While reshaping their microenvironment, tumors are also capable of influencing systemic processes including myeloid cell production. Therefore, the tumor-induced myeloid cells, such as ...myeloid-derived suppressor cells (MDSCs), which are characterized with pro-cancer properties, became another target in order to increase the success of the therapy. This study evaluated the capacity of a novel dendrimeric drug delivery platform to eliminate tumor-induced myeloid cells. As described in a previous study by our research group, the anti-Flt1 antibody-conjugated polyethylene glycol (PEG)-cored poly(amidoamine) (PAMAM) dendrimers improved the efficacy of gemcitabine against pancreatic cancer. Here, the biodistribution studies showed that these dendrimeric structures accumulated in the compartments that became rich in myeloid cells in the pancreatic tumor-bearing mice. When gemcitabine was loaded into the dendrimer complexes, the number of myeloid cells was significantly reduced while the percentage distribution of granulocytic and monocytic myeloid cells was not always significantly altered. The CD11b+Ly6G–Ly6C+ monocytes were more severely affected by the treatments than CD11b+Ly6G+Ly6C+ granulocytes. Immune infiltration levels in the tumor tissue were also altered. Myeloid cells in the spleen and F4/80+ macrophages of the liver were protected. The compartments, such as the liver and the bone marrow, which are known to have high vascular endothelial growth factor (VEGF)–Flt1 pathway activity, were particularly targeted by gemcitabine when delivered through anti-Flt1 antibody-conjugated PAMAM dendrimers. In conclusion, chemotherapeutic agents complexed with dendrimers not only improve anticancer efficacy, but they also assist in the elimination of the tumor-induced myeloid cells.
Starting from Ethylenediamine (EDA) or poly(ethylene glycol) tetra amine (4-arm-PEG) cores, two different peripheral methylester (-COOCH
3
) or amine (-NH
2
) PAMAM dendrimers have been synthesized. ...In the growth phase of dendrimers, two important building blocks, methyl acrylate for the half generation and EDA for the full generations, have been used. In order to improve the yield and decrease the time for the aminolysis step, a microwave-assisted technique was applied. Both of these dendrimers with different cores were grown up to 4.5 generations where surface modification, i.e. PEGylation, with 10% Poly(ethylene glycol) bis(amine) was performed. In order to increase the solubility of dendrimers, esteric surfaces were converted to carboxylic acid groups. Accordingly, the dendrimers were soluble in water or in water-methanol mixture which enabled their purification by liquid-phase polymer-based retention in each step. Finally, the resulting products that were characterized with (NMR and FTIR) spectroscopy were evaluated in vitro and in vivo. The analytical grade dendrimers were not cytotoxic to mouse fibroblasts and their biodistribution was mainly determined in the site of injection (peritoneum), liver and kidneys.
Here, we describe a protocol combining functional metrics with genomic data to elucidate drivers of within-cell-type heterogeneity via the phenotype-to-genotype link. This technique involves using ...fluorescence tagging to label and isolate cells grown in 3D culture, enabling high-throughput enrichment of phenotypically defined cell subpopulations by fluorescence-activated cell sorting. We then perform a validated phenotypically supervised single-cell analysis pipeline to reveal unique functional cell states, including genes and pathways that contribute to cellular heterogeneity and were undetectable by unsupervised analysis.
For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).
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•Sorting of cells by the phenotype from 3D culture is achieved through photoconversion•The photolabeling technique is adaptable to other systems, cells, and phenotypes•Phenotypically supervised analysis reveals novel insights into cellular heterogeneity•A GenePattern notebook facilitates phenotypically supervised scRNAseq analysis
Here, we describe a protocol combining functional metrics with genomic data to elucidate drivers of within-cell-type heterogeneity via the phenotype-to-genotype link. This technique involves using fluorescence tagging to label and isolate cells grown in 3D culture, enabling high-throughput enrichment of phenotypically defined cell subpopulations by fluorescence-activated cell sorting. We then perform a validated phenotypically supervised single-cell analysis pipeline to reveal unique functional cell states, including genes and pathways that contribute to cellular heterogeneity and were undetectable by unsupervised analysis.
To better understand cellular communication driving diverse behaviors, we need to uncover the molecular mechanisms of within-cell-type functional heterogeneity. While single-cell RNA sequencing ...(scRNAseq) has advanced our understanding of cell heterogeneity, linking individual cell phenotypes to transcriptomic data remains challenging. Here, we used a phenotypic cell sorting technique to ask whether phenotypically supervised scRNAseq analysis (pheno-scRNAseq) can provide more insight into heterogeneous cell behaviors than unsupervised scRNAseq. Using a simple 3D in vitro breast cancer (BRCA) model, we conducted pheno-scRNAseq on invasive and non-invasive cells and compared the results to phenotype-agnostic scRNAseq analysis. Pheno-scRNAseq identified unique and more selective differentially expressed genes than unsupervised scRNAseq analysis. Functional studies validated the utility of pheno-scRNAseq in understanding within-cell-type functional heterogeneity and revealed that migration phenotypes were coordinated with specific metabolic, proliferation, stress, and immune phenotypes. This approach lends new insight into the molecular systems underlying BRCA cell phenotypic heterogeneity.
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•Graph-based clusters do not identify collective migration phenotypes•Collective invasion is associated with stress response and proliferation•Non-invasive behavior is associated with plasticity and immunomodulation•Signatures corresponding to migration phenotypes are conserved across species
Cell Biology; Complex System Biology; Transcriptomics
Abstract
The objective of this study is to make comprehensive cytotoxicity evaluation and in vitro characterization of Jeffamine-cored polyamidoamine (PAMAM) dendrimers on L929 cell lines for oral ...drug delivery purposes. Ester-, amine- and carboxylic acid-terminated PAMAMs were investigated for their cytotoxicity on L929 cells at different generations and concentrations. Cationic surface charge caused highest cytotoxicity on L929 cells, while ester-terminated PAMAMs showed generation- and concentration-dependent toxicity. Anionic dendrimers were determined as the lowest cytotoxic group, and highest generation number presented lowest cellular toxicity. Encapsulation studies were performed with anionic PAMAMs at 2.5, 3.5 and 4.5 generations and different concentrations. Increasing generation number provides greater loaded naproxen amounts and larger particle size. Moreover, formulations provide controlled release at simulated terminal ileum conditions. Consequently, Jeffamine-cored carboxylic acid-terminated PAMAMs can be a promising option for oral drug delivery of poorly water-soluble drugs.
More reliable and cheaper sequencing technologies have revealed the vast mutational landscapes characteristic of many phenotypes. The analysis of such genetic variants has led to successful ...identification of altered proteins underlying many Mendelian disorders. Nevertheless the simple one‐variant one‐phenotype model valid for many monogenic diseases does not capture the complexity of polygenic traits and disorders. Although experimental and computational approaches have improved detection of functionally deleterious variants and important interactions between gene products, the development of comprehensive models relating genotype and phenotypes remains a challenge in the field of genomic medicine. In this context, a new view of the pathologic state as significant perturbation of the network of interactions between biomolecules is crucial for the identification of biochemical pathways associated with complex phenotypes. Seminal studies in systems biology combined the analysis of genetic variation with protein–protein interaction networks to demonstrate that even as biological systems evolve to be robust to genetic variation, their topologies create disease vulnerabilities. More recent analyses model the impact of genetic variants as changes to the “wiring” of the interactome to better capture heterogeneity in genotype–phenotype relationships. These studies lay the foundation for using networks to predict variant effects at scale using machine‐learning or algorithmic approaches. A wealth of databases and resources for the annotation of genotype–phenotype relationships have been developed to support developments in this area. This overview describes how study of the molecular interactome has generated insights linking the organization of biological systems to disease mechanism, and how this information can enable precision medicine.
This article is categorized under:
Translational, Genomic, and Systems Medicine > Translational Medicine
Biological Mechanisms > Cell Signaling
Models of Systems Properties and Processes > Mechanistic Models
Analytical and Computational Methods > Computational Methods
Conceptual representation of genome interpretation using biological networks.
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Nanoparticles are promising drug delivery systems which are flexible for targeting specific tissues to reduce therapeutic doses and minimize side effects. Nanoparticles should be ...maintained with high stability and uniformity; however, aggregation is a major challenge which commonly impairs stability and efficacy of nanocarriers. In this study, we revisited the factors that influence the stability of chitosan (Protasan™ UP CL113) nanoparticles prepared with ionotropic gelation, widely recognized to be prone to aggregation, and proposed a model to overcome the negative influence of aggregation while testing in vitro efficacy. Decrease in pH due to cell proliferation, 37 °C cell culture temperature, serum in culture media, and incubation time were considered as factors causing chitosan nanoparticles’ aggregation which deteriorates cell culture assay readouts, increases optical density values and leads to false-positive results. Size and stability studies were not sufficient to avoid misleading results in cell culture. The chitosan nanoparticle aggregation was almost inevitable under standard culture conditions; nevertheless, the removal of nanoparticles before aggregation but after an incubation period long enough for efficient cellular uptake was determined as a feasible and inexpensive method for testing the in vitro efficacy of polymeric nanoformulations. This approach was used with blank and gemcitabine-loaded chitosan nanoparticles on pancreatic cancer cells and proved to be useful for reliable cytotoxicity results.
Single-gene missense mutations remain challenging to interpret. Here, we deploy scalable functional screening by sequencing (SEUSS), a Perturb-seq method, to generate mutations at protein interfaces ...of RUNX1 and quantify their effect on activities of downstream cellular programs. We evaluate single-cell RNA profiles of 115 mutations in myelogenous leukemia cells and categorize them into three functionally distinct groups, wild-type (WT)-like, loss-of-function (LoF)-like, and hypomorphic, that we validate in orthogonal assays. LoF-like variants dominate the DNA-binding site and are recurrent in cancer; however, recurrence alone does not predict functional impact. Hypomorphic variants share characteristics with LoF-like but favor protein interactions, promoting gene expression indicative of nerve growth factor (NGF) response and cytokine recruitment of neutrophils. Accessible DNA near differentially expressed genes frequently contains RUNX1-binding motifs. Finally, we reclassify 16 variants of uncertain significance and train a classifier to predict 103 more. Our work demonstrates the potential of targeting protein interactions to better define the landscape of phenotypes reachable by missense mutations.
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•scRNA-seq profiling of 115 variants at protein interfaces of the RUNX1 transcription factor•Clustering identifies transcriptionally distinct groups and uncovers hypomorphic variants•Hypomorphic variants alter gene programs associated with cytokine and growth factor signaling•scRNA-seq-based assay informs reclassification of variants of uncertain significance
Deciphering distinct effects of single-gene missense mutations is challenging. Ozturk et al. design an interface-guided Perturb-seq library and measure the impact of >100 RUNX1 variants on >40,000 single-cell RNA profiles. The assay identifies functionally distinct groups, demonstrating the potential of scRNA-seq to characterize cellular phenotypes reachable by individual mutations.
Novel advances in targeted drug delivery Öztürk, Kıvılcım; Eroğlu, Hakan; Çalış, Sema
Journal of drug targeting,
09/2018, Letnik:
26, Številka:
8
Journal Article
Recenzirano
Developing a new drug molecule is not only time-consuming and expensive, but also mostly a failing process. However, improving bioavailability, targetability, efficacy or safety of old drugs could be ...more effective way to use them in clinic. For these purposes, so many strategies including individualising drug therapy, nanoparticle-based drug delivery systems, drug conjugates, therapeutic drug monitoring, stimuli-sensitive targeted therapy are investigated intensely. Depending on the desired application or targeted site, nanoparticles can be administrated as orally, locally, topically and systemically. Currently, the Food and Drug Administration and the European Medicines Agency approved nanoparticles are mostly aimed to treat cancer. Although some of these formulations were approved by Food and Drug Administration and/or European Medicines Agency to use in clinic, most of them have fell down to pass either pre-clinical or clinical trials. To have high approval rate, failure reasons need to be better understand.
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