Sharon S. Hori

Mathematical model identifies blood biomarker-based early cancer detection strategies and limitations.

Early cancer detection using current clinical blood-based biomarker assays may not be possible within the first 10 years of tumor growth. The Early Bird Doesn’t Always Catch the Worm The oft-quoted dictum “timing is everything” has special meaning in the realm of cancer diagnosis. Early detection can greatly improve patient prognosis: The longer the cancer goes unnoticed, the bigger it may get and the greater chance it has to spread, making treatment much more difficult. Because patients may not notice any symptoms until the cancer has already progressed, there’s been a huge push to identify cancer biomarkers. Ideally, these biological hallmarks may indicate the presence of disease well before symptoms appear. Hori et al. now develop a mathematical model to predict how early doctors can catch disease by screening blood for cancer biomarkers. Starting with a single ovarian tumor cell, the authors modeled the release of cancer-related molecules (putative biomarkers) into the bloodstream (“shedding”) on the basis of well-characterized tumor growth and shedding rates for ovarian cancer. Hori et al. found that the tumor could grow for more than 10 years—to about the size of an olive—before it was detected by current clinical blood-based biomarker assays. These data suggest that, although identifying new biomarkers is critical for early cancer diagnosis, the sensitivity of biomarker detection must be optimized further if it is to have an effect on disease outcome in the clinic. Most clinical blood biomarkers lack the necessary sensitivity and specificity to reliably detect cancer at an early stage, when it is best treatable. It is not yet clear how early a clinical blood assay can be used to detect cancer or how biomarker-based strategies can be improved to enable earlier detection of smaller tumors. To address these issues, we developed a mathematical model describing dynamic plasma biomarker kinetics in relation to the growth of a tumor, beginning with a single cancer cell. To exemplify a realistic scenario in which biomarker is shed by both cancerous and noncancerous cells, we primed the model on ovarian tumor growth and CA125 shedding data, for which tumor growth parameters and shedding rates are readily available in published literature. We found that a tumor could grow unnoticed for more than 10.1 years and reach a volume of about π/6(25.36 mm)3, corresponding to a spherical diameter of about 25.36 mm, before becoming detectable by current clinical blood assays. Model parameters were perturbed over log orders of magnitude to quantify ideal shedding rates and identify other blood-based strategies required for early submillimeter tumor detectability. The detection times we estimated are consistent with recently published tumor progression time lines based on clinical genomic sequencing data for several cancers. Here, we rigorously showed that shedding rates of current clinical blood biomarkers are likely 104-fold too low to enable detection of a developing tumor within the first decade of tumor growth. The model presented here can be extended to virtually any solid cancer and associated biomarkers.

Role of Endosomal Trafficking Dynamics on the Regulation of Hepatic Insulin Receptor Activity: Models for Fao Cells

Evidence indicates that endosomal insulin receptor (IR) trafficking plays a role in regulating insulin signal transduction. To evaluate its importance, we developed a series of biokinetic models for quantifying activated surface and endosomal IR dynamics from published experimental data. Starting with a published two-compartment Fao hepatoma model, a four-pool model was formulated that depicts IR autophosphorylation after receptor binding, IR endosomal internalization/trafficking, insulin dissociation from and dephosphorylation of internalized IR, and recycling of unliganded, dephosphorylated IR to the plasma membrane. Quantification required three additional data sets, two measured, but unmodeled by the same group. A five-pool model created to include endosomal trafficking of the nonphosphorylated insulin-IR complex was fitted using the same data sets, augmented with another published data set. Creation of a six-pool model added the physiologically relevant dissociation of insulin ligand from the activated endosomal IR. More importantly, all three models, validated against additional data not used in model fitting, predict that, mechanistically, internalization of activated IR is a rate-limiting step, at least under the receptor saturating conditions of the fitting data. This rate includes the transit time to a site where insulin dissociation from and/or dephosphorylation of the IR occurs by docking with protein-tyrosine phosphatases (PTPases), or where a sufficient conformational change occurs in the IR, perhaps due to insulin-IR dissociation, where associated PTPases may complete IR dephosphorylation. Our new models indicate that key events in endosomal IR trafficking have significance in mediating IR activity, possibly serving to regulate insulin signal transduction.

Detecting cancers through tumor-activatable minicircles that lead to a detectable blood biomarker

Significance Blood-based cancer diagnosis is highly attractive, but current strategies suffer because they rely on the detection of endogenous molecules that often are secreted into the circulation by both malignant and nonmalignant cells. One solution to this problem is to avoid nonmalignant tissue expression by artificially engineering tumor cells to express a unique reporter not normally expressed by any tissue. This study shows that systemic administration of nonviral safe vectors we call “tumor-activatable minicircles” allows one to distinguish tumor-bearing from tumor-free subjects reliably and to assess tumor burden simply by measuring blood levels of such a reporter. Our system represents an alternative paradigm for improved cancer detection and could enable more timely interventions to combat this devastating disease. Earlier detection of cancers can dramatically improve the efficacy of available treatment strategies. However, despite decades of effort on blood-based biomarker cancer detection, many promising endogenous biomarkers have failed clinically because of intractable problems such as highly variable background expression from nonmalignant tissues and tumor heterogeneity. In this work we present a tumor-detection strategy based on systemic administration of tumor-activatable minicircles that use the pan-tumor–specific Survivin promoter to drive expression of a secretable reporter that is detectable in the blood nearly exclusively in tumor-bearing subjects. After systemic administration we demonstrate a robust ability to differentiate mice bearing human melanoma metastases from tumor-free subjects for up to 2 wk simply by measuring blood reporter levels. Cumulative change in reporter levels also identified tumor-bearing subjects, and a receiver operator-characteristic curve analysis highlighted this test’s performance with an area of 0.918 ± 0.084. Lung tumor burden additionally correlated (r2 = 0.714; P < 0.05) with cumulative reporter levels, indicating that determination of disease extent was possible. Continued development of our system could improve tumor detectability dramatically because of the temporally controlled, high reporter expression in tumors and nearly zero background from healthy tissues. Our strategy’s highly modular nature also allows it to be iteratively optimized over time to improve the test’s sensitivity and specificity. We envision this system could be used first in patients at high risk for tumor recurrence, followed by screening high-risk populations before tumor diagnosis, and, if proven safe and effective, eventually may have potential as a powerful cancer-screening tool for the general population.

Photoacoustic Tomography Detects Early Vessel Regression and Normalization During Ovarian Tumor Response to the Antiangiogenic Therapy Trebananib

The primary aim of this study was to assess the potential of in vivo photoacoustic tomography for direct functional measurement of ovarian tumor response to antiangiogenic therapy. Methods: In vivo studies were performed with institutional animal care and use committee approval. We used an orthotopic mouse model of ovarian cancer treated with trebananib (n = 9) or vehicle (n = 9). Tumor-bearing mice were randomized into trebananib or vehicle groups at day 10 and dosed on days 12, 15, and 18 after implantation. Photoacoustic tomography and blood draws were performed at day 10 and then 24 h after each drug dose. Tumors were excised for histopathology after the final studies on day 19. Data analysis to test for statistical significance was performed blinded. Results: Blockade of angiopoietin signaling using trebananib resulted in reduced total hemoglobin–weighted photoacoustic signal (n = 9, P = 0.01) and increased oxyhemoglobin-weighted photoacoustic signal (n = 9, P < 0.01). The latter observation indicated normalization of the residual tumor vessels, which was also implied by low levels of angiopoietin 1 in serum biomarker profiling (0.76 ± 0.12 ng/mL). These noninvasive measures reflected a 30% reduction in microvessel density and increased vessel maturation in ex vivo sections. Conclusion: Photoacoustic tomography is able to evaluate both vessel regression and normalization in response to trebananib. Noninvasive imaging data were supported by modulation of serum markers in vitro and ex vivo histopathology.

Development and Validation of an Immuno-PET Tracer as a Companion Diagnostic Agent for Antibody-Drug Conjugate Therapy to Target the CA6 Epitope

PURPOSE To develop and compare three copper 64 ((64)Cu)-labeled antibody fragments derived from a CA6-targeting antibody (huDS6) as immuno-positron emission tomography (immuno-PET)-based companion diagnostic agents for an antibody-drug conjugate by using huDS6. MATERIALS AND METHODS Three antibody fragments derived from huDS6 were produced, purified, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and evaluated in the following ways: (a) the affinity of the fragments and the DOTA conjugates was measured via flow cytometry, (b) the stability of the labeled fragments was determined ex vivo in human serum over 24 hours, and (c) comparison of the in vivo imaging potential of the fragments was evaluated in mice bearing subcutaneous CA6-positive and CA6-negative xenografts by using serial PET imaging and biodistribution. Isotype controls with antilysozyme and anti-DM4 B-Fabs and blocking experiments with an excess of either B-Fab or huDS6 were used to determine the extent of the antibody fragment (64)Cu-DOTA-B-Fab binding specificity. Immunoreactivity and tracer kinetics were evaluated by using cellular uptake and 48-hour imaging experiments, respectively. Statistical analyses were performed by using t tests, one-way analysis of variance, and Wilcoxon and Mann-Whitney tests. RESULTS The antibody fragment (64)Cu-DOTA-B-Fab was more than 95% stable after 24 hours in human serum, had an immunoreactivity of more than 70%, and allowed differentiation between CA6-positive and CA6-negative tumors in vivo as early as 6 hours after injection, with a 1.7-fold uptake ratio between tumors. Isotype and blocking studies experiments showed tracer-specific uptake in antigen-positive tumors, despite some nonspecific uptake in both tumor models. CONCLUSION Three antibody fragments were produced and examined as potential companion diagnostic agents. (64)Cu-DOTA-B-Fab is a stable and effective immuno-PET tracer for CA6 imaging in vivo.

Detection and Quantitation of Circulating Tumor Cell Dynamics by Bioluminescence Imaging in an Orthotopic Mammary Carcinoma Model

Circulating tumor cells (CTCs) have been detected in the bloodstream of both early-stage and advanced cancer patients. However, very little is know about the dynamics of CTCs during cancer progression and the clinical relevance of longitudinal CTC enumeration. To address this, we developed a simple bioluminescence imaging assay to detect CTCs in mouse models of metastasis. In a 4T1 orthotopic metastatic mammary carcinoma mouse model, we demonstrated that this quantitative method offers sensitivity down to 2 CTCs in 0.1–1mL blood samples and high specificity for CTCs originating from the primary tumor, independently of their epithelial status. In this model, we simultaneously monitored blood CTC dynamics, primary tumor growth, and lung metastasis progression over the course of 24 days. Early in tumor development, we observed low numbers of CTCs in blood samples (10–15 cells/100 µL) and demonstrated that CTC dynamics correlate with viable primary tumor growth. To our knowledge, these data represent the first reported use of bioluminescence imaging to detect CTCs and quantify their dynamics in any cancer mouse model. This new assay is opening the door to the study of CTC dynamics in a variety of animal models. These studies may inform clinical decision on the appropriate timing of blood sampling and value of longitudinal CTC enumeration in cancer patients.

A Model-Based Personalized Cancer Screening Strategy for Detecting Early-Stage Tumors Using Blood-Borne Biomarkers

An effective cancer blood biomarker screening strategy must distinguish aggressive from nonaggressive tumors at an early, intervenable time. However, for blood-based strategies to be useful, the quantity of biomarker shed into the blood and its relationship to tumor growth or progression must be validated. To study how blood biomarker levels correlate with early-stage viable tumor growth in a mouse model of human cancer, we monitored early tumor growth of engineered human ovarian cancer cells (A2780) implanted orthotopically into nude mice. Biomarker shedding was monitored by serial blood sampling, whereas tumor viability and volume were monitored by bioluminescence imaging and ultrasound imaging. From these metrics, we developed a mathematical model of cancer biomarker kinetics that accounts for biomarker shedding from tumor and healthy cells, biomarker entry into vasculature, biomarker elimination from plasma, and subject-specific tumor growth. We validated the model in a separate set of mice in which subject-specific tumor growth rates were accurately predicted. To illustrate clinical translation of this strategy, we allometrically scaled model parameters from mouse to human and used parameters for PSA shedding and prostate cancer. In this manner, we found that blood biomarker sampling data alone were capable of enabling the detection and discrimination of simulated aggressive (2-month tumor doubling time) and nonaggressive (18-month tumor doubling time) tumors as early as 7.2 months and 8.9 years before clinical imaging, respectively. Our model and screening strategy offers broad impact in their applicability to any solid cancer and associated biomarkers shed, thereby allowing a distinction between aggressive and nonaggressive tumors using blood biomarker sampling data alone. Cancer Res; 77(10); 2570-84. ©2017 AACR.

Engineering Intracellularly Retained Gaussia Luciferase Reporters for Improved Biosensing and Molecular Imaging Applications

Gaussia luciferase (GLUC) is a bioluminescent reporter protein of increasing importance. As a secretory protein, it has increased sensitivity in vitro and in vivo (∼20 000-fold, and ∼1000-fold, respectively) over its competitor, secreted alkaline phosphatase. Unfortunately, this same advantageous secretory nature of GLUC limits its usefulness for many other possible intracellular applications, e.g., imaging signaling pathways in intact cells, in vivo imaging, and in developing molecular imaging biosensors to study protein-protein interactions and protein folding. Hence, to widen the research applications of GLUC, we developed engineered variants that increase its intracellular retention both by modifying the N-terminal secretory signal peptide and by tagging additional sequences to its C-terminal region. We found that when GLUC was expressed in mammalian cells, its N-terminal secretory signal peptide comprising amino acids 1-16 was essential for GLUC folding and functional activity in addition to its inherent secretory property. Modification of the C-terminus of GLUC by tagging a four amino acid (KDEL) endoplasmic reticulum targeting peptide in multiple repeats significantly improved its intracellular retention, with little impact on its folding and enzymatic activity. We used stable cells expressing this engineered GLUC with KDEL repeats to monitor chemically induced endoplasmic reticulum stress on cells. Additionally, we engineered an apoptotic sensor using modified variants of GLUC containing a four amino acid caspase substrate peptide (DEVD) between the GLUC protein and the KDEL repeats. Its use in cell culture resulted in increased GLUC secretion in the growth medium when cells were treated with the chemotherapeutic drugs doxorubicin, paclitaxel, and carboplatin. We thus successfully engineered a new variant GLUC protein that is retained inside cells rather than secreted extracellularly. We validated this novel reporter by incorporating it in biosensors for detection of cellular endoplasmic reticulum stress and caspase activation. This new molecularly engineered enzymatic reporter has the potential for widespread applications in biological research.

Abstract 873: Correlation of plasma biomarker levels with early-stage tumor viability in an orthotopic ovarian cancer mouse model

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: The detection of proteins or other biomarkers secreted or released (shed) from tumor cells into blood may facilitate early cancer screening, particularly for lethal cancers such as ovarian carcinoma, which typically remains asymptomatic and undetected until advanced stages. But the utility of blood-based assays is confounded because it is unknown whether cancer biomarker levels correlate with viable tumor burden, and it is unclear how highly shed a biomarker must be to enable detection of early (< 1 cm diameter) tumors. No clinical or animal data is available to correlate blood biomarker levels with cancer state, before and after disease diagnosis, for multiple time points in the same subject, without intervention or treatment. Aim: To determine whether cancer blood biomarkers may be used as measures of tumor viability in an early-stage ovarian cancer mouse model. Methods: We genetically engineered human ovarian cancer cell line 2008 to stably coexpress a secretory reporter (secreted alkaline phosphatase, SEAP) not produced endogenously in any living animal, and a bifusion optical imaging reporter (firefly luciferase-eGFP, FL-eGFP) to enable in vivo bioluminescence imaging of tumor viability. We orthotopically implanted as few as 3×103 2008-SEAP-FL-eGFP cells into the ovary of 6-week-old female Nu/Nu mice, and monitored subsequent tumor growth for up to 28 days using serial 25-μl plasma SEAP sampling to assess biomarker shedding, in vivo bioluminescence imaging to quantify tumor viability, and in vivo ultrasound imaging to assess tumor volume. Plasma SEAP concentration was measured using a chemiluminescence assay (Clontech). Mice were sacrificed when tumor volume reached 1-cm diameter (or by Day 28) and tumors were excised for histological analysis. Results and Discussion: This is the first study to show that cancer plasma biomarker measurements may be used as surrogates of tumor viability during the early tumor growth stages in a living animal model. Orthotopic implantation of at least 3×104 transfected cells in the ovary resulted in tumor growth (confirmed by ultrasound imaging) that was detectable by plasma SEAP levels and FL imaging within 4 hours and up to 28 days. Clinical translation of this preclinical work was aided by the use of mathematical modeling, which yielded novel estimates of biomarker shedding rates never before measured in vivo (2×10-5 ng/day/cell) and were consistent with predicted rates from cell culture. Plasma SEAP levels correlated well with increasing FL average radiance during the first 15 days post-implantation (R2=0.86), indicating that a cancer-specific biomarker, when shed highly, may reflect early tumor viability. This integrative mouse model and mathematical modeling approach may be used to further elucidate the biology of cancer biomarker shedding in vivo, thereby helping to define guidelines for selecting better biomarkers for early cancer detection. Citation Format: Sharon S. Hori, Amelie M. Lutz, Ramasamy Paulmurugan, Sanjiv S. Gambhir. Correlation of plasma biomarker levels with early-stage tumor viability in an orthotopic ovarian cancer mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 873. doi:10.1158/1538-7445.AM2014-873

Abstract 2047: Molecular photoacoustic imaging and serum diagnostics rapidly detect response to angiopoietin 1 and 2 blockade in ovarian cancer

Introduction: There is an urgent clinical need to develop noninvasive biomarkers that early detect tumor responses to anti-angiogenic therapy. Treatment response in ovarian cancer patients is assessed with transvaginal ultrasound imaging of tumor size and CA125 screening. We hypothesized that combining photoacoustic (PA) imaging to non-invasively visualize tumor vascular architecture, with serum diagnostics using endothelial biomarkers, would yield a sensitive and readily translatable approach for monitoring response to anti-angiogenic therapy. Methods: Ovarian tumors were established in nude mice by orthotopic injection of OV2008 cells at day 0. At day 10 mice were randomized into groups of at least n = 10. Baseline PA imaging and submandibular blood draw were performed, then repeated 24h after dosing with a peptibody that prevents the interaction of secreted angiopoietin 1 (Ang1; agonist) and 2 (Ang2; antagonist) with the receptor tyrosine kinase Tie2, or vehicle, on days 13, 16 and 19. Mice were then sacrificed and tumors excised for histology. Results: PA imaging uses endogenous hemoglobin light absorption to generate contrast. At 797nm, we found a 1.5-fold increase in hemoglobin-weighted signal in vehicle mice by day 19, while treated mice remained close to the baseline (n=9; p=0.008). The oxyhemoglobin-weighted PA signal (837nm/797nm) was elevated by 20% in treated mice but decreased 15% in vehicle mice (n=9; p Conclusions: We have shown for the first time that PA imaging and serum angiopoietin levels measure complementary parameters that indicate response to angiopoietin blockade in ovarian cancer. PA imaging noninvasively detected drug-induced vessel regression and normalization, giving a functional readout of response. This modality can be readily combined with transvaginal ultrasound. The drug action was mediated by normalization of mAng1 levels, indicated by serum measures and IF. Importantly, this work supports the combined use of in vitro and in vivo diagnostics to monitor response to anti-angiogenic therapy in clinical trials. Citation Format: Sarah E. Bohndiek, Laura Sasportas, Steven Machtaler, Jesse V. Jokerst, Sharon Hori, Sanjiv S. Gambhir. Molecular photoacoustic imaging and serum diagnostics rapidly detect response to angiopoietin 1 and 2 blockade in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2047. doi:10.1158/1538-7445.AM2014-2047