Veronica Estrella

Acidity generated by the tumor microenvironment drives local invasion

The pH of solid tumors is acidic due to increased fermentative metabolism and poor perfusion. It has been hypothesized that acid pH promotes local invasive growth and metastasis. The hypothesis that acid mediates invasion proposes that H(+) diffuses from the proximal tumor microenvironment into adjacent normal tissues where it causes tissue remodeling that permits local invasion. In the current work, tumor invasion and peritumoral pH were monitored over time using intravital microscopy. In every case, the peritumoral pH was acidic and heterogeneous and the regions of highest tumor invasion corresponded to areas of lowest pH. Tumor invasion did not occur into regions with normal or near-normal extracellular pH. Immunohistochemical analyses revealed that cells in the invasive edges expressed the glucose transporter-1 and the sodium-hydrogen exchanger-1, both of which were associated with peritumoral acidosis. In support of the functional importance of our findings, oral administration of sodium bicarbonate was sufficient to increase peritumoral pH and inhibit tumor growth and local invasion in a preclinical model, supporting the acid-mediated invasion hypothesis. Cancer Res; 73(5); 1524-35. ©2012 AACR.

Multiscale Modelling of Vascular Tumour Growth in 3D: The Roles of Domain Size and Boundary Conditions

We investigate a three-dimensional multiscale model of vascular tumour growth, which couples blood flow, angiogenesis, vascular remodelling, nutrient/growth factor transport, movement of, and interactions between, normal and tumour cells, and nutrient-dependent cell cycle dynamics within each cell. In particular, we determine how the domain size, aspect ratio and initial vascular network influence the tumours growth dynamics and its long-time composition. We establish whether it is possible to extrapolate simulation results obtained for small domains to larger ones, by constructing a large simulation domain from a number of identical subdomains, each subsystem initially comprising two parallel parent vessels, with associated cells and diffusible substances. We find that the subsystem is not representative of the full domain and conclude that, for this initial vessel geometry, interactions between adjacent subsystems contribute to the overall growth dynamics. We then show that extrapolation of results from a small subdomain to a larger domain can only be made if the subdomain is sufficiently large and is initialised with a sufficiently complex vascular network. Motivated by these results, we perform simulations to investigate the tumours response to therapy and show that the probability of tumour elimination in a larger domain can be extrapolated from simulation results on a smaller domain. Finally, we demonstrate how our model may be combined with experimental data, to predict the spatio-temporal evolution of a vascular tumour.

Cotreatment with vorinostat (suberoylanilide hydroxamic acid) enhances activity of dasatinib (BMS-354825) against imatinib mesylate-sensitive or imatinib mesylate-resistant chronic myelogenous leukemia cells

Purpose: We determined the effects of vorinostat [suberoylanilide hydroxamic acid (SAHA)] and/or dasatinib, a dual Abl/Src kinase (tyrosine kinase) inhibitor, on the cultured human (K562 and LAMA-84) or primary chronic myelogenous leukemia (CML) cells, as well as on the murine pro-B BaF3 cells with ectopic expression of the unmutated and kinase domain-mutant forms of Bcr-Abl. Experimental Design: Following exposure to dasatinib and/or vorinostat, apoptosis, loss of clonogenic survival, as well as the activity and levels of Bcr-Abl and its downstream signaling proteins were determined. Results: Treatment with dasatinib attenuated the levels of autophosphorylated Bcr-Abl, p-CrkL, phospho-signal transducer and activator of transcription 5 (p-STAT5), p-c-Src, and p-Lyn; inhibited the activity of Lyn and c-Src; and induced apoptosis of the cultured CML cells. Combined treatment of cultured human CML and BaF3 cells with vorinostat and dasatinib induced more apoptosis than either agent alone, as well as synergistically induced loss of clonogenic survival, which was associated with greater depletion of Bcr-Abl, p-CrkL, and p-STAT5 levels. Cotreatment with dasatinib and vorinostat also attenuated the levels of Bcr-AblE255K and Bcr-AblT315I and induced apoptosis of BaF3 cells with ectopic expression of the mutant forms of Bcr-Abl. Finally, cotreatment of the primary CML cells with vorinostat and dasatinib induced more loss of cell viability and depleted Bcr-Abl or Bcr-AblT315I, p-STAT5, and p-CrkL levels than either agent alone. Conclusions: As shown here, the preclinical in vitro activity of vorinostat and dasatinib against cultured and primary CML cells supports the in vivo testing of the combination in imatinib mesylate–sensitive and imatinib mesylate–resistant CML cells.

Hyaluronic Acid Synthase-1 Expression Regulates Bladder Cancer Growth, Invasion, and Angiogenesis through CD44

Hyaluronic acid (HA) promotes tumor metastasis and is an accurate diagnostic marker for bladder cancer. HA is synthesized by HA synthases HAS1, HAS2, or HAS3. We have previously shown that HAS1 expression in tumor tissues is a predictor of bladder cancer recurrence and treatment failure. In this study, we stably transfected HT1376 bladder cancer cells with HAS1-sense (HAS1-S), HAS1-antisense (HAS1-AS), or vector cDNA constructs. Whereas HAS1-S transfectants produced approximately 1.7-fold more HA than vector transfectants, HA production was reduced by approximately 70% in HAS1-AS transfectants. HAS1-AS transfectants grew 5-fold slower and were approximately 60% less invasive than vector and HAS1-S transfectants. HAS1-AS transfectants were blocked in G(2)-M phase of the cell cycle due to down-regulation of cyclin B1, cdc25c, and cyclin-dependent kinase 1 levels. These transfectants were also 5- to 10-fold more apoptotic due to the activation of the Fas-Fas ligand-mediated extrinsic pathway. HAS1-AS transfectants showed a approximately 4-fold decrease in ErbB2 phosphorylation and down-regulation of CD44 variant isoforms (CD44-v3, CD44-v6, and CD44-E) both at the protein and mRNA levels. However, no decrease in RHAMM levels was observed. The decrease in CD44-v mRNA levels was not due to increased mRNA degradation. Whereas CD44 small interfering RNA (siRNA) transfection decreased cell growth and induced apoptosis in HT1376 cells, HA addition modestly increased CD44 expression and cell growth in HAS1-AS transfectants, which could be blocked by CD44 siRNA. In xenograft studies, HAS1-AS tumors grew 3- to 5-fold slower and had approximately 4-fold lower microvessel density. These results show that HAS1 regulates bladder cancer growth and progression by modulating HA synthesis and HA receptor levels.

Chronic acidosis in the tumour microenvironment selects for overexpression of LAMP2 in the plasma membrane

Early cancers are avascular and hence, profoundly acidic. Pre-malignant cells must adapt to acidosis to thrive in this hostile microenvironment. Here, we investigate MCF-7 cells that are adapted to grow in acidic conditions using SILAC proteomics and we reveal a significant upregulation of lysosomal proteins. Prominent among these is LAMP2 that functions to protect lysosomal membranes from acid proteolysis. LAMP2 upregulation by acidosis is confirmed both in vitro and in vivo. Furthermore, we show that the depletion of LAMP2 is sufficient to increase acidosis-mediated toxicity. In breast cancer patient samples, there is a high correlation of LAMP2 mRNA and protein expression with progression. We also observe that LAMP2 is located at the plasma membrane in clinical samples and this redistribution is acid-induced in vitro. Our findings suggest a potential adaptive mechanism, wherein cells chronically exposed to an acidic environment translocate lysosomal proteins to their surface, thus protecting the plasmalemma from acid-induced hydrolysis.

The Role of Tumor Tissue Architecture in Treatment Penetration and Efficacy: An Integrative Study

Despite the great progress that has been made in understanding cancer biology and the potential molecular targets for its treatment, the majority of drugs fail in the clinical trials. This may be attributed (at least in part) to the complexity of interstitial drug transport in the patient’s body, which is hard to test experimentally. Similarly, recent advances in molecular imaging have led to the development of targeted biomarkers that can predict pharmacological responses to therapeutic interventions. However, both the drug and biomarker molecules need to access the tumor tissue and be taken up into individual cells in concentrations sufficient to exert the desired effect. To investigate the process of drug penetration at the mesoscopic level we developed a computational model of interstitial transport that incorporates the biophysical properties of the tumor tissue, including its architecture and interstitial fluid flow, as well as the properties of the agents. This model is based on the method of regularized Stokeslets to describe the fluid flow coupled with discrete diffusion-advection-reaction equations to model the dynamics of the drugs. Our results show that the tissue cellular porosity and density influence the depth of penetration in a non-linear way, with sparsely packed tissues being traveled through more slowly than the denser tissues. We demonstrate that irregularities in the cell spatial configurations result in the formation of interstitial corridors that are followed by agents leading to the emergence of tissue zones with less exposure to the drugs. We describe how the model can be integrated with in vivo experiments to test the extravasation and penetration of the targeted biomarkers through the tumor tissue. A better understanding of tissue- or compound-specific factors that limit the penetration through the tumors is important for non-invasive diagnoses, chemotherapy, the monitoring of treatment responses, and the detection of tumor recurrence.

Free Base Lysine Increases Survival and Reduces Metastasis in Prostate Cancer Model

BACKGROUND Malignant tumor cells typically metabolize glucose anaerobically to lactic acid even under normal oxygen tension, a phenomenon called aerobic glycolysis or the Warburg effect. This results in increased acid production and the acidification of the extracellular microenvironment in solid tumors. H+ ions tend to flow along concentration gradients into peritumoral normal tissue causing extracellular matrix degradation and increased tumor cell motility thus promoting invasion and metastasis. We have shown that reducing this acidity with sodium bicarbonate buffer decreases the metastatic fitness of circulating tumor cells in prostate cancer and other cancer models. Mathematical models of the tumor-host dynamics predicted that buffers with a pka around 7 will be more effective in reducing intra- and peri-tumoral acidosis and, thus, and possibly more effective in inhibiting tumor metastasis than sodium bicarbonate which has a pKa around 6. Here we test this prediction the efficacy of free base lysine; a non-bicarbonate/non-volatile buffer with a higher pKa (~10), on prostate tumor metastases model. METHODS Oxygen consumption and acid production rate of PC3M prostate cancer cells and normal prostate cells were determined using the Seahorse Extracellular Flux (XF-96) analyzer. In vivo effect of 200 mM lysine started four days prior to inoculation on inhibition of metastasis was examined in PC3M-LUC-C6 prostate cancer model using SCID mice. Metastases were followed by bioluminescence imaging. RESULTS PC3M prostate cancer cells are highly acidic in comparison to a normal prostate cell line indicating that reduction of intra- and perit-tumoral acidosis should inhibit metastases formation. In vivo administration of 200 mM free base lysine increased survival and reduced metastasis. CONCLUSION PC3M prostate cancer cells are highly glycolytic and produce large amounts of acid when compared to normal prostate cells. Administration of non-volatile buffer decreased growth of metastases and improved survival indicating acidity plays a significant role in growth and invasion in-vivo.

Intracellular Electric Field and pH Optimize Protein Localization and Movement

Mammalian cell function requires timely and accurate transmission of information from the cell membrane (CM) to the nucleus (N). These pathways have been intensively investigated and many critical components and interactions have been identified. However, the physical forces that control movement of these proteins have received scant attention. Thus, transduction pathways are typically presented schematically with little regard to spatial constraints that might affect the underlying dynamics necessary for protein-protein interactions and molecular movement from the CM to the N. We propose messenger protein localization and movements are highly regulated and governed by Coulomb interactions between: 1. A recently discovered, radially directed E-field from the NM into the CM and 2. Net protein charge determined by its isoelectric point, phosphorylation state, and the cytosolic pH. These interactions, which are widely applied in elecrophoresis, provide a previously unknown mechanism for localization of messenger proteins within the cytoplasm as well as rapid shuttling between the CM and N. Here we show these dynamics optimize the speed, accuracy and efficiency of transduction pathways even allowing measurement of the location and timing of ligand binding at the CM –previously unknown components of intracellular information flow that are, nevertheless, likely necessary for detecting spatial gradients and temporal fluctuations in ligand concentrations within the environment. The model has been applied to the RAF-MEK-ERK pathway and scaffolding protein KSR1 using computer simulations and in-vitro experiments. The computer simulations predicted distinct distributions of phosphorylated and unphosphorylated components of this transduction pathway which were experimentally confirmed in normal breast epithelial cells (HMEC).

In Vivo and in Silico Pharmacokinetics and Biodistribution of a Melanocortin Receptor 1 Targeted Agent in Preclinical Models of Melanoma

The melanocortin 1 receptor (MC1R) is overexpressed in most melanoma metastases, making it a promising target for imaging of melanomas. In this study, the expression of MC1R in a large fraction of patients with melanoma was confirmed using mRNA and tissue microarray. Here, we have characterized the in vivo tumor and tissue distribution and pharmacokinetics (PK) of uptake and clearance of a MC1R specific peptidomimetic ligand conjugated to a near-infrared fluorescent dye. We propose an interdisciplinary framework to bridge the different time and space scales of ligand-tumor-host interactions: intravital fluorescence microscopy to quantify probe internalization at the cellular level, a xenograft tumor model for whole body pharmacokinetics, and a computational pharmacokinetic model for integration and interpretation of experimental data. Administration of the probe into mice bearing tumors with high and low MC1R expression demonstrated normalized image intensities that correlated with expression levels (p < 0.05). The biodistribution study showed high kidney uptake as early as 30 min postinjection. The PK computational model predicted the presence of receptors in the kidneys with a lower affinity, but at higher numbers than in the tumors. As the mouse kidney is known to express the MC5R, this hypothesis was confirmed by both coinjection of a ligand with higher MC5R affinity compared to MC1R and by injection of lower probe concentrations (e.g., 1 nmol/kg), both leading to decreased kidney accumulation of the MC1R ligand. In addition, through this interdisciplinary approach we could predict the rates of ligand accumulation and clearance into and from organs and tumors, and the amount of injected ligand required to have maximum specific retention in tumors. These predictions have potential to aid in the translation of a targeted agent from lab to the clinic. In conclusion, the characterized MC1R-specific probe has excellent potential for in vivo detection of melanoma metastases. The process of cell-surface marker validation, targeted imaging probe development, and in vitro, in vivo, and in silico characterization described in this study can be generally applied to preclinical development of targeted agents.

Tumor Targeting and Pharmacokinetics of a Near-Infrared Fluorescent-Labeled δ-Opioid Receptor Antagonist Agent, Dmt-Tic-Cy5.

Fluorescence molecular imaging can be employed for the development of novel cancer targeting agents. Herein, we investigated the pharmacokinetics (PK) and cellular uptake of Dmt-Tic-Cy5, a delta-opioid receptor (δOR) antagonist-fluorescent dye conjugate, as a tumor-targeting molecular imaging agent. δOR expression is observed normally in the CNS, and pathologically in some tumors, including lung liver and breast cancers. In vitro, in vivo, and ex vivo experiments were conducted to image and quantify the fluorescence signal associated with Dmt-Tic-Cy5 over time using in vitro and intravital fluorescence microscopy and small animal fluorescence imaging of tumor-bearing mice. We observed specific retention of Dmt-Tic-Cy5 in tumors with maximum uptake in δOR-expressing positive tumors at 3 h and observable persistence for >96 h; clearance from δOR nonexpressing negative tumors by 6 h; and systemic clearance from normal organs by 24 h. Live-cell and intravital fluorescence microscopy demonstrated that Dmt-Tic-Cy5 had sustained cell-surface binding lasting at least 24 h with gradual internalization over the initial 6 h following administration. Dmt-Tic-Cy5 is a δOR-targeted agent that exhibits long-lasting and specific signal in δOR-expressing tumors, is rapidly cleared from systemic circulation, and is not retained in non-δOR-expressing tissues. Hence, Dmt-Tic-Cy5 has potential as a fluorescent tumor imaging agent.