Mathematical descriptions of Spodoptera frugiperda insect cell culture kinetics and recombinant protein yields

Vita.Optimal design and operation of bioreactors for insect cell culture is facilitated by functional relations providing quantitative information on metabolite consumption kinetics of the cells, as well as on the specific cell growth rates ($murmsb G$). We have investigated the effects of three important carbon sources, glucose, malate and glutamine, on the metabolism of Spodoptera frugiperda (Sf9) cells in batch suspension culture. Specific consumption rates of glucose, malate and oxygen were measured in infected and uninfected Sf9 cells grown in TNM-FH medium containing 7-35mM glucose, 0-16mM malate and 4-16mM glutamine. In addition, the effects of glucose, malate and glutamine on cell growth and recombinant $beta$-galactosidase productivity in Sf9 cells were analyzed. The initial specific glucose consumption rate (q$rmsb G$) of the uninfected cells was related to glucose, malate and glutamine concentrations by means of a modified Michaelis-Menten type equation treating malate as a "competitive inhibitor" and glutamine as a "non-competitive inhibitor" of q$rmsb G$. The initial specific malate consumption rate (q$rmsb M$) of uninfected cells was described by an equation treating both glucose and glutamine as "competitive inhibitors" of q$rmsb M$. All three carbon sources were found to increase $murmsb G$ in a saturable manner. The fitted value of the "Monod constant" for malate was more than an order of magnitude smaller than those for glucose and glutamine. The variation of initial specific oxygen consumption rate (q$rmsb{O2}$) in Sf9 cells was found to be related to $murmsb G$ in a manner consistent with the maintenance energy model. It was found that under typical culture conditions, about 15-20% of the cellular energy demand comes from functions not related to growth. Experiments conducted on Sf9 cells infected with a recombinant baculovirus ($beta$gal-AcMNPV) revealed that recombinant protein yields were lowered by the presence of high concentrations (16mM) of malate, while high medium glucose (35mM) and glutamine (16mM) content lead to a two-fold increase in the specific yield of $beta$-galactosidase. Based on this study, it is recommended that medium with high concentrations of glucose and glutamine, and no malate, be employed for fed-batch or repeated-batch cultivation of Sf9 cells and subsequent production of recombinant proteins

Tumor acidity, ion trapping, and chemotherapeutics. II. pH-Dependent partition coefficients predict importance of ion trapping on pharmacokinetics of weakly basic chemotherapeutic agents. Biochem Pharmacol 2003;66:1219–1229

Abstract Resistance to anti-cancer chemotherapies often leads to regional failure, and can be caused by biochemical and/or physiological mechanisms. Biochemical mechanisms include the overexpression of resistance-conferring proteins. In contrast, physiological resistance involves the tumor microenvironment, and can be caused by poor perfusion, hypoxia and/or acidity. This communication investigates the role of tumor acidity in resistance to a panel of chemotherapeutic agents commonly used against breast cancer, such as anthracyclines, taxanes, anti-metabolites and alkylating agents. The effects of pH on the cytotoxicity of these agents were determined, and ion trapping was confirmed by monitoring the effect of pH on the cellular uptake of radiolabeled anthracyclines. Furthermore, pH-dependent cytotoxicity and uptake were compared between parental drug sensitive MCF-7 cells and variants overexpressing p-glycoprotein (MDR-1) and Breast Cancer Resistance Protein. These data indicate that the magnitude of physiological resistance from pH-dependent ion trapping is comparable to biochemical resistance caused by overexpression of drug efflux pumps. Hence, microenvironment-based ion trapping is a significant barrier to anthracycline-based chemotherapy and can itself be a therapeutic target to enhance the efficacy of existing chemotherapies.

Causes and Effects of Heterogeneous Perfusion in Tumors

A characteristic of solid tumors is their heterogeneous distribution of blood flow, with significant hypoxia and acidity in low-flow regions. We review effects of heterogeneous tumor perfusion are reviewed and propose a conceptual model for its cause. Hypoxic-acidic regions are resistant to chemo- and radiotherapy and may stimulate progression to a more metastatic phenotype. In normal tissues, hypoxia and acidity induce angiogenesis, which is expected to improve perfusion. However, aggressive tumors can have high local microvessel density simultaneously with significant regions of hypoxia and acidosis. A possible explanation for this apparent contradiction is that the mechanisms regulating growth and adaptation of vascular networks are impaired. According to a recent theory for structural adaptation of vascular networks, four interrelated adaptive responses can work as a self-regulating system to produce a mature and efficient blood distribution system in normal tissues. It is proposed that heterogeneous perfusion in tumors may result from perturbation of this system. Angiogenesis may increase perfusion heterogeneity in tumors by increasing the disparity between parallel low- and high-resistance flow pathways. This conceptual model provides a basis for future rational therapies. For example, it indicates that selective destruction of tumor vasculature may increase perfusion efficiency and improve therapeutic efficacy

Acute Metabolic Alkalosis Enhances Response of C3H Mouse Mammary Tumors to the Weak Base Mitoxantrone

Uptake of weak acid and weak base chemotherapeutic drugs by tumors is greatly influenced by the tumor extracellular/interstitial pH (pHe), the intracellular pH (pHi) maintained by the tumor cells, and by the ionization properties of the drug itself. The acid-outside plasmalemmal pH gradient in tumors acts to exclude weak base drugs like the anthracyclines, anthraquinones, and vinca alkaloids from the cells, leading to a substantial degree of “physiological drug resistance” in tumors. We have induced acute metabolic alkalosis in C3H tumor-bearing C3H/hen mice, by gavage and by intraperitoneal (i.p.) administration of NaHCO3. 31P magnetic resonance spectroscopic measurements of 3-aminopropylphosphonate show increases of up to 0.6 pH units in tumor pHe, and 0.2 to 0.3 pH units in hind leg tissue pHe, within 2 hours of i.p. administration of NaHCO3. Theoretical calculations of mitoxantrone uptake into tumor and normal (hind leg) tissue at the measured pH, and pHI values indicate that a gain in therapeutic index of up to 3.3-fold is possible with NaHCO3 pretreatment. Treatment of C3H tumor-bearing mice with 12 mg/kg mitoxantrone resulted in a tumor growth delay of 9 days, whereas combined NaHCO3mitoxantrone therapy resulted in an enhancement of the TGD to 16 days

The thioredoxin-1 inhibitor 1-methylpropyl 2-imidazolyl disulfide (PX-12) decreases vascular permeability in tumor xenografts monitored by dynamic contrast enhanced magnetic resonance Imaging

Purpose: The purpose of this study was to use dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to measure changes in tumor xenograft permeability produced by the antitumor thioredoxin-1 (Trx-1) inhibitor 1-methylpropyl 2-imidazolyl disulfide (PX-12) and to assess the relationship to Trx-1 and vascular endothelial growth factor (VEGF) levels. Experimental Design: DCE-MRI was used to monitor the dynamics of gadolinium-diethylenetriaminepentaacetic acid coupled bovine serum albumin as a macromolecular contrast reagent to measure hemodynamic changes in HT-29 human colon xenografts in immunodeficient mice treated with PX-12. Blood vessel permeability was estimated from the slope of the enhancement curves, and tumor vascular volume fraction from the ordinate. Tumor Trx-1 and VEGF was also measured. Results: PX-12 caused a rapid 63% decrease in the average tumor blood vessel permeability within 2 hours of administration. The decrease lasted 24 hours and had returned to pretreatment values by 48 hours. The changes in vascular permeability were not accompanied by alterations in average tumor vascular volume fraction. There was a decrease in tumor and tumor-derived VEGF in plasma at 24 hours after treatment with PX-12, but not at earlier time points. However, tumor redox active Trx-1 showed a rapid decline within 2 hours following PX-12 administration that was maintained for 24 hours. Conclusion: The rapid decrease in tumor vascular permeability caused by PX-12 administration coincided with a decrease in tumor redox active Trx-1 and preceded a decrease in VEGF. DCE-MRI responses to PX-12 in patients of Trx-1 inhibition at early time points and decreased VEGF at later times. may be useful to follow tumor response and even therapeutic benefit

Dynamic contrast-enhanced and diffusion MRI show rapid and dramatic changes in tumor microenvironment in response to inhibition of HIF-1 alpha using PX-478

PX-478 is a new agent known to inhibit the hypoxia-responsive transcription factor, HIF-1 alpha, in experimental tumors. The current study was undertaken in preparation for clinical trials to determine which noninvasive imaging endpoint(s) is sensitive to this drug's actions. Dynamic contrast-enhanced (DCE) and diffusion-weighted (DW) magnetic resonance imaging (MRI) were used to monitor acute effects on tumor hemodynamics and cellularity, respectively. Mice bearing human xenografts were treated either with PX-478 or vehicle, and imaged over time. DW imaging was performed at three b values to generate apparent diffusion coefficient of water (ADCw) maps. For DCE-MRI, a macromolecular contrast reagent, BSA-Gd-DTPA, was used to determine vascular permeability and vascular volume fractions. PX-478 induced a dramatic reduction in tumor blood vessel permeability within 2 hours after treatment, which returned to baseline by 48 hours. The anti-VEGF antibody, Avastin, reduced both the permeability and vascular volume. PX-478 had no effect on the perfusion behavior of a drug-resistant tumor system, A-549. Tumor cellularity, estimated from ADCw, was significantly decreased 24 and 36 hours after treatment. This is the earliest significant response of ADC to therapy yet reported. Based on these preclinical findings, both of these imaging endpoints will be included in the clinical trial of PX-478

Dynamic Contrast-Enhanced and Diffusion MRI Show Rapid and Dramatic Changes in Tumor Microenvironment in Response to Inhibition of HIF-1α Using PX-478

PX-478 is a new agent known to inhibit the hypoxia-responsive transcription factor, HIF-1α, in experimental tumors. The current study was undertaken in preparation for clinical trials to determine which noninvasive imaging endpoint(s) is sensitive to this drug's actions. Dynamic contrast-enhanced (DCE) and diffusion-weighted (DW) magnetic resonance imaging (MRI) were used to monitor acute effects on tumor hemodynamics and cellularity, respectively. Mice bearing human xenografts were treated either with PX-478 or vehicle, and imaged over time. DW imaging was performed at three b values to generate apparent diffusion coefficient of water (ADCw) maps. For DCE-MRI, a macromolecular contrast reagent, BSA-Gd-DTPA, was used to determine vascular permeability and vascular volume fractions. PX-478 induced a dramatic reduction in tumor blood vessel permeability within 2 hours after treatment, which returned to baseline by 48 hours. The anti-VEGF antibody, Avastin, reduced both the permeability and vascular volume. PX-478 had no effect on the perfusion behavior of a drug-resistant tumor system, A-549. Tumor cellularity, estimated from ADCw, was significantly decreased 24 and 36 hours after treatment. This is the earliest significant response of ADC to therapy yet reported. Based on these preclinical findings, both of these imaging endpoints will be included in the clinical trial of PX-478

Magnetic Resonance Imaging Identifies Differential Response to Pro-Oxidant Chemotherapy in a Xenograft Model

AbstractInduction of oxidative stress is a key component of cancer therapy. Pro-oxidant drugs have been demonstrated to enhance the efficacy of radiotherapy and chemotherapy. An emerging concept is that therapeutic outcomes are dictated by the differential redox buffering reserve in subpopulations of malignant cells, indicating the need for noninvasive biomarkers of tumor redox that can be used for dose identification and response assessment in a longitudinal setting. Magnetic resonance imaging (MRI) enhanced with the thiol-binding contrast agent Gd-LC6-SH, and hemodynamic response imaging (HRI) in combination with hypercapnia and hyperoxia were investigated as biomarkers of the pharmacodynamics of the small molecule pro-oxidant imexon (IMX). Human multiple myeloma cell lines 8226/S and an IMX-resistant variant, 8226/IM10, were established as contralateral tumors in SCID mice. T1slope, an MRI measure of the washout rate of Gd-LC6-SH, was significantly lower post-IMX therapy in 8226/S tumors compared with vehicle controls, indicating treatment-related oxidization of the tumor microenvironment, which was confirmed by analysis of tumor tissue for thiols. T1slope and ex vivo assays for thiols both indicated a more reduced microenvironment in 8226/IM10 tumors following IMX therapy. HRI with hypercapnia challenge revealed IMX inhibition of vascular dilation in 8226/S tumors but not 8226/IM10 tumors, consistent with decreased immunohistochemical staining for smooth muscle actin in treated 8226/S tumors. MRI enhanced with Gd-LC6-SH, and HRI coupled with a hypercapnic challenge provide noninvasive biomarkers of tumor response to the redox modulator imexon

Magnetic resonance imaging of RRx-001 pharmacodynamics in preclinical tumors

RRx-001 is an anticancer agent that subjects cancer cells to reactive oxygen/nitrogen species (ROS/RNS) and acts as an epigenetic modifier. We have used a thiol-bearing MRI contrast agent, Gd-LC7-SH, to investigate the pharmacodynamics of RRx-001 in CHP-100 Ewing's Sarcoma, HT-29 colorectal carcinoma, and PANC-1 pancreatic carcinoma xenografts in SCID mice. Binding of Gd-LC7-SH to the Cys34 residue on plasma albumin prolongs retention in the tumor microenvironment and increases tumor enhancement on MRI. Mice were imaged by MRI and in vivo T1 maps acquired 50 min (T1(50 min)) after injection of 0.05 mmol/kg Gd-LC7-SH (i.v.) at baseline and 1, 24, and 72 h post-treatment with 10 mg/kg RRx-001 (i.v.). Consistent with an indirect thiol-modifying activity of RRx-001, tumor T150 min at 1 h post-drug was significantly longer than pre-drug tumor T150 min in all three tumor models, with the T150 min remaining significantly longer than baseline through 72 h post-drug in the HT-29 and PANC-1 tumors. The T150 min of CHP-100 tumors recovered to baseline by 24 h post-drug, suggesting a robust anti-oxidant response to the RRx-001 challenge that was presaged by a marked increase in perfusion at 1 h post-drug measured by DCE-MRI. MRI enhanced with Gd-LC7-SH provides a mechanistically rational biomarker of RRx-001 pharmacodynamics.EpicentRx; National Institutes of Health [R01-CA118359, P30-CA023074]This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]