New Ref-1/APE1 targeted inhibitors demonstrating improved potency for clinical applications in multiple cancer types.

AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein that is overexpressed in most aggressive cancers and impacts various cancer cell signaling pathways. Ref-1's redox activity plays a significant role in activating transcription factors (TFs) such as NFκB, HIF1α, STAT3 and AP-1, which are crucial contributors to the development of tumors and metastatic growth. Therefore, development of potent, selective inhibitors to target Ref-1 redox function is an appealing approach for therapeutic intervention. A first-generation compound, APX3330 successfully completed phase I clinical trial in adults with progressing solid tumors with favorable response rate, pharmacokinetics (PK), and minimal toxicity. These positive results prompted us to develop more potent analogs of APX3330 to effectively target Ref-1 in solid tumors. In this study, we present structure-activity relationship (SAR) identification and validation of lead compounds that exhibit a greater potency and a similar or better safety profile to APX3330. In order to triage and characterize the most potent and on-target second-generation Ref-1 redox inhibitors, we assayed for PK, mouse and human S9 fraction metabolic stability, in silico ADMET properties, ligand-based WaterLOGSY NMR measurements, pharmacodynamic markers, cell viability in multiple cancer cell types, and two distinct 3-dimensional (3D) cell killing assays (Tumor-Microenvironment on a Chip and 3D spheroid). To characterize the effects of Ref-1 inhibition in vivo, global proteomics was used following treatment with the top four analogs. This study identified and characterized more potent inhibitors of Ref-1 redox function (that outperformed APX3330 by 5-10-fold) with PK studies demonstrating efficacious doses for translation to clinic.

Physical constraints on accuracy and persistence during breast cancer cell chemotaxis.

Directed cell motion in response to an external chemical gradient occurs in many biological phenomena such as wound healing, angiogenesis, and cancer metastasis. Chemotaxis is often characterized by the accuracy, persistence, and speed of cell motion, but whether any of these quantities is physically constrained by the others is poorly understood. Using a combination of theory, simulations, and 3D chemotaxis assays on single metastatic breast cancer cells, we investigate the links among these different aspects of chemotactic performance. In particular, we observe in both experiments and simulations that the chemotactic accuracy, but not the persistence or speed, increases with the gradient strength. We use a random walk model to explain this result and to propose that cells' chemotactic accuracy and persistence are mutually constrained. Our results suggest that key aspects of chemotactic performance are inherently limited regardless of how favorable the environmental conditions are.

Tumor-Microenvironment-on-Chip Platform for Assessing Drug Response in 3D Dynamic Culture.

Microphysiological systems involving microfluidic 3D culture of cancer cells have emerged as a versatile toolkit to study tumor biological problems and evaluate potential treatment strategies. Incorporation of microfluidic technologies in 3D tissue culture offers opportunities for realistic simulation of tumor microenvironment in vitro by facilitating a dynamic culture environment mimicking features of human physiology such as reconstituted ECM, interstitial flow, and gradients of drugs and biomacromolecules. This protocol describes development of 3D microfluidic cell culture based on Tumor-Microenvironment-on-Chip (T-MOC) platform modeling tumor blood and lymphatic capillary vessels and the interstitial space in between. Based on earlier applications of T-MOC for transport characteristics, drug response, and tumor-stroma interactions in mammary carcinoma and pancreatic adenocarcinoma, this protocol provides detailed description of device fabrication, on-chip 3D culture, and drug treatment assays. This protocol can easily be adapted for applications involving other cancer types.

Guidelines for Manufacturing and Application of Organoids: Liver.

Recent amendments to regulatory frameworks have placed a greater emphasis on the utilization of in vitro testing platforms for preclinical drug evaluations and toxicity assessments. This requires advanced tissue models capable of accurately replicating liver functions for drug efficacy and toxicity predictions. Liver organoids, derived from human cell sources, offer promise as a reliable platform for drug evaluation. However, there is a lack of standardized quality evaluation methods, which hinders their regulatory acceptance. This paper proposes comprehensive quality standards tailored for liver organoids, addressing cell source validation, organoid generation, and functional assessment. These guidelines aim to enhance reproducibility and accuracy in toxicity testing, thereby accelerating the adoption of organoids as a reliable alternative or complementary tool to animal testing in drug development. The quality standards include criteria for size, cellular composition, gene expression, and functional assays, thus ensuring a robust hepatotoxicity testing platform.

Cells function as a ternary logic gate to decide migration direction under integrated chemical and fluidic cues.

Cells sense various environmental cues and subsequently process intracellular signals to decide their migration direction in many physiological and pathological processes. Although several signaling molecules and networks have been identified in these directed migrations, it still remains ambiguous to predict the migration direction under multiple and integrated cues, specifically chemical and fluidic cues. Here, we investigated the cellular signal processing machinery by reverse-engineering directed cell migration under integrated chemical and fluidic cues. We imposed controlled chemical and fluidic cues to cells using a microfluidic platform and analyzed the extracellular coupling of the cues with respect to the cellular detection limit. Then, the cell's migratory behavior was reverse-engineered to build a cellular signal processing system as a logic gate, which is based on a "selection" gate. This framework is further discussed with a minimal intracellular signaling network of a shared pathway model. The proposed framework of the ternary logic gate suggests a systematic view to understand how cells decode multiple cues and make decisions about the migration direction.

Microphysiological systems as reliable drug discovery and evaluation tools: Evolution from innovation to maturity.

Microphysiological systems (MPSs), also known as organ-on-chip or disease-on-chip, have recently emerged to reconstitute the in vivo cellular microenvironment of various organs and diseases on in vitro platforms. These microfluidics-based platforms are developed to provide reliable drug discovery and regulatory evaluation testbeds. Despite recent emergences and advances of various MPS platforms, their adoption of drug discovery and evaluation processes still lags. This delay is mainly due to a lack of rigorous standards with reproducibility and reliability, and practical difficulties to be adopted in pharmaceutical research and industry settings. This review discusses the current and potential use of MPS platforms in drug discovery processes while considering the context of several key steps during drug discovery processes, including target identification and validation, preclinical evaluation, and clinical trials. Opportunities and challenges are also discussed for the broader dissemination and adoption of MPSs in various drug discovery and regulatory evaluation steps. Addressing these challenges will transform long and expensive drug discovery and evaluation processes into more efficient discovery, screening, and approval of innovative drugs.

Optically induced electrothermal microfluidic tweezers in bio-relevant media.

Non-contact micro-manipulation tools have enabled invasion-free studies of fragile synthetic particles and biological cells. Rapid electrokinetic patterning (REP) traps target particles/cells, suspended in an electrolyte, on an electrode surface. This entrapment is electrokinetic in nature and thus depends strongly on the suspension medium's properties. REP has been well characterized for manipulating synthetic particles suspended in low concentration salt solutions (~ 2 mS/m). However, it is not studied as extensively for manipulating biological cells, which introduces an additional level of complexity due to their limited viability in hypotonic media. In this work, we discuss challenges posed by isotonic electrolytes and suggest solutions to enable REP manipulation in bio-relevant media. Various formulations of isotonic media (salt and sugar-based) are tested for their compatibility with REP. REP manipulation is observed in low concentration salt-based media such as 0.1× phosphate buffered saline (PBS) when the device electrodes are passivated with a dielectric layer. We also show manipulation of murine pancreatic cancer cells suspended in a sugar-based (8.5% w/v sucrose and 0.3% w/v dextrose) isotonic medium. The ability to trap mammalian cells and deposit them in custom patterns enables high-impact applications such as determining their biomechanical properties and 3D bioprinting for tissue scaffolding.

Inkjet-printed morphogenesis of tumor-stroma interface using bi-cellular bioinks of collagen-poly(N-isopropyl acrylamide-co-methyl methacrylate) mixture.

Recent advances in biomaterials and 3D printing/culture methods enable various tissue-engineered tumor models. However, it is still challenging to achieve native tumor-like characteristics due to lower cell density than native tissues and prolonged culture duration for maturation. Here, we report a new method to create tumoroids with a mechanically active tumor-stroma interface at extremely high cell density. This method, named "inkjet-printed morphogenesis" (iPM) of the tumor-stroma interface, is based on a hypothesis that cellular contractile force can significantly remodel the cell-laden polymer matrix to form densely-packed tissue-like constructs. Thus, differential cell-derived compaction of tumor cells and cancer-associated fibroblasts (CAFs) can be used to build a mechanically active tumor-stroma interface. In this methods, two kinds of bioinks are prepared, in which tumor cells and CAFs are suspended respectively in the mixture of collagen and poly (N-isopropyl acrylamide-co-methyl methacrylate) solution. These two cellular inks are inkjet-printed in multi-line or multi-layer patterns. As a result of cell-derived compaction, the resulting structure forms tumoroids with mechanically active tumor-stroma interface at extremely high cell density. We further test our working hypothesis that the morphogenesis can be controlled by manipulating the force balance between cellular contractile force and matrix stiffness. Furthermore, this new concept of "morphogenetic printing" is demonstrated to create more complex structures beyond current 3D bioprinting techniques.

Deduction of signaling mechanisms from cellular responses to multiple cues.

Cell signaling networks are complex and often incompletely characterized, making it difficult to obtain a comprehensive picture of the mechanisms they encode. Mathematical modeling of these networks provides important clues, but the models themselves are often complex, and it is not always clear how to extract falsifiable predictions. Here we take an inverse approach, using experimental data at the cell level to deduce the minimal signaling network. We focus on cells' response to multiple cues, specifically on the surprising case in which the response is antagonistic: the response to multiple cues is weaker than the response to the individual cues. We systematically build candidate signaling networks one node at a time, using the ubiquitous ingredients of (i) up- or down-regulation, (ii) molecular conversion, or (iii) reversible binding. In each case, our method reveals a minimal, interpretable signaling mechanism that explains the antagonistic response. Our work provides a systematic way to deduce molecular mechanisms from cell-level data.

Signal processing capacity of the cellular sensory machinery regulates the accuracy of chemotaxis under complex cues.

Chemotaxis is ubiquitous in many biological processes, but it still remains elusive how cells sense and decipher multiple chemical cues. In this study, we postulate a hypothesis that the chemotactic performance of cells under complex cues is regulated by the signal processing capacity of the cellular sensory machinery. The underlying rationale is that cells in vivo should be able to sense and process multiple chemical cues, whose magnitude and compositions are entangled, to determine their migration direction. We experimentally show that the combination of transforming growth factor-ß and epidermal growth factor suppresses the chemotactic performance of cancer cells using independent receptors to sense the two cues. Based on this observation, we develop a biophysical framework suggesting that the antagonism is caused by the saturation of the signal processing capacity but not by the mutual repression. Our framework suggests the significance of the signal processing capacity in the cellular sensory machinery.

Engineering of a functional pancreatic acinus with reprogrammed cancer cells by induced PTF1a expression.

A pancreatic acinus is a functional unit of the exocrine pancreas producing digest enzymes. Its pathobiology is crucial to pancreatic diseases including pancreatitis and pancreatic cancer, which can initiate from pancreatic acini. However, research on pancreatic acini has been significantly hampered due to the difficulty of culturing normal acinar cells in vitro. In this study, an in vitro model of the normal acinus, named pancreatic acinus-on-chip (PAC), is developed using reprogrammed pancreatic cancer cells. The developed model is a microfluidic platform with an epithelial duct and acinar sac geometry microfabricated by a newly developed two-step controlled "viscous-fingering" technique. In this model, human pancreatic cancer cells, Panc-1, reprogrammed to revert to the normal state upon induction of PTF1a gene expression, are cultured. Bioinformatic analyses suggest that, upon induced PTF1a expression, Panc-1 cells transition into a more normal and differentiated acinar phenotype. The microanatomy and exocrine functions of the model are characterized to confirm the normal acinus phenotypes. The developed model provides a new and reliable testbed to study the initiation and progression of pancreatic cancers.

Ref-1 redox activity alters cancer cell metabolism in pancreatic cancer: exploiting this novel finding as a potential target.

BACKGROUND: Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy. Redox factor-1 (Ref-1), a redox signaling protein, regulates the conversion of several transcription factors (TFs), including HIF-1α, STAT3 and NFκB from an oxidized to reduced state leading to enhancement of their DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia. METHODS: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model and validated using proteomics and qRT-PCR. The identified Ref-1's role in mitochondrial function was confirmed using mitochondrial function assays, qRT-PCR, western blotting and NADP assay. Further, the effect of Ref-1 redox function inhibition against pancreatic cancer metabolism was assayed using 3D co-culture in vitro and xenograft studies in vivo. RESULTS: Distinct transcriptional variation in central metabolism, cell cycle, apoptosis, immune response, and genes downstream of a series of signaling pathways and transcriptional regulatory factors were identified in Ref-1 knockdown vs Scrambled control from the scRNA-seq data. Mitochondrial DEG subsets downregulated with Ref-1 knockdown were significantly reduced following Ref-1 redox inhibition and more dramatically in combination with Devimistat in vitro. Mitochondrial function assays demonstrated that Ref-1 knockdown and Ref-1 redox signaling inhibition decreased utilization of TCA cycle substrates and slowed the growth of pancreatic cancer co-culture spheroids. In Ref-1 knockdown cells, a higher flux rate of NADP + consuming reactions was observed suggesting the less availability of NADP + and a higher level of oxidative stress in these cells. In vivo xenograft studies demonstrated that tumor reduction was potent with Ref-1 redox inhibitor similar to Devimistat. CONCLUSION: Ref-1 redox signaling inhibition conclusively alters cancer cell metabolism by causing TCA cycle dysfunction while also reducing the pancreatic tumor growth in vitro as well as in vivo.

An engineered pancreatic cancer model with intra-tumoral heterogeneity of driver mutations.

Pancreatic ductal adenocarcinoma (PDAC) is a complex disease with significant intra-tumoral heterogeneity (ITH). Currently, no reliable PDAC tumor model is available that can present ITH profiles in a controlled manner. We develop an in vitro microfluidic tumor model mimicking the heterogeneous accumulation of key driver mutations of human PDAC using cancer cells derived from genetically engineered mouse models. These murine pancreatic cancer cell lines have KPC (Kras and Trp53 mutations) and KIC genotypes (Kras mutation and Cdkn2a deletion). Also, the KIC genotypes have two distinct phenotypes - mesenchymal or epithelial. The tumor model mimics the ITH of human PDAC to study the effects of ITH on the gemcitabine response. The results show gemcitabine resistance induced by ITH. Remarkably, it shows that cancer cell-cell interactions induce the gemcitabine resistance potentially through epithelial-mesenchymal-transition. The tumor model can provide a useful testbed to study interaction mechanisms between heterogeneous cancer cell subpopulations.

Subtype-specific characterization of breast cancer invasion using a microfluidic tumor platform.

Understanding progression of breast cancers to invasive ductal carcinoma (IDC) can significantly improve breast cancer treatments. However, it is still difficult to identify genetic signatures and the role of tumor microenvironment to distinguish pathological stages of pre-invasive lesion and IDC. Presence of multiple subtypes of breast cancers makes the assessment more challenging. In this study, an in-vitro microfluidic assay was developed to quantitatively assess the subtype-specific invasion potential of breast cancers. The developed assay is a microfluidic platform in which a ductal structure of epithelial cancer cells is surrounded with a three-dimensional (3D) collagen matrix. In the developed platform, two triple negative cancer subtypes (MDA-MB-231 and SUM-159PT) invaded into the surrounding matrix but the luminal A subtype, MCF-7, did not. Among invasive subtypes, SUM-159PT cells showed significantly higher invasion and degradation of the surrounding matrix than MDA-MB-231. Interestingly, the cells cultured on the platform expressed higher levels of CD24 than in their conventional 2D cultures. This microfluidic platform may be a useful tool to characterize and predict invasive potential of breast cancer subtypes or patient-derived cells.

In vitro microfluidic models of tumor microenvironment to screen transport of drugs and nanoparticles.

Advances in nanotechnology have enabled numerous types of nanoparticles (NPs) to improve drug delivery to tumors. While many NP systems have been proposed, their clinical translation has been less than anticipated primarily due to failure of current preclinical evaluation techniques to adequately model the complex interactions between the NP and physiological barriers of tumor microenvironment. This review focuses on microfluidic tumor models for characterization of delivery efficacy and toxicity of cancer nanomedicine. Microfluidics offer significant advantages over traditional macroscale cell cultures by enabling recapitulation of tumor microenvironment through precise control of physiological cues such as hydrostatic pressure, shear stress, oxygen, and nutrient gradients. Microfluidic systems have recently started to be adapted for screening of drugs and NPs under physiologically relevant settings. So far the two primary application areas of microfluidics in this area have been high-throughput screening using traditional culture settings such as single cells or multicellular tumor spheroids, and mimicry of tumor microenvironment for study of cancer-related cell-cell and cell-matrix interactions. These microfluidic technologies are also useful in modeling specific steps in NP delivery to tumor and characterize NP transport properties and outcomes by systematic variation of physiological conditions. Ultimately, it will be possible to design drug-screening platforms uniquely tailored for individual patient physiology using microfluidics. These in vitro models can contribute to development of precision medicine by enabling rapid and patient-specific evaluation of cancer nanomedicine. WIREs Nanomed Nanobiotechnol 2017, 9:e1460. doi: 10.1002/wnan.1460 For further resources related to this article, please visit the WIREs website.

Anticoagulating activities of low-molecular weight fuco-oligosaccharides prepared by enzymatic digestion of fucoidan from the sporophyll of Korean Undaria pinnatifida.

In spite of their potential as biologically active compounds, the high molecular mass and viscous natures of fucoidans have hampered their applications especially as a therapeutic agent. Herein the fucoidan-degrading enzyme activities were partially purified from the cultured cells of Sphingomonas paucimobilis PF-1 mainly by ammonium sulfate precipitation. This enzyme preparation degraded fucoidans from the Korean Undaria pinnatifida sporophyll into several low-molecular weight fuco-oligosaccharides (LMFOs) with less than 3,749 Da. The FTIR spectra of intact fucoidan and mixture of LMFOs (1,389-3,749 Da) showed no significant structural difference except for about 10% reduced level of sulfate esters in LMFOs. The LMFOs have exerted strong anticoagulating activities at which the activated partial thromboplastin time (APTT) and thrombin time (TT) were significantly prolonged, although 3 approximately 20 times weaker activities were observed than those of intact fucoidan. In addition, unlike intact fucoidan, LMFOs did not affect significantly to the prothrombin time (PT). These results suggest that the partially purified fucoidan-degrading enzyme preparation is valuable for the production of fuco-oligosaccharides having anticoagulating activities, and that the molecular weight and/or sulfate content of the fucoidan from the Korean Undaria pinnatifida sporophyll could be important factors for its anticoagulating activity.