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.

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.

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.

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.

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.

A Biomimetic Tumor Model of Heterogeneous Invasion in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a complex, heterogeneous, and genetically unstable disease. Its tumor microenvironment (TME) is complicated by heterogeneous cancer cell populations and strong desmoplastic stroma. This complex and heterogeneous environment makes it challenging to discover and validate unique therapeutic targets. Reliable and relevant in vitro PDAC tumor models can significantly advance the understanding of the PDAC TME and may enable the discovery and validation of novel drug targets. In this study, an engineered tumor model is developed to mimic the PDAC TME. This biomimetic model, named ductal tumor-microenvironment-on-chip (dT-MOC), permits analysis and experimentation on the epithelial-mesenchymal transition (EMT) and local invasion with intratumoral heterogeneity. This dT-MOC is a microfluidic platform where a duct of murine genetically engineered pancreatic cancer cells is embedded within a collagen matrix. The cancer cells used carry two of the three mutations of KRAS, CDKN2A, and TP53, which are key driver mutations of human PDAC. The intratumoral heterogeneity is mimicked by co-culturing these cancer cells. Using the dT-MOC model, heterogeneous invasion characteristics, and response to transforming growth factor-beta1 are studied. A mechanism of EMT and local invasion caused by the interaction between heterogeneous cancer cell populations is proposed.

Engineering of biomaterials for tumor modeling.

Development of biomaterials mimicking tumor and its microenvironment has recently emerged for the use of drug discovery, precision medicine, and cancer biology. These biomimetic models have developed by reconstituting tumor and stroma cells within the 3D extracellular matrix. The models are recently extended to recapitulate the in vivo tumor microenvironment, including biological, chemical, and mechanical conditions tailored for specific cancer type and its microenvironment. In spite of the recent emergence of various innovative engineered tumor models, many of these models are still early stage to be adapted for cancer research. In this article, we review the current status of biomaterials engineering for tumor models considering three main aspects - cellular engineering, matrix engineering, and engineering for microenvironmental conditions. Considering cancer-specific variability in these aspects, our discussion is focused on pancreatic cancer, specifically pancreatic ductal adenocarcinoma (PDAC). In addition, we further discussed the current challenges and future opportunities to create reliable and relevant tumor models.

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.

Analysis of adhesion kinetics of cancer cells on inflamed endothelium using a microfluidic platform.

Metastasis is the ultimate cause of death among the vast majority of cancer patients. This process is comprised of multiple steps, including the migration of circulating cancer cells across microvasculature. This trans-endothelial migration involves the adhesion and eventual penetration of cancer cells to the vasculature of the target organ. Many of these mechanisms remain poorly understood due to poor control of pathophysiological conditions in tumor models. In this work, a microfluidic device was developed to support the culture and observation of engineered microvasculature with systematic control of the environmental characteristics. This device was then used to study the adhesion of circulating cancer cells to an endothelium under varying conditions to delineate the effects of hemodynamics and inflammations. The resulting understanding will help to establish a quantitative and biophysical mechanism of interactions between cancer cells and endothelium.

Active Antioxidizing Particles for On-Demand Pressure-Driven Molecular Release.

Overproduced reactive oxygen species (ROS) are closely related to various health problems including inflammation, infection, and cancer. Abnormally high ROS levels can cause serious oxidative damage to biomolecules, cells, and tissues. A series of nano- or microsized particles has been developed to reduce the oxidative stress level by delivering antioxidant drugs. However, most systems are often plagued by slow molecular discharge, driven by diffusion. Herein, this study demonstrates the polymeric particles whose internal pressure can increase upon exposure to H2O2, one of the ROS, and in turn, discharge antioxidants actively. The on-demand pressurized particles are assembled by simultaneously encapsulating water-dispersible manganese oxide (MnO2) nanosheets and green tea derived epigallocatechin gallate (EGCG) molecules into a poly(lactic-co-glycolic acid) (PLGA) spherical shell. In the presence of H2O2, the MnO2 nanosheets in the PLGA particle generate oxygen gas by decomposing H2O2 and increase the internal pressure. The pressurized PLGA particles release antioxidative EGCG actively and, in turn, protect vascular and brain tissues from oxidative damage more effectively than the particles without MnO2 nanosheets. This H2O2 responsive, self-pressurizing particle system would be useful to deliver a wide array of molecular cargos in response to the oxidation level.

Differential response to doxorubicin in breast cancer subtypes simulated by a microfluidic tumor model.

Successful drug delivery and overcoming drug resistance are the primary clinical challenges for management and treatment of cancer. The ability to rapidly screen drugs and delivery systems within physiologically relevant environments is critically important; yet is currently limited due to lack of appropriate tumor models. To address this problem, we developed the Tumor-microenvironment-on-chip (T-MOC), a new microfluidic tumor model simulating the interstitial flow, plasma clearance, and transport of the drug within the tumor. We demonstrated T-MOC's capabilities by assessing the delivery and efficacy of doxorubicin in small molecular form versus hyaluronic acid nanoparticle (NP) formulation in MCF-7 and MDA-MB-231, two cell lines representative of different molecular subtypes of breast cancer. Doxorubicin accumulated and penetrated similarly in both cell lines while the NP accumulated more in MDA-MB-231 than MCF-7 potentially due to binding of hyaluronic acid to CD44 expressed by MDA-MB-231. However, the penetration of the NP was less than the molecular drug due to its larger size. In addition, both cell lines cultured on the T-MOC showed increased resistance to the drug compared to 2D culture where MDA-MB-231 attained a drug-resistant tumor-initiating phenotype indicated by increased CD44 expression. When grown in immunocompromised mice, both cell lines exhibited cell-type-dependent resistance and phenotypic changes similar to T-MOC, confirming its predictive ability for in vivo drug response. This initial characterization of T-MOC indicates its transformative potential for in vitro testing of drug efficacy towards prediction of in vivo outcomes and investigation of drug resistance mechanisms for advancement of personalized medicine.

Enzyme-Induced Matrix Softening Regulates Hepatocarcinoma Cancer Cell Phenotypes.

The progression of cancer is often accompanied by changes in the mechanical properties of an extracellular matrix. However, limited efforts have been made to reproduce these biological events in vitro. To this end, this study demonstrates that matrix remodeling caused by matrix metalloproteinase (MMP)-1 regulates phenotypic activities and modulates radiosensitivity of cancer cells exclusively in a 3D matrix. In this study, hepatocarcinoma cells are cultured in a collagen-based gel tailored to present an elastic modulus of ≈4.0 kPa. The subsequent exposure of the gel to MMP-1 decreases the elastic modulus from 4.0 to 0.5 kPa. In response to MMP-1, liver cancer cells undergo active proliferation, downregulation of E-cadherin, and the loss of detoxification capacity. The resulting spheroids are more sensitive to radiation than the spheroids cultured in the stiffer gel not exposed to MMP-1. Overall, this study serves to better understand and control the effects of MMP-induced matrix remodeling.

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.

Collective Chemotaxis through Noisy Multicellular Gradient Sensing.

Collective cell migration in response to a chemical cue occurs in many biological processes such as morphogenesis and cancer metastasis. Clusters of migratory cells in these systems are capable of responding to gradients of <1% difference in chemical concentration across a cell length. Multicellular systems are extremely sensitive to their environment, and although the limits to multicellular sensing are becoming known, how this information leads to coherent migration remains poorly understood. We develop a computational model of multicellular sensing and migration in which groups of cells collectively measure noisy chemical gradients. The output of the sensing process is coupled to the polarization of individual cells to model migratory behavior. Through the use of numerical simulations, we find that larger clusters of cells detect the gradient direction with higher precision and thus achieve stronger polarization bias, but larger clusters also induce more drag on collective motion. The trade-off between these two effects leads to an optimal cluster size for most efficient migration. We discuss how our model could be validated using simple, phenomenological experiments.

Characterization of Cell-Type-Specific Drug Transport and Resistance of Breast Cancers Using Tumor-Microenvironment-on-Chip.

Heterogeneous response and resistance of cancer cells to chemotherapeutic drugs pose a significant challenge for successful cancer treatments. In this study, an integrated experimental and theoretical analysis of cellular drug transport was developed. The experimental platform, called tumor-microenvironment-on-chip (T-MOC), is a microfluidic platform where cancer cells were cultured within a three-dimensional extracellular matrix perfused with interstitial fluid. Three types of human breast cancer cell lines (MCF-7, MDA-MB-231, and SUM-159PT) were cultured on this T-MOC platform, and their drug response and resistance to doxorubicin were characterized by time-lapse quantitative fluorescence microscopy. To study the effects of nanoparticle-mediated drug delivery, the transport and action of doxorubicin encapsulated nanoparticles were also examined. Based on the experimental data obtained, a theoretical model was developed to quantify and ultimately predict the cellular transport processes of drugs cell-type specifically. The results demonstrate that the cellular drug transport can be cell-type-specifically quantified by rate constants representing the uptake and efflux of doxorubicin across the cellular membrane.