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BackgroundPancreatic 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.MethodsscRNA-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.ResultsDistinct 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.ConclusionRef-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.

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.

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.

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.

Tumor models are essential in cancer research and treatment to discover novel therapeutic targets and design personalized treatment strategies. A wide spectrum of tumor models has been developed ranging from cancer cell lines, spheroids/organoids, and small and large animal models. These models provide either the convenience of handling or relevance of mimicking human tumor microenvironment (TME). However, most of the currently available models are not capable of providing both. Recently the emergence of engineered tumor models based on microfluidics and tissue engineering technology may be able to address these challenges. In this chapter, we briefly review the complexities of the TME and discuss the development of tumor models to mimic the TME. Our discussion is focused on engineered tumor models based on microfluidics and tissue engineering, so-called tumor-microenvironment-on-a-chip, and related challenges and opportunities as also discussed.

Directed cancer cell migration induced by the environmental signals is a critical process in cancer metastasis. Cancer cells are exposed to complex chemical and mechanical signals stimulating directed migration in the tumor microenvironment, where the physical nature is highly complex. It is still barely understood how cells sense and process the complex environmental signals through the complex intercellular signaling networks to execute the cell responses. This study explores the migratory response of cancer cells under a single and combined signal. The driving hypothesis is that the cell innate capability constraints the signal stimulations physically in inducing directed cell migration. We assess the hypothesis by engineering the microenvironment in the microfluidic platform, exposing a single or combined signal environment. The combined signal environment is established by 1) two different chemoattractants (TGF-β1 and EGF) and 2) the convection-driven signal environment (TGF-β1 and interstitial flow). The results show that the performance of cancer cell directed migration is physically constrained when the environmental stimulation meets the cell’s innate physical limit. We illustrate the results in a physical and quantitative manner. This approach provides a novel insight to understand the cellular process and eventually enables to predict the cellular response under the complex environmental signals.

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.

Summary 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-β 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., Graphical abstract, Highlights • Cellular sensory machinery can sense and decipher multiple chemical cues simultaneously • Combination of TGF-β and EGF suppresses the chemotactic accuracy of cancer cells • Cells have the physical capacity in signal processing with the shared pathway • The saturation of the shared pathway can disturb the cells' accurate chemotaxis, Cell biology; Mathematical biosciences; Systems biology

Targeted therapy for cancer using small molecules has progressed exponentially, but agents that can affect cancer cells rather than non-tumorigenic cells are crucial to avoid pernicious side effects. AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein with DNA repair activity and redox signaling activity as major functions. The DNA repair function is indispensable for cell survival. Its role as a redox factor that stimulates the DNA binding activity of numerous transcription factors (TFs) such as HIF-1α, NFΚB, STAT3, and AP-1 tends to be dysregulated in cancer cells. Ref-1 is overexpressed in many cancers including, aggressive and invasive pancreatic ductal adenocarcinoma (PDAC), which is the 3rd leading cause of death in U.S. The dense stroma of PDAC primarily constituting cancer-associated fibroblasts (CAFs) makes it highly hypoxic, nutrient poor, and drug resistant. Current treatment regimens offer only modest improvement in survival. Targeting the PDAC stroma or ECM did not result in clinical benefit thus far presumably due to complex interactions between PDAC and its TME. Better treatments options that selectively target the tumor within its protective stroma are hence needed. Our group was able to generate a Ref-1 redox inhibitor, APX3330, that completed Phase I clinical trial (NCT03375086) with a good safety profile, verified target engagement and a recommended phase II dose. However, its potency in preclinical models is in the high µM range indicating the need for more potent second-generation inhibitors. Based on initial SAR studies, we selected 13 second-generation compounds from > 350 that were further characterized for positive properties including increased efficacy for cell killing, mouse and human S9 fraction metabolic stability, plasma half-life, and in silico ADMET properties. Target engagement studies involving blockade of TF activity via luciferase assay and EMSA as well as validation of direct interaction of these inhibitors with Ref-1 using thermal shift assay are ongoing. In two 3D co-culture models, second-generation Ref-1 redox analogs suppressed tumor survival significantly while sparing the CAFs. These findings were confirmed in vivo with xenografts co-implanted with tumor and CAF lines. Gene expression and mitochondria functional data revealed Ref-1's control of TCA cycle in tumor cells, but not in CAFs. To confirm and compare the effects of Ref-1 redox signaling inhibitors in cells, CRISPR editing was used to generate Ref-1 redox deficient cell lines (Ref-1C65A). As confirmation of significantly reduced Ref-1 redox activity, PDAC cells expressing Ref-1C65A did not induce hypoxia marker (CA9) under hypoxia, similar to when Ref-1 was knocked down or blocked via small molecule inhibitor. Ref-1 redox signaling and validation for selective killing of PDAC cells leaving the stomal cells undisturbed paves the way to improved PDAC treatment. Citation Format: Silpa Gampala, Nayela Chowdhury, Olivia Babb, Rachel A. Caston, Randall S. Wireman, Hye-ran Moon, George Sandusky, Emily Hulsey, Bumsoo Han, Millie M. Georgiadis, Sara K. Quinney, Andi R. Masters, James H. Wikel, Mark R. Kelley, Melissa L. Fishel. Deciphering mechanisms of Ref-1 signaling and its inhibition in aggressive tumor-stroma PDAC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1452.

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., Author summary One of the most ubiquitous and important cell behaviors is chemotaxis: the ability to move in the direction of a chemical gradient. Due to its importance, key aspects of chemotaxis have been quantified for a variety of cells, including the accuracy, persistence, and speed of cell motion. However, whether these aspects are mutually constrained is poorly understood. Can a cell be accurate but not persistent, or vice versa? Here we use theory, simulations, and experiments on cancer cells to uncover mutual constraints on the properties of chemotaxis. Our results suggest that accuracy and persistence are mutually constrained.

Pancreatic cancer can survive under the harshest of conditions including nutrient deprivation and extreme hypoxia. Its complex microenvironment contributes to PDAC’s (pancreatic ductal adenocarcinoma) therapeutic resistance and aggressive metastasis. Previous research shows that PDAC cells highly rely on increased glycolysis for ATP thereby limiting the use of TCA (tricarboxylic acid) cycle and ETC (electron transport chain), especially under hypoxia. However, recent studies indicate a dynamic exchange of nutrients between PCCs (Pancreatic Cancer cells) and its TME (tumor microenvironment) thus allowing the tumor to rely on either aerobic glycolysis or traditional oxidative phosphorylation (OXPHOS) depending on the oxygen tension and glucose availability. With HIF-1a being a central player in PDAC’s hypoxic response, and Redox factor-1 (Ref-1) controlling HIF-1a along with several other transcription factors (TFs) like NFkB and STAT3, Ref-1 proffers to be an ideal target. Ref-1 reduces oxidized TFs, thus enhancing their DNA binding and transcription. Several studies show that perturbation of Ref-1 levels affect the function of mitochondria, warranting the investigation of its role in metabolism. We conducted single cell RNA-seq (scRNA-seq) with Ref-1 knockdown (KD) in PDAC patient-derived cells under hypoxia and identified TCA cycle to be the most downregulated pathway with glycolysis and OXPHOS also significantly down. These results were validated with proteomics analysis. From the most downregulated DEGs (differentially expressed genes), a subset of genes confined to mitochondrial ETC and a subset of metabolite intermediates from TCA cycle were chosen to study and validate the role of Ref-1 in PDAC mitochondrial metabolic signaling. PCCs as well as CAFs (cancer-associated fibroblasts) were subjected to Ref-1 KD or redox inhibition using a potent second-generation inhibitor, APX2009. CAFs were chosen with the intent of understanding the differences in metabolic pathways between tumor and TME cells. As observed from scRNA-seq data, qPCR results demonstrated significant downregulation of mitochondrial DEG subsets with Ref-1 KD and redox inhibition. Mitochondrial plate-based functional assays revealed that Ref-1 inhibition caused decreased uptake of TCA cycle substrates in PCCs, but no change in CAFs. These effects of mitochondrial inhibition on cell growth were confirmed with reduction in growth of PCCs, but not CAFs in 3D co-culture spheroid assays. CPI-613 (mitochondrial metabolic inhibitor) was used as a comparator compound and showed similar results. Likewise, in-vivo xenograft studies with co-implantation of PCCs and CAFs confirmed reduction of tumor growth similar to CPI-613. These results confirm Ref-1’s redox role in tumor cells’ ability to utilize TCA cycle substrates and that we can detect differences in the metabolic phenotype between PCCs and CAFs. To conclude, suppression of tumor growth with minimal impact on CAFs, suggests a significant therapeutic benefit of Ref-1 redox inhibition. Citation Format: Silpa Gampala, Fenil Shah, Xiaoyu Lu, Hye-ran Moon, George E. Sandusky, Emily Hulsey, Amber L. Mosley, Bamsoo Han, Chi Zhang, Mark R. Kelley, Melissa L. Fishel. Ref-1 redox function identified as mitochondrial metabolic regulator in pancreatic cancer cells but not in CAFs [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO025.

Various 2-aminobenzophenones were synthesized from readily available 2-arylindoles in DMSO under O2 balloon atmosphere. The synthesis was carried out without the aid of a transition metal catalyst or moisture-sensitive organometallic reagents from 2-arylindoles.

Various dihydrobenzo[c]azepines were synthesized in good yields via Pictet–Spengler cyclization protocol from tosylamide derivatives of Morita–Baylis–Hillman adducts and 1,3,5-trioxane. The synthesis was carried out in the presence of easily removable montmorillonite K-10 in short time in high yields.

A transition metal and base-free synthesis of 3,3-diaryl-2-oxindoles has been developed from 2,2,N-triarylacetamides in the presence of montmorillonite K-10 in 1,2-dichlorobenzene under O2 balloon atmosphere.

Various spirofluorenyl oxindoles and spiroindeno[1,2- b ]indolyl oxindoles were synthesized in good yields in one-pot reaction by TiCl 4 -catalyzed sequential inter- and intramolecular Friedel–Crafts reactions of isatins with meta -arylphenols and 2-arylindoles in 1,2-dichloroethane.

Thermal cyclization of E,E,E-trienes prepared from isatines and Morita—Baylis—Hillman bromides results in highly diastereoselective 6π-electrocyclization to form spirocyclic oxindole derivatives (II) and (V).

Various pyrazolo[1,5-a]quinolines were synthesized via the intramolecular Friedel-Crafts reaction of 1-aryl-5-styrylpyrazoles and the following base-catalyzed aerobic oxidation process. The required 1-aryl-5-styrylpyrazoles were readily prepared from the corresponding conjugated dienones and arylhydrazines in a one-pot process.

The title reaction follows an oxidative Heck arylation, allylic palladium migration, aryl C—H bond functionalization/arylation sequence.

Various 3-(γ,δ-disubstituted)allylidene-2-oxindoles were synthesized by the Wittig reaction between isatins and the phosphorous ylide derived in situ from the Morita–Baylis–Hillman bromides. The 3-allylidene-2-oxindole could be used as an efficient synthetic precursor of 1-thiacarbazole derivatives. During the conversion of 2-oxindole moiety to 2-thiooxindole by Lawesson's reagent, 1-thiacarbazole derivative was obtained in good yield.

The synthesis of pyrazolo[4,3-c]quinolines has been carried out from the Morita–Baylis–Hillman bromides of 2-bromobenzaldehydes and methyl vinyl ketone. The synthesis involved a novel one-pot preparation of functionalized pyrazoles from α,β-enones, a one-pot Cu-mediated intramolecular N-arylation, and the elimination of p-toluenesulfinic acid.

E-mail: kimjn@chonnam.ac.krReceived July 1, 2014, Accepted July 21, 2014Intramolecular Diels-Alder (IMDA) reaction of vinylarenes bearing a Z-alkenyl tether, prepared from Morita-Baylis-Hillman (MBH) adducts, afforded arene-fused isoindoline derivatives in good yields. Vinylfurans,vinylthiophenes, and vinylnaphthalenes could be used successfully as dienes, while vinylbenzene failed underthe same reaction conditions.Key Words : Intramolecular Diels-Alder reaction, Morita-Baylis-Hillman adducts, Vinylarenes, IsoindolinesIntroductionThe construction of a cyclohexene ring by using vinyl-arene (such as styrenyl) as a 1,3-diene moiety in both inter-and intramolecular Diels-Alder reactions has been welldocumented.

E-mail: kimjn@chonnam.ac.krReceived January 27, 2014, Accepted February 12, 2014A practical and efficient one-pot synthesis of 1,3,5-trisubstituted pyrazoles from α,β-enones and arylhydrazinehydrochlorides has been developed. The pyrazoles were formed via a tandem formation of the correspondingpyrazolines and an acid-catalyzed aerobic oxidation process.Key Words : Pyrazoles, One-pot synthesis, Aerobic oxidation, α,β-Enones Introduction1,3,5-Trisubstituted pyrazolines are important heterocycliccompounds which can be prepared from substituted hydra-zines and α,β-enones. The oxidation of these pyrazolinesprovides the corresponding pyrazoles, which are known topossess diverse biological activities.

Various 2-aminobenzophenones were synthesized from readily available 2-arylindoles in DMSO under O2 balloon atmosphere. The synthesis was carried out without the aid of a transition metal catalyst or moisture-sensitive organometallic reagents from 2-arylindoles.

Various dihydrobenzo[c]azepines were synthesized in good yields via Pictet–Spengler cyclization protocol from tosylamide derivatives of Morita–Baylis–Hillman adducts and 1,3,5-trioxane. The synthesis was carried out in the presence of easily removable montmorillonite K-10 in short time in high yields.

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.

A transition metal and base-free synthesis of 3,3-diaryl-2-oxindoles has been developed from 2,2,N-triarylacetamides in the presence of montmorillonite K-10 in 1,2-dichlorobenzene under O2 balloon atmosphere.

Various spirofluorenyl oxindoles and spiroindeno[1,2- b ]indolyl oxindoles were synthesized in good yields in one-pot reaction by TiCl 4 -catalyzed sequential inter- and intramolecular Friedel–Crafts reactions of isatins with meta -arylphenols and 2-arylindoles in 1,2-dichloroethane.

Acorn-derived foods are commonly marketed in Korea, but their beneficial health effects have not been extensively explored. The aim of this study was to investigate the anti-inflammatory and antiasthmatic effects of an ethanol extract (AEx) from acorn (Quercus acutissima CARR.) in murine macrophage RAW264.7 cells and ovalbumin (OVA)-induced asthma model mice. The AEx at concentrations less than 100 µg/mL significantly, dose dependently attenuated the production of nitric oxide and the expression of inducible nitric oxide synthase, cyclooxygenase-2 and interleukin-1β (IL-1β) in RAW264.7 cells. The AEx (100 or 300 mg/kg/day) also inhibited the production of tumor necrosis factor-α and T-helper 2 (Th2) type cytokines (IL-4 and IL-13) in both the lung tissues and bronchoalveolar lavage fluid of the OVA-induced asthma model mice. These results suggest that the AEx has suppressive effects on asthma through its anti-inflammatory action both in vitro and in vivo. PRACTICAL APPLICATIONS AEx may be useful for preventing inflammatory diseases, such as allergic asthma, and may thus be effectively used as a natural anti-inflammatory ingredient in health food or pharmaceuticals.