Simulating Interclonal Interactions in Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is one of the most common types of cancers, accounting for 37% of B-cell tumor cases globally. DLBCL is known to be a heterogeneous disease, resulting in variable clinical presentations and the development of drug resistance. One underexplored aspect of drug resistance is the evolving dynamics between parental and drug-resistant clones within the same microenvironment. In this work, the effects of interclonal interactions between two cell populations-one sensitive to treatment and the other resistant to treatment-on tumor growth behaviors were explored through a mathematical model. In vitro cultures of mixed DLBCL populations demonstrated cooperative interactions and revealed the need for modifying the model to account for complex interactions. Multiple best-fit models derived from in vitro data indicated a difference in steady-state behaviors based on therapy administrations in simulations. The model and methods may serve as a tool for understanding the behaviors of heterogeneous tumors and identifying the optimal therapeutic regimen to eliminate cancer cell populations using computer-guided simulations.

Pharmacodynamic Model of the Dynamic Response of Pseudomonas aeruginosa Biofilms to Antibacterial Treatments.

Accurate pharmacokinetic-pharmacodynamic (PK-PD) models of biofilm treatment could be used to guide formulation and administration strategies to better control bacterial lung infections. To this end, we developed a detailed pharmacodynamic model of P. aeruginosa treatment with the front-line antibiotics, tobramycin and colistin, and validated it on a detailed dataset of killing dynamics. A compartmental model structure was developed in which the key features are the diffusion of the drug through a boundary layer to the bacteria, concentration-dependent interactions with bacteria, and the passage of the bacteria through successive transit states before death. The number of transit states employed was greater for tobramycin, which is a ribosomal inhibitor, than for colistin, which disrupts bacterial membranes. For both drugs, the experimentally observed delay in the killing of bacteria following drug exposure was consistent with the sum of the diffusion time and the time for passage through the transit states. For each drug, the PD model with a single set of parameters described data across a ten-fold range of concentrations and for both continuous and transient exposure protocols, as well as for combined drug treatments. The ability to predict drug response over a range of administration protocols allows this PD model to be integrated with PK descriptions to describe in vivo antibiotic response dynamics and to predict drug delivery strategies for the improved control of bacterial lung infections.

Shortwave infrared emitting multicolored nanoprobes for biomarker-specific cancer imaging in vivo.

BACKGROUND: The ability to detect tumor-specific biomarkers in real-time using optical imaging plays a critical role in preclinical studies aimed at evaluating drug safety and treatment response. In this study, we engineered an imaging platform capable of targeting different tumor biomarkers using a multi-colored library of nanoprobes. These probes contain rare-earth elements that emit light in the short-wave infrared (SWIR) wavelength region (900-1700 nm), which exhibits reduced absorption and scattering compared to visible and NIR, and are rendered biocompatible by encapsulation in human serum albumin. The spectrally distinct emissions of the holmium (Ho), erbium (Er), and thulium (Tm) cations that constitute the cores of these nanoprobes make them attractive candidates for optical molecular imaging of multiple disease biomarkers. METHODS: SWIR-emitting rare-earth-doped albumin nanocomposites (ReANCs) were synthesized using controlled coacervation, with visible light-emitting fluorophores additionally incorporated during the crosslinking phase for validation purposes. Specifically, HoANCs, ErANCs, and TmANCs were co-labeled with rhodamine-B, FITC, and Alexa Fluor 647 dyes respectively. These Rh-HoANCs, FITC-ErANCs, and 647-TmANCs were further conjugated with the targeting ligands daidzein, AMD3100, and folic acid respectively. Binding specificities of each nanoprobe to distinct cellular subsets were established by in vitro uptake studies. Quantitative whole-body SWIR imaging of subcutaneous tumor bearing mice was used to validate the in vivo targeting ability of these nanoprobes. RESULTS: Each of the three ligand-functionalized nanoprobes showed significantly higher uptake in the targeted cell line compared to untargeted probes. Increased accumulation of tumor-specific nanoprobes was also measured relative to untargeted probes in subcutaneous tumor models of breast (4175 and MCF-7) and ovarian cancer (SKOV3). Preferential accumulation of tumor-specific nanoprobes was also observed in tumors overexpressing targeted biomarkers in mice bearing molecularly-distinct bilateral subcutaneous tumors, as evidenced by significantly higher signal intensities on SWIR imaging. CONCLUSIONS: The results from this study show that tumors can be detected in vivo using a set of targeted multispectral SWIR-emitting nanoprobes. Significantly, these nanoprobes enabled imaging of biomarkers in mice bearing bilateral tumors with distinct molecular phenotypes. The findings from this study provide a foundation for optical molecular imaging of heterogeneous tumors and for studying the response of these complex lesions to targeted therapy.

Gene silencing of HIF-2α disrupts glioblastoma stem cell phenotype.

AIM: Improved treatment strategies are desperately needed for eradicating cancer stem cells (CSCs), which drive malignancy and recurrence in glioblastoma multiforme. Hypoxic regions within the tumor microenvironment help maintain and promote the proliferation of CSCs. Here, we explored the effects of silencing hypoxia inducible factor-2α (HIF-2α) because of its specificity for CSCs within the hypoxic environment. METHODS: Cancer stem cell neurospheres were formed by enriching from both the glioblastoma cell line U87 and from brain tumor stem cells isolated directly from human brain tumors. Silencing of human HIF-2α was performed using both commercial and in-house transfection of a validated short interfering RNA, with all results compared to an established non-silencing control short interfering RNA. Silencing of HIF-2α was established by Western blotting, and phenotypic effects were assayed by cell migration assays, cell viability measurements, and immunofluorescence staining of differentiation markers. RESULTS: Transfection with either our previously reported pH-sensitive, cationic amphiphilic macromolecule-based delivery system or Lipofectamine was similarly effective in silencing HIF-2α. The chemotherapeutic resistance and neurosphere formation were reduced when HIF-2α was silenced. Migratory capacities in the presence of macrophage conditioned media were modulated. HIF-2α silencing was complementary to temozolomide treatment in producing phenotypic rather than cytotoxic effects. CONCLUSION: HIF-2α silencing under hypoxia inhibited CSC phenotypes while promoting differentiated cell phenotypes and is complementary to existing DNA alkylating treatments in inhibiting glioma CSC activity.

Multiscale optical imaging of rare-earth-doped nanocomposites in a small animal model.

Rare-earth-doped nanocomposites have appealing optical properties for use as biomedical contrast agents, but few systems exist for imaging these materials. We describe the design and characterization of (i) a preclinical system for whole animal in vivo imaging and (ii) an integrated optical coherence tomography/confocal microscopy system for high-resolution imaging of ex vivo tissues. We demonstrate these systems by administering erbium-doped nanocomposites to a murine model of metastatic breast cancer. Short-wave infrared emissions were detected in vivo and in whole organ imaging ex vivo. Visible upconversion emissions and tissue autofluorescence were imaged in biopsy specimens, alongside optical coherence tomography imaging of tissue microstructure. We anticipate that this work will provide guidance for researchers seeking to image these nanomaterials across a wide range of biological models.

Crosstalk between M2 macrophages and glioma stem cells.

PURPOSE: Given its extremely poor prognosis, there is a pressing need for an improved understanding of the biology of glioblastoma multiforme (GBM), including the roles of tumor subpopulations that may contribute to their growth rate and therapy resistance. The most malignant phenotypes of GBM have been ascribed to the presence of subpopulations of cancer stem cells (CSCs), which are resistant to chemotherapeutic drugs and ionizing radiation and which promote invasiveness and metastasis. The mechanisms by which the CSC state is obtained and by which it promotes tumor maintenance are only beginning to emerge. We hypothesize that M2 polarized macrophages may affect CSC phenotypes via cell-cell communication. METHODS: We investigated the interplay between glioma CSCs and macrophages via co-culture. The invasiveness of CSCs in the absence and presence of macrophages was assessed using collagen degradation and Transwell migration assays. The role of STAT3 as a CSC phenotypic mediator was assessed using siRNA-mediated gene silencing. RESULTS: We found that the levels of a M2 macrophage-specific secreted cytokine, TGF-ß1, were elevated in the presence of CSCs, regardless of whether the cells were plated as contacting or non-contacting co-cultures. In addition, we found that the co-culture resulted in enhanced expression of M2 markers in macrophages that were previously polarized to the M1 phenotype. siRNA-mediated STAT3 silencing was found to reduce the chemo-responsiveness and migratory abilities of the CSCs. Combination treatment of STAT3 siRNA and DNA alkylating agents was found to further abrogate CSC functions. CONCLUSIONS: Our data indicate that the co-culture of CSCs and macrophages results in bi-directional signaling that alters the phenotypes of both cell types. These results provide an explanation for recently observed effects of macrophages on GBM tumor cell growth, motility and therapeutic resistance, and suggest potential therapeutic strategies to disrupt the CSC phenotype by impairing its communication with macrophages.

Surveillance nanotechnology for multi-organ cancer metastases.

The identification and molecular profiling of early metastases remains a major challenge in cancer diagnostics and therapy. Most in vivo imaging methods fail to detect small cancerous lesions, a problem that is compounded by the distinct physical and biological barriers associated with different metastatic niches. Here, we show that intravenously injected rare-earth-doped albumin-encapsulated nanoparticles emitting short-wave infrared light (SWIR) can detect targeted metastatic lesions in vivo, allowing for the longitudinal tracking of multi-organ metastases. In a murine model of basal human breast cancer, the nanoprobes enabled whole-body SWIR detection of adrenal gland microlesions and bone lesions that were undetectable via contrast-enhanced magnetic resonance imaging (CE-MRI) as early as, respectively, three weeks and five weeks post-inoculation. Whole-body SWIR imaging of nanoprobes functionalized to differentially target distinct metastatic sites and administered to a biomimetic murine model of human breast cancer resolved multi-organ metastases that showed varied molecular profiles at the lungs, adrenal glands and bones. Real-time surveillance of lesions in multiple organs should facilitate pre-therapy and post-therapy monitoring in preclinical settings.

Line-scanning confocal microscopy for high-resolution imaging of upconverting rare-earth-based contrast agents.

Rare-earth (RE) doped nanocomposites emit visible luminescence when illuminated with continuous wave near-infrared light, making them appealing candidates for use as contrast agents in biomedical imaging. However, the emission lifetime of these materials is much longer than the pixel dwell times used in scanning intravital microscopy. To overcome this limitation, we have developed a line-scanning confocal microscope for high-resolution, optically sectioned imaging of samples labeled with RE-based nanomaterials. Instrument performance is quantified using calibrated test objects. NaYF4 : Er,Yb nanocomposites are imaged in vitro, and in ex vivo tissue specimens, with direct comparison to point-scanning confocal microscopy. We demonstrate that the extended pixel dwell time of line-scanning confocal microscopy enables subcellular-level imaging of these nanomaterials while maintaining optical sectioning. The line-scanning approach thus enables microscopic imaging of this emerging class of contrast agents for preclinical studies, with the potential to be adapted for real-time in vivo imaging in the clinic.

CXCR-4 Targeted, Short Wave Infrared (SWIR) Emitting Nanoprobes for Enhanced Deep Tissue Imaging and Micrometastatic Cancer Lesion Detection.

Realizing the promise of precision medicine in cancer therapy depends on identifying and tracking cancerous growths to maximize treatment options and improve patient outcomes. This goal of early detection remains unfulfilled by current clinical imaging techniques that fail to detect lesions due to their small size and suborgan localization. With proper probes, optical imaging techniques can overcome this by identifying the molecular phenotype of tumors at both macroscopic and microscopic scales. In this study, the first use of nanophotonic short wave infrared technology is proposed to molecularly phenotype small lesions for more sensitive detection. Here, human serum albumin encapsulated rare-earth nanoparticles (ReANCs) with ligands for targeted lesion imaging are designed. AMD3100, an antagonist to CXCR4 (a classic marker of cancer metastasis) is adsorbed onto ReANCs to form functionalized ReANCs (fReANCs). fReANCs are able to preferentially accumulate in receptor positive lesions when injected intraperitoneally in a subcutaneous tumor model. fReANCs can also target subtissue microlesions at a maximum depth of 10.5 mm in a lung metastatic model of breast cancer. Internal lesions identified with fReANCs are 2.25 times smaller than those detected with ReANCs. Thus, an integrated nanoprobe detection platform is presented, which allows target-specific identification of subtissue cancerous lesions.

Targeting tumor metastases: Drug delivery mechanisms and technologies.

Primary sites of tumor are the focal triggers of cancers, yet it is the subsequent metastasis events that cause the majority of the morbidity and mortality. Metastatic tumor cells exhibit a phenotype that differs from that of the parent cells, as they represent a resistant, invasive subpopulation of the original tumor, may have acquired additional genetic or epigenetic alterations under exposure to prior chemotherapeutic or radiotherapeutic treatments, and reside in a microenvironment differing from that of its origin. This combination of resistant phenotype and distal location make tracking and treating metastases particularly challenging. In this review, we highlight some of the unique biological traits of metastasis, which in turn, inspire emerging strategies for targeted imaging of metastasized tumors and metastasis-directed delivery of therapeutics.

Delivery of antisense oligonucleotides using poly(alkylene oxide)-poly(propylacrylic acid) graft copolymers in conjunction with cationic liposomes.

The clinical application of gene silencing is hindered by poor stability and low delivery efficiency of naked oligonucleotides. Here, we present the in vitro and in vivo behaviors of a rationally designed, ternary, self-assembled nanoparticle complex, consisting of an anionic copolymer, cationic DOTAP liposome, and antisense oligonucleotide (AON). The multifunctional copolymers are based on backbone poly(propylacrylic acid) (PPAA), a pH-sensitive hydrophobic polymer, with grafted poly(alkylene oxides) (PAOs) varying in extent of grafting and PAO chemistry. The nanoparticle complexes with PPAA-g-PAO copolymers enhance antisense gene silencing effects in A2780 human ovarian cancer cells. A greater amount of AON is delivered to ovarian tumor xenografts using the ternary copolymer-stabilized delivery system, compared to a binary DOTAP/AON complex, following intraperitoneal injection in mice. Further, intratumoral injection of the nanoparticle complexes containing 1 mol% grafted PAO reduced tumoral bcl-2 expression by up to 60%. The data for complexes across the set of PAO polymers support a strong role for the hydrophilic-lipophilic balance of the graft copolymer in achieving serum stability and cellular uptake. Based upon these results, we anticipate that this novel nanoparticle delivery system can be extended to the delivery of plasmid DNA, siRNA, or aptamers for preclinical and clinical development.

Cationic amphiphilic macromolecule (CAM)-lipid complexes for efficient siRNA gene silencing.

The accumulated evidence has shown that lipids and polymers each have distinct advantages as carriers for siRNA delivery. Composite materials comprising both lipids and polymers may present improved properties that combine the advantage of each. Cationic amphiphilic macromolecules (CAMs) containing a hydrophobic alkylated mucic acid segment and a hydrophilic poly(ethylene glycol) (PEG) tail were non-covalently complexed with two lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), to serve as a siRNA delivery vehicle. By varying the weight ratio of CAM to lipid, cationic complexes with varying compositions were obtained in aqueous media and their properties evaluated. CAM-lipid complex sizes were relatively independent of composition, ranging from 100 to 200nm, and zeta potentials varied from 10 to 30mV. Transmission electron microscopy confirmed the spherical morphology of the complexes. The optimal N/P ratio was 50 as determined by electrophoretic mobility shift assay. The ability to achieve gene silencing was evaluated by anti-luciferase siRNA delivery to a U87-luciferase cell line. Several weight ratios of CAM-lipid complexes were found to have similar delivery efficiency compared to the gold standard, Lipofectamine. Isothermal titration calorimetry revealed that siRNA binds more tightly at pH=7.4 than pH=5 to CAM-lipid (1:10 w/w). Further intracellular trafficking studies monitored the siRNA escape from the endosomes at 24h following transfection of cells. The findings in the paper indicate that CAM-lipid complexes can serve as a novel and efficient siRNA delivery vehicle.

Multifunctional albumin nanoparticles as combination drug carriers for intra-tumoral chemotherapy.

Current cancer therapies are challenged by weakly soluble drugs and by drug combinations that exhibit non-uniform biodistribution and poor bioavailability. In this study, we have presented a new platform of advanced healthcare materials based on albumin nanoparticles (ANPs) engineered as tumor penetrating, delivery vehicles of combinatorially applied factors to solid tumors. These materials were designed to overcome three sequential key barriers: tissue level transport across solid tumor matrix; uptake kinetics into individual cancer cells; therapeutic resistance to single chemotherapeutic drugs. The ANPs were designed to penetrate deeper into solid tumor matrices using collagenase decoration and evaluated using a three-dimensional multicellular melanoma tumor spheroid model. Collagenase modified ANPs exhibited 1-2 orders of magnitude greater tumor penetration than unmodified ANPs into the spheroid mass after 96 hours, and showed preferential uptake into individual cancer cells for smaller sized ANPs (<100 nm). For enhanced efficacy, collagenase coated ANPs were modified with two therapeutic agents, curcumin and riluzole, with complementary mechanisms of action for combined cell cycle arrest and apoptosis in melanoma. The collagenase coated, drug loaded nanoparticles induced significantly more cell death within 3-D tumor models than the unmodified, dual drug loaded ANP particles and the kinetics of cytotoxicity was further influenced by the ANP size. Thus, multifunctional nanoparticles can be imbued with complementary size and protease activity features that allow them to penetrate solid tumors and deliver combinatorial therapeutic payload with enhanced cancer cytotoxicity but minimal collateral damage to healthy primary cells.

Delivery of siRNA silencing Runx2 using a multifunctional polymer-lipid nanoparticle inhibits osteogenesis in a cell culture model of heterotopic ossification.

Heterotopic ossification (HO) associated with traumatic neurological or musculoskeletal injuries remains a major clinical challenge. One approach to understanding better and potentially treating this condition is to silence one or more genes believed to be responsible for osteogenesis by small interfering RNA (siRNA) post-injury. Improved methods of delivering siRNA to myoprogenitor cells as well as relevant cell culture models of HO are needed to advance this approach. We utilize a model of HO featuring C2C12 myoprogenitor cells stimulated to the osteogenic phenotype by addition of BMP-2. For siRNA delivery, we utilize a nanocomposite consisting of DOTAP-based cationic liposomes coated with a graft copolymer of poly(propylacrylic acid) grafted with polyetheramine (Jeffamine), as this system has been shown previously to deliver antisense oligonucleotides safely into cells and out of endosomes for gene silencing in vitro and in vivo. Delivery of siRNA targeting Runx2, a transcription factor downstream of BMP-2, to stimulated C2C12 cells produced greater than 60% down-regulation of the Runx2 gene. This level of gene silencing was sufficient to inhibit alkaline phosphatase activity over the course of several days and calcium phosphate deposition over the course of 2 weeks. These results show the utility of the BMP-2/C2C12 model for capturing the cellular cell-fate decision in HO. Further, they suggest DOTAP/PPAA-g-Jeffamine as a promising delivery system for siRNA-based therapy for HO.

Nanoscale drug delivery systems for enhanced drug penetration into solid tumors: current progress and opportunities.

Poor penetration of anticancer drags into solid tumors significantly limits their efficacy. This phenomenon has long been observed for small-molecule chemotherapeutics, and it can be even more pronounced for nanoscale therapies. Nanoparticles have enormous potential for the treatment of cancer due to their wide applicability as drug delivery and imaging vehicles and their size-dependent accumulation into solid tumors by the enhanced permeability and retention (EPR) effect. Further, synthetic nanoparticles can be engineered to overcome barriers to drag delivery. Despite their promise for the treatment of cancer, relatively little work has been done to study and improve their ability to diffuse into solid tumors following passive accumulation in the tumor vasculature. In this review, we present the complex issues governing efficient penetration of nanoscale therapies into solid tumors. The current methods available to researchers to study nanoparticle penetration into malignant tumors are described, and the most recent works studying the penetration of nanoscale materials into solid tumors are summarized. We conclude with an overview of the important nanoparticle design parameters governing their tumor penetration, as well as by highlighting critical directions in this field.

Poly(alkylene oxide) copolymers for nucleic acid delivery.

The advancement of gene-based therapeutics to the clinic is limited by the ability to deliver physiologically relevant doses of nucleic acids to target tissues safely and effectively. Over the last couple of decades, researchers have successfully employed polymer and lipid based nanoassemblies to deliver nucleic acids for the treatment of a variety of diseases. Results of phase I/II clinical studies to evaluate the efficacy and biosafety of these gene delivery vehicles have been encouraging, which has promoted the design of more efficient and biocompatible systems. Research has focused on designing carriers to achieve biocompatibility, stability in the circulatory system, biodistribution to target the disease site, and intracellular delivery, all of which enhance the resulting therapeutic effect. The family of poly(alkylene oxide) (PAO) polymers includes random, block, and branched structures, among which the ABA type triblocks copolymers of ethylene oxide (EO) and propylene oxide (PO) (commercially known as Pluronic) have received the greatest consideration. In this Account, we highlight examples of polycation-PAO conjugates, liposome-PAO formulations, and PAO micelles for nucleic acid delivery. Among the various polymer design considerations, which include molecular weight of polymer, molecular weight of blocks, and length of blocks, the overall hydrophobic-lipophilic balance (HLB) is a critical parameter in defining the behavior of the polymer conjugates for gene delivery. We discuss the effects of varying this parameter in the context of improving gene delivery processes, such as serum stability and association with cell membranes. Other innovative macromolecular modifications discussed in this category include our work to enhance the serum stability and efficiency of lipoplexes using PAO graft copolymers, the development of a PAO gel-based carrier for sustained and stimuli responsive delivery, and the development of biodegradable PAO-based amphiphilic block copolymers.

Efficient intracellular siRNA delivery by ethyleneimine-modified amphiphilic macromolecules.

New materials that can bind and deliver oligonucleotides such as short interfering RNA (siRNA) without toxicity are greatly needed to fulfill the promise of therapeutic gene silencing. Amphiphilic macromolecules (AMs) were functionalized with linear ethyleneimines to create cationic AMs capable of complexing with siRNA. Structurally, the parent AM is formed from a mucic acid backbone whose tetra-hydroxy groups are alkylated with 12-carbon aliphatic chains to form the hydrophobic component of the macromolecule. This alkylated mucic acid is then mono-functionalized with poly(ethylene glycol) (PEG) as a hydrophilic component. The resulting AM contains a free carboxylic acid within the hydrophobic domain. In this work, linear ethyleneimines were conjugated to the free carboxylic acid to produce an AM with one primary amine (1N) or one primary amine and four secondary amines (5N). Further, an AM with amine substitution both to the free carboxylic acid in the hydrophobic domain and also to the adjacent PEG was synthesized to produce a polymer with one primary amine and eight secondary amines (9N), four located on each side of the AM hydrophobic domain. All amine-functionalized AMs formed nanoscale micelles but only the 5N and 9N AMs had cationic zeta potentials, which increased with increasing number of amines. All AMs exhibited less inherent cytotoxicity than linear polyethyleneimine (L-PEI) at concentrations of 10 µM and above. By increasing the length of the cationic ethyleneimine chain and the total number of amines, successful siRNA complexation and cellular siRNA delivery was achieved in a malignant glioma cell line. In addition, siRNA-induced silencing of firefly luciferase was observed using complexes of siRNA with the 9N AM and comparable to L-PEI, yet showed better cell viability at higher concentrations (above 10 µM). This work highlights the promise of cationic AMs as safe and efficient synthetic vectors for siRNA delivery. Specifically, a novel polymer (9N) was identified for efficient siRNA delivery to cancer cells and will be further evaluated.

Binding and transport of PAMAM-RGD in a tumor spheroid model: the effect of RGD targeting ligand density.

The mechanisms governing the efficient tumor spheroid penetration and transport by poly(amidoamine) (PAMAM) dendrimers displaying varying numbers of cyclic RGD targeting peptides (2, 3, 7, or 10) were evaluated in this work. The cell-free binding affinities and cellular internalization kinetics of PAMAM-RGD conjugates to malignant glioma cells were determined experimentally, and the results were incorporated into a mathematical model to predict the transport of these materials through a multicellular tumor spheroid. The theoretical analysis demonstrated that greater RGD crosslinking may improve transport through tumor spheroids due to their decreased integrin-binding affinity. This study provides evidence that altering the density of tumor-targeting ligands from a drug delivery platform is a feasible way to optimize the tumor-penetration efficiency of an anticancer agent, and provides insight into the physicochemical mechanisms governing the relative effectiveness of these conjugates.

Mathematical modeling to distinguish cell cycle arrest and cell killing in chemotherapeutic concentration response curves.

Concentration response experiments are utilized widely to characterize the response of tumor cell lines to chemotherapeutic drugs, but the assay methods are non-standardized and their analysis based on phenomenological equations. To provide a framework for better interpretation of these experiments, we have developed a mathematical model in which progression through the tumor cell cycle is inhibited by drug treatment via either cell cycle arrest or entrance into cell death pathways. By fitting concentration response data, preferably over a dynamic range, the contributions of these mechanisms can be delineated. The model was shown to fit well experimental data for three glioma cell lines treated with either carmustine or etoposide. In each cell line, the major mechanism of tumor cell inhibition was cell death for carmustine in contrast to cell cycle arrest for etoposide. The model also provides a possible interpretation for the acquired in vitro resistance of U87 cells to carmustine as an accelerated desensitization to cell killing effects. This approach will aid in understanding better the action of chemotherapeutic agents on tumor cells.

Analysis of amino acid supplementation effects on hepatocyte cultures using flux balance analysis.

When cultured hepatocytes are exposed to challenging environments such as plasma, they frequently suffer a decline in liver-specific functions. Media supplements are sought to reduce or eliminate this effect. A rational design approach for amino acid supplementation in hepatocyte culture has been developed in our prior work, and designed amino acid supplementation (DAA) was found to increase urea and albumin production. To fully characterize the metabolic state of hepatocytes under different amino acid supplementations, a number of metabolite measurements are performed in this work and used in a metabolic network flexibility analysis framework including thermodynamic constraints to determine the range of values for the intracellular fluxes. A metabolic objective prediction model is used to infer the metabolic objectives of the hepatocytes and to quantify the intracellular flux distribution for three different amino acid supplementations. The results illustrate that DAA leads to greater fluxes in the tricarboxylic acid cycle (TCA) cycle, urea cycle, and fatty acid oxidation concomitant with lower fluxes in intracellular lipid metabolism compared with empirical amino acid and no amino acid supplementation for hepatocytes during plasma exposure. It is also found that hepatocytes exhibit flexibility in their metabolic objectives depending on the composition of the amino acid supplementations. By incorporating both experimental data and thermodynamic constraints into the mathematical model, the proposed approach leads to identification of metabolic objectives and characterization of fluxes' variability and pathway changes due to different cultured conditions.