Immune cell-based screening assay for response to anticancer agents: applications in pharmacogenomics

ABSTRACTPeptides bound to class I major histocompatibility complexes (MHC) play a critical role in immune cell recognition and can trigger an antitumor immune response in cancer. Surface MHC levels can be modulated by anticancer agents, altering immunity. However, understanding the peptide repertoire’s response to treatment remains challenging and is limited by quantitative mass spectrometry-based strategies lacking robust normalization controls. We describe a novel approach that leverages recombinant heavy isotope-coded peptide MHCs (hipMHCs) and multiplex isotope tagging to quantify peptide repertoire alterations using low sample input. HipMHCs improve quantitative accuracy of peptide repertoire changes by normalizing for variation across analyses and enable absolute quantification using internal calibrants to determine copies per cell of MHC antigens, which can inform immunotherapy design. Applying this platform in melanoma to profile the immunopeptidome response to CDK4/6 inhibition and interferon gamma, known modulators of antigen presentation, we uncovered treatment-specific alterations, connecting the intracellular response to extracellular immune presentation.

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The Potential of Combining Tubulin-Targeting Anticancer Therapeutics and Immune Therapy

International Journal of Molecular Sciences ◽

10.3390/ijms20030586 ◽

2019 ◽

Vol 20 (3) ◽

pp. 586 ◽

Cited By ~ 7

Author(s):

Alexis Fong ◽

Amanda Durkin ◽

Hoyun Lee

Keyword(s):

Anticancer Agents ◽

Immune Cell ◽

Cytotoxic T Cells ◽

Immune Therapy ◽

Cell Types ◽

Treg Cells ◽

Dendritic Cell Maturation ◽

Microtubule Inhibitors ◽

M1 Macrophage ◽

Effective Option

Cancer immune therapy has recently shown tremendous promise to combat many different cancers. The microtubule is a well-defined and very effective cancer therapeutic target. Interestingly, several lines of evidence now suggest that microtubules are intimately connected to the body’s immune responses. This raises the possibility that the combination of microtubule inhibitors and immune therapy can be a highly effective option for cancer treatments. However, our understanding on this potentially important aspect is still very limited, due in part to the multifaceted nature of microtubule functions. Microtubules are not only involved in maintaining cell morphology, but also a variety of cellular processes, including the movement of secretory vesicles and organelles, intracellular macromolecular assembly, signaling pathways, and cell division. Microtubule inhibitors may be subdivided into two classes: Anti-depolymerization agents such as the taxane family, and anti-polymerization agents such as colchicine and vinka alkaloids. These two different classes may have different effects on immune cell subtypes. Anti-depolymerization agents can not only induce NK cells, but also appear to inhibit T regulatory (Treg) cells. However, different inhibitors may have different functions even among the same class. For example, the doxetaxel anti-depolymerization agent up-regulates cytotoxic T cells, while paclitaxel down-regulates them. Certain anti-polymerization agents such as colchicine appear to down-regulate most immune cell types, while inducing dendritic cell maturation and increasing M1 macrophage population. In contrast, the vinblastine anti-polymerization agent activates many of these cell types, albeit down-regulating Treg cells. In this review, we focus on the various effects of tubulin inhibitors on the activities of the body’s immune system, in the hope of paving the way to develop an effective cancer therapy by combining tubulin-targeting anticancer agents and immune therapy.

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Immune subtype of mismatch repair deficient colorectal carcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e15194 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e15194-e15194

Author(s):

Ning He ◽

Qingzhu Jia ◽

Ge Jin ◽

Bo Zhu

Keyword(s):

Colorectal Carcinoma ◽

Inflammatory Response ◽

Immune Cell ◽

Wnt Signaling Pathway ◽

Enrichment Analysis ◽

Underlying Mechanism ◽

Interferon Γ ◽

Gene Expression Omnibus ◽

Gene Set Enrichment Analysis ◽

Screening Assay

e15194 Background: Half of microsatellite instability-high (MSI-H) colorectal carcinoma (CRCs) patients do not response to checkpoint blockade (ICB) immunotherapy. We aimed to unleash the underlying mechanism of distinct efficacy to ICB by sophisticated depicting the immune microenvironment of MSI-H CRC. Methods: Public omics and clinical data of patients with MSI labels were collected from TCGA data portal (364 samples) and Gene Expression Omnibus (GSE39582, GSE13294, GSE13067, GSE35896, GSE24514, GSE25071, GSE79959, GSE103340, GSE29638, GSE33113 cohorts, 1119 samples). GEO datasets were used as discovering sets, and TCGA dataset was used as validating sets. The immune cell infiltrations and signaling pathways in the tumor microenvironment were quantified by single sample Gene Set Enrichment Analysis (ssGSEA), and the bio-similarities were estimated by t-Distributed Stochastic Neighbor Embedding (tSNE). Clustering was identified by k-mean unsupervised strategy with optimal k. Statistical analysis and data visualization were all carried out using the R. Results: Based on the whole transcriptome and immune cell infiltration profiling, both GEO and TCGA cohorts showed that MSI-H patients harbored unique pattern which significantly differed from the MSS counterpart. Unsupervised clustering further classified the MSI-H patients into “Hot” and “Cold” subgroups with remarkable different IFNG-related signatures, inflammatory response score, interferon-γ signaling score, and survival outcome. Screening assay identified 23 genes ( ADAMTSL3, DLGAP4, DNAH11, FFAR3, GABRA4, KCNA6, KCTD9, KIAA1033, KLHL6, LARGE, MORC1, NDST3, OR5H14, PAX5, PHF3, PIK3R5, RNF25, SPATA20, STAT4, TLR9, TMC3, TTC12, ZNF790) enriched and 5 genes ( CELSR3, DGKD, HECTD4, RECQL4, SLC9A4) spared in immune “hot” tumors. Moreover, “hot” tumor showed less WNT signaling pathway score, which was negatively correlated with the IFNG signaling and inflammatory response within the MSI-H CRC tumors. Conclusions: We firstly identified immune hot and cold groups from MSI-H CRCs, which may be related to different immunotherapy response. This result may help further understanding the anti-immunotherapy of MSI-H CRCs.

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A High-Throughput Screening Assay Using a Photoconvertable Protein for Identifying Inhibitors of Transcription, Translation, or Proteasomal Degradation

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555216684333 ◽

2017 ◽

Vol 22 (4) ◽

pp. 399-407 ◽

Cited By ~ 3

Author(s):

David K. Heidary ◽

Ashley Fox ◽

Chris I. Richards ◽

Edith C. Glazer

Keyword(s):

Protein Degradation ◽

High Throughput ◽

Anticancer Agents ◽

High Throughput Screening ◽

Protein Production ◽

Fluorescent Proteins ◽

Light Emitting Diode ◽

Screening Assay ◽

High Throughput Screening Assay ◽

Ratiometric Detection

Dysregulated transcription, translation, and protein degradation are common features of cancer cells, regardless of specific genetic profiles. Several clinical anticancer agents take advantage of this characteristic vulnerability and interfere with the processes of transcription and translation or inhibit protein degradation. However, traditional assays that follow the process of protein production and removal require multistep processing and are not easily amenable to high-throughput screening. The use of recombinant fluorescent proteins provides a convenient solution to this problem, and moreover, photoconvertable fluorescent proteins allow for ratiometric detection of both new protein production and removal of existing proteins. Here, the photoconvertable protein Dendra2 is used in the development of in-cell assays of protein production and degradation that are optimized and validated for high-throughput screening. Conversion from the green to red emissive form can be achieved using a high-intensity light-emitting diode array, producing a stable pool of the red fluorescent form of Dendra2. This allows for rates of protein production or removal to be quantified in a plate reader or by fluorescence microscopy, providing a means to measure the potencies of inhibitors that affect these key processes.

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Cardiac Glycosides as Immune System Modulators

Biomolecules ◽

10.3390/biom11050659 ◽

2021 ◽

Vol 11 (5) ◽

pp. 659

Author(s):

Jan Škubník ◽

Vladimíra Pavlíčková ◽

Silvie Rimpelová

Keyword(s):

Immune Response ◽

Anticancer Agents ◽

Cardiac Glycosides ◽

Immune Cell ◽

Current Knowledge ◽

Inflammatory Diseases ◽

Large Area ◽

Strongly Connected ◽

Autoimmune And Inflammatory Diseases ◽

Multiple Signaling

Cardiac glycosides (CGs) are natural steroid compounds occurring both in plants and animals. They are known for long as cardiotonic agents commonly used for various cardiac diseases due to inhibition of Na+/K+-ATPase (NKA) pumping activity and modulating heart muscle contractility. However, recent studies show that the portfolio of diseases potentially treatable with CGs is much broader. Currently, CGs are mostly studied as anticancer agents. Their antiproliferative properties are based on the induction of multiple signaling pathways in an NKA signalosome complex. In addition, they are strongly connected to immunogenic cell death, a complex mechanism of induction of anticancer immune response. Moreover, CGs exert various immunomodulatory effects, the foremost of which are connected with suppressing the activity of T-helper cells or modulating transcription of many immune response genes by inhibiting nuclear factor kappa B. The resulting modulations of cytokine and chemokine levels and changes in immune cell ratios could be potentially useful in treating sundry autoimmune and inflammatory diseases. This review aims to summarize current knowledge in the field of immunomodulatory properties of CGs and emphasize the large area of potential clinical use of these compounds.

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Non-invasive transdermal delivery of chemotherapeutic molecules in vivo using superparamagnetic iron oxide nanoparticles

Cancer Nanotechnology ◽

10.1186/s12645-021-00079-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Vanisri Raviraj ◽

Binh T. T. Pham ◽

Byung J. Kim ◽

Nguyen T. H. Pham ◽

Lai F. Kok ◽

...

Keyword(s):

Iron Oxide ◽

Fluorescence Microscopy ◽

Iron Oxide Nanoparticles ◽

Anticancer Agents ◽

Transdermal Delivery ◽

Immune Cell ◽

Superparamagnetic Iron Oxide ◽

Superparamagnetic Iron Oxide Nanoparticles ◽

Oxide Nanoparticles

Abstract Background The skin is both a target and a potential conduit for the delivery of drugs, but its cornified cell layer resists penetration by most molecules. This study investigated the potential of superparamagnetic iron oxide nanoparticles to facilitate the transdermal delivery of anticancer agents. Results Chemotherapeutic cancer drugs were applied with or without nanoparticles to the skin of hairless mice, and their ability to penetrate the skin was assessed using fluorescence microscopy and tumor growth. Nanoparticles enhanced the penetration of the skin by doxorubicin and 5-fluorouracil as determined by fluorescence microscopy and growth retardation of experimental melanoma in immunocompetent, syngeneic mice. This drug enhancement did not require conjugation or encapsulation of the drugs by the nanoparticles—simple co-administration sufficed. Nanoparticles applied topically to melanomas increased the cytotoxicity and immune cell infiltration induced by co-administered 5-fluorouracil, and also reduced vascularization of the tumors independently of 5-fluorouracil. Conclusion Correctly formulated superparamagnetic iron oxide nanoparticles can facilitate the chemotherapeutic effectiveness of cytotoxic drugs on skin tumors by both increasing their transdermal penetration and ameliorating host–tumor interactions. This enhancement of skin penetration occurs without the need for conjugation or encapsulation of the co-administered drugs, and so will likely be applicable to other drugs, also.

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Targeting Host Cellular Factors as a Strategy of Therapeutic Intervention for Herpesvirus Infections

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.603309 ◽

2021 ◽

Vol 11 ◽

Author(s):

Kumari Asha ◽

Neelam Sharma-Walia

Keyword(s):

Transcription Factors ◽

Anticancer Agents ◽

Cell Activation ◽

Immune Cell ◽

Viral Reactivation ◽

Signal Pathways ◽

Immune Cell Activation ◽

Cellular Signal ◽

Cellular Machinery ◽

Therapeutic Compounds

Herpesviruses utilize various host factors to establish latent infection, survival, and spread disease in the host. These factors include host cellular machinery, host proteins, gene expression, multiple transcription factors, cellular signal pathways, immune cell activation, transcription factors, cytokines, angiogenesis, invasion, and factors promoting metastasis. The knowledge and understanding of host genes, protein products, and biochemical pathways lead to discovering safe and effective antivirals to prevent viral reactivation and spread infection. Here, we focus on the contribution of pro-inflammatory, anti-inflammatory, and resolution lipid metabolites of the arachidonic acid (AA) pathway in the lifecycle of herpesvirus infections. We discuss how various herpesviruses utilize these lipid pathways to their advantage and how we target them to combat herpesvirus infection. We also summarize recent development in anti-herpesvirus therapeutics and new strategies proposed or under clinical trials. These anti-herpesvirus therapeutics include inhibitors blocking viral life cycle events, engineered anticancer agents, epigenome influencing factors, immunomodulators, and therapeutic compounds from natural extracts.

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Three-Dimensional Aggregated Spheroid Model of Hepatocellular Carcinoma Using a 96-Pillar/Well Plate

Molecules ◽

10.3390/molecules26164949 ◽

2021 ◽

Vol 26 (16) ◽

pp. 4949

Author(s):

Sang-Yun Lee ◽

Yvonne Teng ◽

Miseol Son ◽

Bosung Ku ◽

Hyun Ju Hwang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Drug Screening ◽

Immune Cell ◽

Single Cells ◽

Three Dimensional ◽

Cell Interaction ◽

Screening Assay ◽

Cytokine Activation

A common method of three-dimensional (3D) cell cultures is embedding single cells in Matrigel. Separated cells in Matrigel migrate or grow to form spheroids but lack cell-to-cell interaction, which causes difficulty or delay in forming mature spheroids. To address this issue, we proposed a 3D aggregated spheroid model (ASM) to create large single spheroids by aggregating cells in Matrigel attached to the surface of 96-pillar plates. Before gelling the Matrigel, we placed the pillar inserts into blank wells where gravity allowed the cells to gather at the curved end. In a drug screening assay, the ASM with Hepatocellular carcinoma (HCC) cell lines showed higher drug resistance compared to both a conventional spheroid model (CSM) and a two-dimensional (2D) cell culture model. With protein expression, cytokine activation, and penetration analysis, the ASM showed higher expression of cancer markers associated with proliferation (p-AKT, p-Erk), tight junction formation (Fibronectin, ZO-1, Occludin), and epithelial cell identity (E-cadherin) in HCC cells. Furthermore, cytokine factors were increased, which were associated with immune cell recruitment/activation (MIF-3α), extracellular matrix regulation (TIMP-2), cancer interaction (IL-8, TGF-β2), and angiogenesis regulation (VEGF-A). Compared to CSM, the ASM also showed limited drug penetration in doxorubicin, which appears in tissues in vivo. Thus, the proposed ASM better recapitulated the tumor microenvironment and can provide for more instructive data during in vitro drug screening assays of tumor cells and improved prediction of efficacious drugs in HCC patients.

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