Tingan Chen

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.

Delta-Opioid Receptor (δOR) Targeted Near-Infrared Fluorescent Agent for Imaging of Lung Cancer: Synthesis and Evaluation In Vitro and In Vivo.

In the United States, lung cancer is the leading cause of cancer death and ranks second in the number of new cases annually among all types of cancers. Better methods or tools for diagnosing and treating this disease are needed to improve patient outcomes. The delta-opioid receptor (δOR) is reported to be overexpressed in lung cancers and not expressed in normal lung. Thus, we decided to develop a lung cancer-specific imaging agent targeting this receptor. We have previously developed a δOR-targeted fluorescent imaging agent based on a synthetic peptide antagonist (Dmt-Tic) conjugated to a Cy5 fluorescent dye. In this work, we describe the synthesis of Dmt-Tic conjugated to a longer wavelength near-infrared fluorescent (NIRF) dye, Li-cor IR800CW. Binding affinity of Dmt-Tic-IR800 for the δOR was studied using lanthanide time-resolved fluorescence (LTRF) competitive binding assays in cells engineered to overexpress the δOR. In addition, we identified lung cancer cell lines with high and low endogenous expression of the δOR. We confirmed protein expression in these cell lines using confocal fluorescence microscopy imaging and used this technique to estimate the cell-surface receptor number in the endogenously expressing lung cancer cell lines. The selectivity of Dmt-Tic-IR800 for imaging of the δOR in vivo was shown using both engineered cell lines and endogenously expressing lung cancer cells in subcutaneous xenograft models in mice. In conclusion, the δOR-specific fluorescent probe developed in this study displays excellent potential for imaging of lung cancer.

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.

Targeting Ligand Specificity Linked to Tumor Tissue Topological Heterogeneity via Single-Cell Micro-Pharmacological Modeling

Targeted therapy has held promise to be a successful anticancer treatment due to its specificity towards tumor cells that express the target receptors. However, not all targeting drugs used in the clinic are equally effective in tumor eradication. To examine which biochemical and biophysical properties of targeted agents are pivotal for their effective distribution inside the tumor and their efficient cellular uptake, we combine mathematical micro-pharmacological modeling with in vivo imaging of targeted human xenograft tumors in SCID mice. The mathematical model calibrated to experimental data was used to explore properties of the targeting ligand (diffusion and affinity) and ligand release schemes (rates and concentrations) with a goal to identify the properties of cells and ligands that enable high receptor saturation. By accounting for heterogeneities typical of in vivo tumors, our model was able to identify cell- and tissue-level barriers to efficient drug uptake. This work provides a base for utilizing experimentally measurable properties of a ligand-targeted agent and patient-specific attributes of the tumor tissue to support the development of novel targeted imaging agents and for improvement in their delivery to individual tumor cells.

Abstract B21: Using computational modeling to quantify targeted agent binding and internalization in pancreatic cancers

The pancreatic adenocarcinoma is one of the most deadly cancers with only 6% overall 5-year survival rate. Since the current therapies fail to provide successful results, the improvement of techniques for early detection, predicting of treatment efficacy and monitoring of tumor spread during and after surgical procedures remain under focused research. The purpose of this study is to develop a novel method to assess behavior (diffusion and binding within the tissues) of imaging agents targeted to pancreatic cancer cells. Our long-term goal is to design a prospective tool for the prediction of targeted imaging agent peak concentration and cellular uptake based on individual patient data. The toll-like receptor 2 (TLR2) is known to play an important role in the immune system response. In addition, our team reported that TLR2 is a bona fide cell-surface marker for targeting pancreatic cancer. TLR2 recognizes a vast number of biomolecules, including lipoproteins, such as designed by our team, novel TLR2 ligand (TLR2L). Recent development of an intravital fluorescence microscopy method allowed for the real time in vivo imaging of the TLR2L conjugated to near-infrared fluorescent dye, Cyanine 5 (TLR2L-Cy5) and its penetration through the tissue of pancreatic adenocarcinoma tumor xenografts in mice with endogenous expression of TLR2. In order to quantify the space- and time-dependent dynamics of TLR2L we combined intravital dorsal window chamber experiments with computational modeling of TLR2L-Cy5 diffusion and internalization following intravenous administration. Our computational model accounts for an explicitly defined tissue morphology composed of individual tumor cells, extracellular matrix interpenetrated by the interstitial fluid, and tumor vasculature. We also model individual molecules of a fluorescent imaging agent that extravasate via transmural influx from blood capillaries, spread through the tumor and become internalized by the cells. The process of agonist binding to the receptor is modeled by trapping the particle by the near-by cell boundary receptor, which mimics the distance dependent electrostatic and hydrophobic interactions initiating the recognition and binding in vivo. Our studies led to quantification of the TLR2L-Cy5 intratumoral transport including agent extravasation, diffusion and intracellular accumulation in relation to tumor tissue structure and vascular architecture. The performed computational simulations allowed for detailed continuous in time assessment of the targeted imaging agent penetration on the microscopic (cell-to-tissue) level. The calibrated in silico model revealed the time-dependent dynamics of TLR2L-Cy5 agent binding, internalization and intracellular distribution. Our quantitative approach also showed a non-uniform spatial saturation of the TLR2L-Cy5 on the plasma membrane and inside the cell. Moreover, continuous simulations allowed for prediction of the maximum concentration peak in the extracellular matrix and for the uptake peak in individual cells. In addition to quantitative analysis, we provide novel (two- and three-dimensional) visualization tools for imaging agent dynamical penetration, membrane binding and receptor-mediated endocytosis process in pancreatic cells. In conclusion, we present an interdisciplinary approach to quantify diffusion and cellular uptake of an imaging agent targeted to pancreatic cancer cell lines expressing the TLR2 receptor. This integrated approach can be used in the future for the development of other targeted imaging and therapeutic agents, for other solid tumors, and for optimizing the administration schedules and time points for data collection from individual human tumor xenografts in order to improve treatment efficacy. Citation Format: Aleksandra Karolak, Veronica Estrella, Tingan Chen, Amanda Huynh, David Morse, Katarzyna Rejniak. Using computational modeling to quantify targeted agent binding and internalization in pancreatic cancers. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B21.

3D Multiscale Modelling of Angiogenesis and Vascular Tumour Growth

We present a three-dimensional, multiscale model of vascular tumour growth, which couples nutrient/growth factor transport, blood flow, angiogenesis, vascular remodelling, movement of and interactions between normal and tumour cells, and nutrient-dependent cell cycle dynamics within each cell. We present computational simulations which show how a vascular network may evolve and interact with tumour and healthy cells. We also demonstrate how our model may be combined with experimental data, to predict the spatio-temporal evolution of a vascular tumour.

Abstract 4557: Development of a melanocortin receptor 1 targeted probe for molecular imaging of melanoma

Melanoma is curable by means of surgical excision if diagnosed in early stages, but once the metastatic stage is reached, prognosis is poor because the tumor is resistant to most cures. Therefore, a melanoma targeted probe that can deliver therapy to metastases at high dosages could increase the chances of treatment. Melanocortin 1 receptor (MC1R) is overexpressed in most human melanoma metastases, thus making it a promising target for imaging and therapy of melanomas. To confirm that MC1R is a specific melanoma marker, mRNA and protein expression was confirmed and quantified in tumor and normal unaffected patient tissue samples. MC1R mRNA expression was highly and generally expressed among melanoma samples surveyed. In contrast, MC1R expression was not elevated in other skin cancers, normal skin and organs involved in toxicity and clearance, i.e. heart, spleen, liver and kidney. To determine MC1R protein expression in patient samples, immunohistochemistry was performed on a melanoma tissue microarray containing 267 samples. None of the normal skin samples (n = 19) had staining with a pathology score of α4. Benign lesions (n = 65), samples of local invasion to regional lymph nodes (n = 35) and metastatic melanoma (n=40) had moderate to high staining in 15, 33 and 47% of the samples, respectively. We have previously reported the development of a peptidomimetic ligand with high specificity and affinity for MC1R. In this study, we have conjugated this ligand to an infrared dye to generate a MC1R specific optical probe (named ML-800). Our whole-cell binding assay using A375/MC1R human melanoma engineered cells was used to determine the high binding affinity of ML-800 (0.4± 0.1 nM Ki). The cellular uptake of the probe was studied in A375/MC1R cells by fluorescence microscopy both in vitro and in vivo using a dorsal skin-fold window-chamber mouse model. The in vivo tumor targeting of ML-800 was evaluated by intravenous injection of probe into nude mice bearing bilateral subcutaneous tumors of A375 with low number of MC1R receptors and A375/MC1R with high expression of MC1Rs. Fluorescence imaging showed that the agent has higher uptake values in A375/MC1R tumors than those in A375M tumors (P, 0.05), demonstrating differentiation of probe retention in tumors with different levels of expression. In conclusion, the imaging probe designed in this study demonstrates the potential for the development of agents that can deliver imaging contrast and therapy to melanoma metastases that express MC1R. Since radiopeptides have proven their usefulness for diagnostic imaging and radiotherapy, in the future, this ligand could be developed as a targeted delivery vehicle for non-invasive nuclear imaging to detect regional lymph node involvement, or delivery of radiotherapy. Furthermore, by attachment to the MC1R ligand with a cleavable linker, cytotoxins may also be targeted to tumor cells for receptor mediated endocytosis and intracellular release. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4557. doi:1538-7445.AM2012-4557

Abstract 53: Exploiting heterogeneity to develop better treatment strategies using an evolutionary multiscale mathematical model of tumor-vessel interactions

Tumor heterogeneity in cancer is an observed fact, both genetically and phenotypically. Cell-cell variation is seen in almost all aspects of cancer from early development all the way through to invasion and subsequent metastasis. The tumor microenvironment is also very heterogeneous and dynamic with multiple zones of hypoxia, normoxia, acidosis and necrosis. Central to our understanding of this heterogeneity is how the tumor cells interact with each other and with their microenvironment. Since the microenvironment is modulated by the tumor itself, as well as the surrounding stroma and the vasculature supply, these interactions rapidly become complex. In order to better understand these complex interactions, we have developed an integrated approach utilizing a hybrid multiscale mathematical model in parallel with an experimental murine window chamber model. This study aims to understand the development of the vasculature in response to a growing tumor, the effects of a heterogeneous microenvironment, and how interactions between the tumor and its vasculature modulate tumor progression and treatment resistance. By incorporating phenotypic heterogeneity into the model, a range of tumor phenotypes emerges due to the heterogeneous microenvironment. The response of the tumor to a treatment will depend on the distribution of phenotypes as well as the local conditions. At the same time, treatment will alter the phenotypic distribution and the microenvironment, so that subsequent tumor progression will be profoundly different than it was prior to treatment. By using the spatial and temporal projections of the mathematical model, optimal treatment strategies are derived and classified based on tumor and microenvironmental parameters. One particularly intriguing result is that tumor control, as opposed to eradication, may be more readily achieved by modulation of the tumor heterogeneity via treatment-mediated microenvironmental selection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 53. doi:10.1158/1538-7445.AM2011-53