D cell models and can improve the clinical translation of novel treatments. Microfluidics can streamline high-throughput screening and reduce the practical difficulties of conventional organoid and 3D cell culturing. Take-home message Organoids are the most advanced in vitro models of clinical cancers. Microfluidics can streamline and improve traditional laboratory experiments.

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