Digital patient twins for personalized therapeutics and pharmaceutical manufacturing.

Digital twins are virtual models of physical artefacts that may or may not be synchronously connected, and that can be used to simulate their behavior. They are widely used in several domains such as manufacturing and automotive to enable achieving specific quality goals. In the health domain, so-called digital patient twins have been understood as virtual models of patients generated from population data and/or patient data, including, for example, real-time feedback from wearables. Along with the growing impact of data science technologies like artificial intelligence, novel health data ecosystems centered around digital patient twins could be developed. This paves the way for improved health monitoring and facilitation of personalized therapeutics based on management, analysis, and interpretation of medical data via digital patient twins. The utility and feasibility of digital patient twins in routine medical processes are still limited, despite practical endeavors to create digital twins of physiological functions, single organs, or holistic models. Moreover, reliable simulations for the prediction of individual drug responses are still missing. However, these simulations would be one important milestone for truly personalized therapeutics. Another prerequisite for this would be individualized pharmaceutical manufacturing with subsequent obstacles, such as low automation, scalability, and therefore high costs. Additionally, regulatory challenges must be met thus calling for more digitalization in this area. Therefore, this narrative mini-review provides a discussion on the potentials and limitations of digital patient twins, focusing on their potential bridging function for personalized therapeutics and an individualized pharmaceutical manufacturing while also looking at the regulatory impacts.

The Role of Proteoglycans in Cancer Metastasis and Circulating Tumor Cell Analysis.

Circulating tumor cells (CTCs) are accessible by liquid biopsies via an easy blood draw. They represent not only the primary tumor site, but also potential metastatic lesions, and could thus be an attractive supplement for cancer diagnostics. However, the analysis of rare CTCs in billions of normal blood cells is still technically challenging and novel specific CTC markers are needed. The formation of metastasis is a complex process supported by numerous molecular alterations, and thus novel CTC markers might be found by focusing on this process. One example of this is specific changes in the cancer cell glycocalyx, which is a network on the cell surface composed of carbohydrate structures. Proteoglycans are important glycocalyx components and consist of a protein core and covalently attached long glycosaminoglycan chains. A few CTC assays have already utilized proteoglycans for both enrichment and analysis of CTCs. Nonetheless, the biological function of proteoglycans on clinical CTCs has not been studied in detail so far. Therefore, the present review describes proteoglycan functions during the metastatic cascade to highlight their importance to CTCs. We also outline current approaches for CTC assays based on targeting proteoglycans by their protein cores or their glycosaminoglycan chains. Lastly, we briefly discuss important technical aspects, which should be considered for studying proteoglycans.

DNMT3B deficiency alters mitochondrial biogenesis and α-ketoglutarate levels in human embryonic stem cells.

Embryonic stem cell renewal and differentiation is regulated by metabolites that serve as cofactors for epigenetic enzymes. An increase of α-ketoglutarate (α-KG), a cofactor for histone and DNA demethylases, triggers multilineage differentiation in human embryonic stem cells (hESCs). To gain further insight into how the metabolic fluxes in pluripotent stem cells can be influenced by inactivating mutations in epigenetic enzymes, we generated hESCs deficient for de novo DNA methyltransferases (DNMTs) 3A and 3B. Our data reveal a bidirectional dependence between DNMT3B and α-KG levels: a-KG is significantly upregulated in cells deficient for DNMT3B, while DNMT3B expression is downregulated in hESCs treated with α-KG. In addition, DNMT3B null hESCs exhibit a disturbed mitochondrial fission and fusion balance and a switch from glycolysis to oxidative phosphorylation. Taken together, our data reveal a novel link between DNMT3B and the metabolic flux of hESCs.

Optimization of rVAR2-Based Isolation of Cancer Cells in Blood for Building a Robust Assay for Clinical Detection of Circulating Tumor Cells.

Early detection and monitoring of cancer progression is key to successful treatment. Therefore, much research is invested in developing technologies, enabling effective and valuable use of non-invasive liquid biopsies. This includes the detection and analysis of circulating tumor cells (CTCs) from blood samples. Recombinant malaria protein VAR2CSA (rVAR2) binds a unique chondroitin sulfate modification present on the vast majority of cancers and thereby holds promise as a near-universal tumor cell-targeting reagent to isolate CTCs from complex blood samples. This study describes a technical approach for optimizing the coupling of rVAR2 to magnetic beads and the development of a CTC isolation platform targeting a range of different cancer cell lines. We investigate both direct and indirect approaches for rVAR2-mediated bead retrieval of cancer cells and conclude that an indirect capture approach is most effective for rVAR2-based cancer cell retrieval.

Capture and Detection of Circulating Glioma Cells Using the Recombinant VAR2CSA Malaria Protein.

Diffuse gliomas are the most common primary malignant brain tumor. Although extracranial metastases are rarely observed, recent studies have shown the presence of circulating tumor cells (CTCs) in the blood of glioma patients, confirming that a subset of tumor cells are capable of entering the circulation. The isolation and characterization of CTCs could provide a non-invasive method for repeated analysis of the mutational and phenotypic state of the tumor during the course of disease. However, the efficient detection of glioma CTCs has proven to be challenging due to the lack of consistently expressed tumor markers and high inter- and intra-tumor heterogeneity. Thus, for this field to progress, an omnipresent but specific marker of glioma CTCs is required. In this article, we demonstrate how the recombinant malaria VAR2CSA protein (rVAR2) can be used for the capture and detection of glioma cell lines that are spiked into blood through binding to a cancer-specific oncofetal chondroitin sulfate (ofCS). When using rVAR2 pull-down from glioma cells, we identified a panel of proteoglycans, known to be essential for glioma progression. Finally, the clinical feasibility of this work is supported by the rVAR2-based isolation and detection of CTCs from glioma patient blood samples, which highlights ofCS as a potential clinical target for CTC isolation.

The Clinically Used Iron Chelator Deferasirox Is an Inhibitor of Epigenetic JumonjiC Domain-Containing Histone Demethylases.

Fe(II)- and 2-oxoglutarate (2OG)-dependent JumonjiC domain-containing histone demethylases (JmjC KDMs) are "epigenetic eraser" enzymes involved in the regulation of gene expression and are emerging drug targets in oncology. We screened a set of clinically used iron chelators and report that they potently inhibit JMJD2A (KDM4A) in vitro. Mode of action investigations revealed that one compound, deferasirox, is a bona fide active site-binding inhibitor as shown by kinetic and spectroscopic studies. Synthesis of derivatives with improved cell permeability resulted in significant upregulation of histone trimethylation and potent cancer cell growth inhibition. Deferasirox was also found to inhibit human 2OG-dependent hypoxia inducible factor prolyl hydroxylase activity. Therapeutic effects of clinically used deferasirox may thus involve transcriptional regulation through 2OG oxygenase inhibition. Deferasirox might provide a useful starting point for the development of novel anticancer drugs targeting 2OG oxygenases and a valuable tool compound for investigations of KDM function.

Turning Skyscrapers into Town Houses: Insights into Barrett's Esophagus.

Barrett's esophagus (BE) is defined as metaplasia of the esophageal squamous epithelium with multiple cell layers into a single layer of intestinal columnar epithelial cells - or, in other words, skyscrapers are turned into town houses. The underlying pathomechanism(s) and the cell of origin of BE lesions have not been defined yet. However, four potential hypotheses for BE development have been suggested. The morphological changes during BE development are associated with rather well-described aberrant gene/protein expression patterns. However, the potential key regulators of this conversion process are still unclear. The process of metaplastic conversion is difficult to monitor in a spatiotemporal manner in vitro, and robust models are lacking. There is therefore a need for novel experimental systems. This review focuses on potential key regulators, microenvironmental influences, epigenetic alterations and experimental research systems related to BE.

Response of esophageal cancer cells to epigenetic inhibitors is mediated via altered thioredoxin activity.

We previously showed that histone deacetylase inhibitor (HDACi) and 5-azacytidine (AZA) treatment selectively induced cell death of esophageal cancer cells. The mechanisms of cancer selectivity, however, remained unclear. Here we examined whether the cancer selectivity of HDACi/AZA treatment is mediated by the thioredoxin (Trx) system and reactive oxygen species (ROS) in esophageal cancer cells. For this, we first analyzed human tissue specimens of 37 esophageal cancer patients by immunohistochemistry for Trx, Trx-interacting protein (TXNIP) and Trx reductase (TXNRD). This revealed a loss or at least reduction of nuclear Trx in esophageal cancer cells, compared with normal epithelial cells (P<0.001). Although no differences were observed for TXNIP, TXNRD was more frequently expressed in cancer cells (P<0.001). In the two main histotypes of esophageal squamous cell carcinomas (ESCCs, n=19) and esophageal adenomcarcinomas (EAC, n=16), similar Trx, TXNIP and TXNRD expression patterns were observed. Also in vitro, nuclear Trx was only detectable in non-neoplastic Het-1A cells, but not in OE21/ESCC or OE33/EAC cell lines. Moreover, the two cancer cell lines showed an increased Trx activity, being significant for OE21 (P=0.0237). After treatment with HDACi and/or AZA, ROS were exclusively increased in both cancer cell lines (P=0.048-0.017), with parallel decrease of Trx activity. This was variably accompanied by increased TXNIP levels upon AZA, MS-275 or MS-275/AZA treatment for 6 or 24 h in OE21, but not in Het-1A or OE33 cells. In summary, this study evaluated Trx and its associated proteins TXNIP and TXNRD for the first time in esophageal cancers. The analyses revealed an altered subcellular localization of Trx and strong upregulation of TXNRD in esophageal cancer cells. Moreover, HDACi and AZA disrupted Trx function and induced accumulation of ROS with subsequent apoptosis in esophageal cancer cells exclusively. Trx function is hence an important cellular mediator conferring non-neoplastic cell resistance for HDACi and/or AZA.

Selective inhibition of esophageal cancer cells by combination of HDAC inhibitors and Azacytidine.

Esophageal cancers are highly aggressive tumors with poor prognosis despite some recent advances in surgical and radiochemotherapy treatment options. This study addressed the feasibility of drugs targeting epigenetic modifiers in esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) cells. We tested inhibition of histone deacetylases (HDACs) by SAHA, MS-275, and FK228, inhibition of DNA methyltransferases by Azacytidine (AZA) and Decitabine (DAC), and the effect of combination treatment using both types of drugs. The drug targets, HDAC1/2/3 and DNMT1, were expressed in normal esophageal epithelium and tumor cells of ESCC or EAC tissue specimens, as well as in non-neoplastic esophageal epithelial (Het-1A), ESCC (OE21, Kyse-270, Kyse-410), and EAC (OE33, SK-GT-4) cell lines. In vitro, HDAC activity, histone acetylation, and p21 expression were similarly affected in non-neoplastic, ESCC, and EAC cell lines post inhibitor treatment. Combined MS-275/AZA treatment, however, selectively targeted esophageal cancer cell lines by inducing DNA damage, cell viability loss, and apoptosis, and by decreasing cell migration. Non-neoplastic Het-1A cells were protected against HDACi (MS-275)/AZA treatment. RNA transcriptome analyses post MS-275 and/or AZA treatment identified novel regulated candidate genes (up: BCL6, Hes2; down: FAIM, MLKL), which were specifically associated with the treatment responses of esophageal cancer cells. In summary, combined HDACi/AZA treatment is efficient and selective for the targeting of esophageal cancer cells, despite similar target expression of normal and esophageal cancer epithelium, in vitro and in human esophageal carcinomas. The precise mechanisms of action of treatment responses involve novel candidate genes regulated by HDACi/AZA in esophageal cancer cells. Together, targeting of epigenetic modifiers in esophageal cancers may represent a potential future therapeutic approach.

Epigenetics in esophageal cancers.

Esophageal cancers are a challenging upper gastrointestinal tract tumor entity for interdisciplinary oncology. For the two main histotypes, namely esophageal squamous cell carcinomas and Barrett's adenocarcinomas, several genetic aberrations have been shown to contribute to carcinogenesis and progression as well as to represent potential novel targets for therapeutic intervention. This is paralleled by growing insight into epigenetic alterations of esophageal cancers. Studies involving the analyses of human tissue specimens predominantly describe altered patterns of miRNA expression, DNA methylation patterns, and histone marks levels. This review provides a critical update on this increasing knowledge of epigenetic alteration in esophageal cancers by specifically focusing on the translational aspects of epigenetic analyses from human tissue specimens.

Double deficiency of cathepsins B and L results in massive secretome alterations and suggests a degradative cathepsin-MMP axis.

Endolysosomal cysteine cathepsins functionally cooperate. Cathepsin B (Ctsb) and L (Ctsl) double-knockout mice die 4 weeks after birth accompanied by (autophago-) lysosomal accumulations within neurons. Such accumulations are also observed in mouse embryonic fibroblasts (MEFs) deficient for Ctsb and Ctsl. Previous studies showed a strong impact of Ctsl on the MEF secretome. Here we show that Ctsb alone has only a mild influence on extracellular proteome composition. Protease cleavage sites dependent on Ctsb were identified by terminal amine isotopic labeling of substrates (TAILS), revealing a prominent yet mostly indirect impact on the extracellular proteolytic cleavages. To investigate the cooperation of Ctsb and Ctsl, we performed a quantitative secretome comparison of wild-type MEFs and Ctsb (-/-) Ctsl (-/-) MEFs. Deletion of both cathepsins led to drastic alterations in secretome composition, highlighting cooperative functionality. While many protein levels were decreased, immunodetection corroborated increased levels of matrix metalloproteinase (MMP)-2. Re-expression of Ctsl rescues MMP-2 abundance. Ctsl and to a much lesser extent Ctsb are able to degrade MMP-2 at acidic and neutral pH. Addition of active MMP-2 to the MEF secretome degrades proteins whose levels were also decreased by Ctsb and Ctsl double deficiency. These results suggest a degradative Ctsl-MMP-2 axis, resulting in increased MMP-2 levels upon cathepsin deficiency with subsequent degradation of secreted proteins such as collagen α-1 (I).