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author:("Li, fuwai")
1.  Targeting Stromal-Cancer Cell Crosstalk Networks in Ovarian Cancer Treatment 
Biomolecules  2016;6(1):3.
Ovarian cancer is a histologically, clinically, and molecularly diverse disease with a five-year survival rate of less than 30%. It has been estimated that approximately 21,980 new cases of epithelial ovarian cancer will be diagnosed and 14,270 deaths will occur in the United States in 2015, making it the most lethal gynecologic malignancy. Ovarian tumor tissue is composed of cancer cells and a collection of different stromal cells. There is increasing evidence that demonstrates that stromal involvement is important in ovarian cancer pathogenesis. Therefore, stroma-specific signaling pathways, stroma-derived factors, and genetic changes in the tumor stroma present unique opportunities for improving the diagnosis and treatment of ovarian cancer. Cancer-associated fibroblasts (CAFs) are one of the major components of the tumor stroma that have demonstrated supportive roles in tumor progression. In this review, we highlight various types of signaling crosstalk between ovarian cancer cells and stromal cells, particularly with CAFs. In addition to evaluating the importance of signaling crosstalk in ovarian cancer progression, we discuss approaches that can be used to target tumor-promoting signaling crosstalk and how these approaches can be translated into potential ovarian cancer treatment.
doi:10.3390/biom6010003
PMCID: PMC4808797  PMID: 26751490
ovarian cancer; cancer-associated fibroblasts; stromal-tumor crosstalk; tumor microenvironment
2.  Computational analysis of image-based drug profiling predicts synergistic drug combinations: applications in triple-negative breast cancer 
Molecular oncology  2014;8(8):1548-1560.
An imaged-based profiling and analysis system was developed to predict clinically effective synergistic drug combinations that could accelerate the identification of effective multi-drug therapies for the treatment of triple-negative breast cancer (TNBC) and other challenging malignancies. The identification of effective drug combinations for the treatment of triple-negative breast cancer was achieved by integrating high-content screening, computational analysis, and experimental biology. The approach was based on altered cellular phenotypes induced by 55 FDA-approved drugs and biologically active compounds, acquired using fluorescence microscopy and retained in multivariate compound profiles. Dissimilarities between compound profiles guided the identification of 5 combinations, which were assessed for qualitative interaction on TNBC cell growth. The combination of the microtubule-targeting drug vinblastine with KSP/Eg5 motor protein inhibitors monastrol or ispinesib showed potent synergism in 3 independent TNBC cell lines, which was not substantiated in normal fibroblasts. The synergistic interaction was mediated by an increase in mitotic arrest with cells demonstrating typical ispinesib-induced monopolar mitotic spindles, which translated into enhanced apoptosis induction. The antitumor activity of the combination vinblastine / ispinesib was confirmed in an orthotopic mouse model of TNBC. Compared to single drug treatment, combination treatment significantly reduced tumor growth without causing increased toxicity. Image-based profiling and analysis led to the rapid discovery of a drug combination effective against TNBC in vitro and in vivo, and has the potential to lead to the development of new therapeutic options in other hard-to-treat cancers.
doi:10.1016/j.molonc.2014.06.007
PMCID: PMC4253311  PMID: 24997502
High-content screening; compound profiling; synergy; triple-negative breast cancer; KSP/Eg5 inhibitors; microtubule-targeting agents
3.  A High-content screen identifies compounds promoting the neuronal differentiation and the midbrain dopamine neuron specification of human neural progenitor cells 
Scientific Reports  2015;5:16237.
Small molecule compounds promoting the neuronal differentiation of stem/progenitor cells are of pivotal importance to regenerative medicine. We carried out a high-content screen to systematically characterize known bioactive compounds, on their effects on the neuronal differentiation and the midbrain dopamine (mDA) neuron specification of neural progenitor cells (NPCs) derived from the ventral mesencephalon of human fetal brain. Among the promoting compounds three major pharmacological classes were identified including the statins, TGF-βRI inhibitors, and GSK-3 inhibitors. The function of each class was also shown to be distinct, either to promote both the neuronal differentiation and mDA neuron specification, or selectively the latter, or promote the former but suppress the latter. We then carried out initial investigation on the possible mechanisms underlying, and demonstrated their applications on NPCs derived from human pluripotent stem cells (PSCs). Our study revealed the potential of several small molecule compounds for use in the directed differentiation of human NPCs. The screening result also provided insight into the signaling network regulating the differentiation of human NPCs.
doi:10.1038/srep16237
PMCID: PMC4635364  PMID: 26542303
4.  A computational method for clinically relevant cancer stratification and driver mutation module discovery using personal genomics profiles 
BMC Genomics  2015;16(Suppl 7):S6.
Background
Personalized genomics instability, e.g., somatic mutations, is believed to contribute to the heterogeneous drug responses in patient cohorts. However, it is difficult to discover personalized driver mutations that are predictive of drug sensitivity owing to diverse and complex mutations of individual patients. To circumvent this problem, a novel computational method is presented to discover potential drug sensitivity relevant cancer subtypes and identify driver mutation modules of individual subtypes by coupling differentially expressed genes (DEGs) based subtyping analysis with the driver mutation network analysis.
Results
The proposed method was applied to breast cancer and lung cancer samples available from The Cancer Genome Atlas (TCGA). Cancer subtypes were uncovered with significantly different survival rates, and more interestingly, distinct driver mutation modules were also discovered among different subtypes, indicating the potential mechanism of heterogeneous drug sensitivity.
Conclusions
The research findings can be used to help guide the repurposing of known drugs and their combinations in order to target these dysfunctional modules and their downstream signaling effectively for achieving personalized or precision medicine treatment.
doi:10.1186/1471-2164-16-S7-S6
PMCID: PMC4474541  PMID: 26099165
5.  Wnt-Responsive Cancer Stem Cells Are Located Close to Distorted Blood Vessels and Not in Hypoxic Regions in a p53-Null Mouse Model of Human Breast Cancer 
High-resolution imaging techniques were used to analyze the relationship between a Wnt-responsive cancer stem cell (CSC)-enriched population and the tumor vasculature using p53-null mouse mammary tumors transduced with a Wnt signaling pathway reporter. The results demonstrate that the combined strategy of monitoring the fluorescently labeled CSCs and vasculature using high-resolution imaging techniques provides a unique opportunity to study the CSC microenvironment.
Cancer stem cells (CSCs, or tumor-initiating cells) may be responsible for tumor formation in many types of cancer, including breast cancer. Using high-resolution imaging techniques, we analyzed the relationship between a Wnt-responsive, CSC-enriched population and the tumor vasculature using p53-null mouse mammary tumors transduced with a lentiviral Wnt signaling reporter. Consistent with their localization in the normal mammary gland, Wnt-responsive cells in tumors were enriched in the basal/myoepithelial population and generally located in close proximity to blood vessels. The Wnt-responsive CSCs did not colocalize with the hypoxia-inducible factor 1α-positive cells in these p53-null basal-like tumors. Average vessel diameter and vessel tortuosity were increased in p53-null mouse tumors, as well as in a human tumor xenograft as compared with the normal mammary gland. The combined strategy of monitoring the fluorescently labeled CSCs and vasculature using high-resolution imaging techniques provides a unique opportunity to study the CSC and its surrounding vasculature.
doi:10.5966/sctm.2013-0088
PMCID: PMC4073819  PMID: 24797826
Cancer stem cells; Stem cell microenvironment; In vivo optical imaging; Microvasculature; Signal transduction; p53
6.  Efficient and Rapid Derivation of Primitive Neural Stem Cells and Generation of Brain Subtype Neurons From Human Pluripotent Stem Cells 
Stem Cells Translational Medicine  2013;2(11):862-870.
This study developed a highly efficient serum-free pluripotent stem cell (PSC) neural induction medium that can induce human PSCs into primitive neural stem cells (NSCs) in 7 days, obviating the need for time-consuming, laborious embryoid body generation or rosette picking. This method of primitive NSC derivation sets the stage for the scalable production of clinically relevant neural cells for cell therapy applications in good manufacturing practice conditions.
Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are unique cell sources for disease modeling, drug discovery screens, and cell therapy applications. The first step in producing neural lineages from hPSCs is the generation of neural stem cells (NSCs). Current methods of NSC derivation involve the time-consuming, labor-intensive steps of an embryoid body generation or coculture with stromal cell lines that result in low-efficiency derivation of NSCs. In this study, we report a highly efficient serum-free pluripotent stem cell neural induction medium that can induce hPSCs into primitive NSCs (pNSCs) in 7 days, obviating the need for time-consuming, laborious embryoid body generation or rosette picking. The pNSCs expressed the neural stem cell markers Pax6, Sox1, Sox2, and Nestin; were negative for Oct4; could be expanded for multiple passages; and could be differentiated into neurons, astrocytes, and oligodendrocytes, in addition to the brain region-specific neuronal subtypes GABAergic, dopaminergic, and motor neurons. Global gene expression of the transcripts of pNSCs was comparable to that of rosette-derived and human fetal-derived NSCs. This work demonstrates an efficient method to generate expandable pNSCs, which can be further differentiated into central nervous system neurons and glia with temporal, spatial, and positional cues of brain regional heterogeneity. This method of pNSC derivation sets the stage for the scalable production of clinically relevant neural cells for cell therapy applications in good manufacturing practice conditions.
doi:10.5966/sctm.2013-0080
PMCID: PMC3808201  PMID: 24113065
Astrocytes; Cell culture; Neural stem cell; Neural induction; Neural differentiation; Oligodendrocytes; Neuron; Nestin
7.  Three-dimensional vasculature reconstruction of tumour microenvironment via local clustering and classification 
Interface Focus  2013;3(4):20130015.
The vasculature inside breast cancers is one important component of the tumour microenvironment. The investigation of its spatial morphology, distribution and interactions with cancer cells, including cancer stem cells, is essential for elucidating mechanisms of tumour development and treatment response. Using confocal microscopy and fluorescent markers, we have acquired three-dimensional images of vasculature within mammary tumours and normal mammary gland of mouse models. However, it is difficult to segment and reconstruct complex vasculature accurately from the in vivo three-dimensional images owing to the existence of uneven intensity and regions with low signal-to-noise ratios (SNR). To overcome these challenges, we have developed a novel three-dimensional vasculature segmentation method based on local clustering and classification. First, images of vasculature are clustered into local regions, whose boundaries well delineate vasculature even in low SNR and uneven intensity regions. Then local regions belonging to vasculature are identified by applying a semi-supervised classification method based on three informative features of the local regions. Comparison of results using simulated and real vasculature images, from mouse mammary tumours and normal mammary gland, shows that the new method outperforms existing methods, and can be used for three-dimensional images with uneven background and low SNR to achieve accurate vasculature reconstruction.
doi:10.1098/rsfs.2013.0015
PMCID: PMC3915834  PMID: 24511379
vasculature reconstruction, tumour microenvironment, vascular imaging
8.  DrugComboRanker: drug combination discovery based on target network analysis 
Bioinformatics  2014;30(12):i228-i236.
Motivation: Currently there are no curative anticancer drugs, and drug resistance is often acquired after drug treatment. One of the reasons is that cancers are complex diseases, regulated by multiple signaling pathways and cross talks among the pathways. It is expected that drug combinations can reduce drug resistance and improve patients’ outcomes. In clinical practice, the ideal and feasible drug combinations are combinations of existing Food and Drug Administration-approved drugs or bioactive compounds that are already used on patients or have entered clinical trials and passed safety tests. These drug combinations could directly be used on patients with less concern of toxic effects. However, there is so far no effective computational approach to search effective drug combinations from the enormous number of possibilities.
Results: In this study, we propose a novel systematic computational tool DrugComboRanker to prioritize synergistic drug combinations and uncover their mechanisms of action. We first build a drug functional network based on their genomic profiles, and partition the network into numerous drug network communities by using a Bayesian non-negative matrix factorization approach. As drugs within overlapping community share common mechanisms of action, we next uncover potential targets of drugs by applying a recommendation system on drug communities. We meanwhile build disease-specific signaling networks based on patients’ genomic profiles and interactome data. We then identify drug combinations by searching drugs whose targets are enriched in the complementary signaling modules of the disease signaling network. The novel method was evaluated on lung adenocarcinoma and endocrine receptor positive breast cancer, and compared with other drug combination approaches. These case studies discovered a set of effective drug combinations top ranked in our prediction list, and mapped the drug targets on the disease signaling network to highlight the mechanisms of action of the drug combinations.
Availability and implementation: The program is available on request.
Contact: stwong@tmhs.org
doi:10.1093/bioinformatics/btu278
PMCID: PMC4058933  PMID: 24931988
9.  Novel Modeling of Cancer Cell Signaling Pathways Enables Systematic Drug Repositioning for Distinct Breast Cancer Metastases 
Cancer research  2013;73(20):6149-6163.
A new type of signaling network element, called cancer signaling bridges (CSB), has been shown to have the potential for systematic and fast-tracked drug repositioning. On the basis of CSBs, we developed a computational model to derive specific downstream signaling pathways that reveal previously unknown target–disease connections and new mechanisms for specific cancer subtypes. The model enables us to reposition drugs based on available patient gene expression data. We applied this model to repurpose known or shelved drugs for brain, lung, and bone metastases of breast cancer with the hypothesis that cancer subtypes have their own specific signaling mechanisms. To test the hypothesis, we addressed specific CSBs for each metastasis that satisfy (i) CSB proteins are activated by the maximal number of enriched signaling pathways specific to a given metastasis, and (ii) CSB proteins are involved in the most differential expressed coding genes specific to each breast cancer metastasis. The identified signaling networks for the three types of breast cancer metastases contain 31, 15, and 18 proteins and are used to reposition 15, 9, and 2 drug candidates for the brain, lung, and bone metastases. We conducted both in vitro and in vivo preclinical experiments as well as analysis on patient tumor specimens to evaluate the targets and repositioned drugs. Of special note, we found that the Food and Drug Administration-approved drugs, sunitinib and dasatinib, prohibit brain metastases derived from breast cancer, addressing one particularly challenging aspect of this disease.
doi:10.1158/0008-5472.CAN-12-4617
PMCID: PMC4005386  PMID: 24097821
10.  A Screen for Morphological Complexity Identifies Regulators of Switch-like Transitions between Discrete Cell Shapes 
Nature cell biology  2013;15(7):860-871.
The way in which cells adopt different morphologies is not fully understood. Cell shape could be a continuous variable or restricted to a set of discrete forms. We developed quantitative methods to describe cell shape and show that Drosophila hemocytes in culture are a heterogeneous mixture of five discrete morphologies. In an RNAi screen of genes affecting the morphological complexity of heterogeneous populations, we found that most genes regulate the transition between discrete shapes rather than generating new morphologies. In particular, we identified a subset of genes, including the tumour suppressor PTEN, that decrease the heterogeneity of the population leading to populations enriched in rounded or elongated forms. We show that these genes have a highly conserved function as regulators of cell shape in both mouse and human metastatic melanoma cells.
doi:10.1038/ncb2764
PMCID: PMC3712499  PMID: 23748611
11.  A 3D multiscale model of cancer stem cell in tumor development 
BMC Systems Biology  2013;7(Suppl 2):S12.
Background
Recent reports indicate that a subgroup of tumor cells named cancer stem cells (CSCs) or tumor initiating cells (TICs) are responsible for tumor initiation, growth and drug resistance. This subgroup of tumor cells has self-renewal capacity and could differentiate into heterogeneous tumor cell populations through asymmetric proliferation. The idea of CSC provides informative insights into tumor initiation, metastasis and treatment. However, the underlying mechanisms of CSCs regulating tumor behaviors are unclear due to the complex cancer system. To study the functions of CSCs in the complex tumor system, a few mathematical modeling studies have been proposed. Whereas, the effect of microenvironment (mE) factors, the behaviors of CSCs, progenitor tumor cells (PCs) and differentiated tumor cells (TCs), and the impact of CSC fraction and signaling heterogeneity, are not adequately explored yet.
Methods
In this study, a novel 3D multi-scale mathematical modeling is proposed to investigate the behaviors of CSCsin tumor progressions. The model integrates CSCs, PCs, and TCs together with a few essential mE factors. With this model, we simulated and investigated the tumor development and drug response under different CSC content and heterogeneity.
Results
The simulation results shown that the fraction of CSCs plays a critical role in driving the tumor progression and drug resistance. It is also showed that the pure chemo-drug treatment was not a successful treatment, as it resulted in a significant increase of the CSC fraction. It further shown that the self-renew heterogeneity of the initial CSC population is a cause of the heterogeneity of the derived tumors in terms of the CSC fraction and response to drug treatments.
Conclusions
The proposed 3D multi-scale model provides a new tool for investigating the behaviors of CSC in CSC-initiated tumors, which enables scientists to investigate and generate testable hypotheses about CSCs in tumor development and drug response under different microenvironments and drug perturbations.
doi:10.1186/1752-0509-7-S2-S12
PMCID: PMC3866259  PMID: 24564919
13.  Chapter 17: Bioimage Informatics for Systems Pharmacology 
PLoS Computational Biology  2013;9(4):e1003043.
Recent advances in automated high-resolution fluorescence microscopy and robotic handling have made the systematic and cost effective study of diverse morphological changes within a large population of cells possible under a variety of perturbations, e.g., drugs, compounds, metal catalysts, RNA interference (RNAi). Cell population-based studies deviate from conventional microscopy studies on a few cells, and could provide stronger statistical power for drawing experimental observations and conclusions. However, it is challenging to manually extract and quantify phenotypic changes from the large amounts of complex image data generated. Thus, bioimage informatics approaches are needed to rapidly and objectively quantify and analyze the image data. This paper provides an overview of the bioimage informatics challenges and approaches in image-based studies for drug and target discovery. The concepts and capabilities of image-based screening are first illustrated by a few practical examples investigating different kinds of phenotypic changes caEditorsused by drugs, compounds, or RNAi. The bioimage analysis approaches, including object detection, segmentation, and tracking, are then described. Subsequently, the quantitative features, phenotype identification, and multidimensional profile analysis for profiling the effects of drugs and targets are summarized. Moreover, a number of publicly available software packages for bioimage informatics are listed for further reference. It is expected that this review will help readers, including those without bioimage informatics expertise, understand the capabilities, approaches, and tools of bioimage informatics and apply them to advance their own studies.
doi:10.1371/journal.pcbi.1003043
PMCID: PMC3635992  PMID: 23633943
14.  Genome-Wide RNAi Screening Identifies Genes Inhibiting the Migration of Glioblastoma Cells 
PLoS ONE  2013;8(4):e61915.
Glioblastoma Multiforme (GBM) cells are highly invasive, infiltrating into the surrounding normal brain tissue, making it impossible to completely eradicate GBM tumors by surgery or radiation. Increasing evidence also shows that these migratory cells are highly resistant to cytotoxic reagents, but decreasing their migratory capability can re-sensitize them to chemotherapy. These evidences suggest that the migratory cell population may serve as a better therapeutic target for more effective treatment of GBM. In order to understand the regulatory mechanism underlying the motile phenotype, we carried out a genome-wide RNAi screen for genes inhibiting the migration of GBM cells. The screening identified a total of twenty-five primary hits; seven of them were confirmed by secondary screening. Further study showed that three of the genes, FLNA, KHSRP and HCFC1, also functioned in vivo, and knocking them down caused multifocal tumor in a mouse model. Interestingly, two genes, KHSRP and HCFC1, were also found to be correlated with the clinical outcome of GBM patients. These two genes have not been previously associated with cell migration.
doi:10.1371/journal.pone.0061915
PMCID: PMC3625150  PMID: 23593504
15.  A computational framework for studying neuron morphology from in vitro high content neuron-based screening 
Journal of neuroscience methods  2010;190(2):299-309.
High content neuron image processing is considered as an important method for quantitative neurobiological studies. The main goal of analysis in this paper is to provide automatic image processing approaches to process neuron images for studying neuron mechanism in high content screening. In the nuclei channel, all nuclei are segmented and detected by applying the gradient vector field based watershed. Then the neuronal nuclei are selected based on the soma region detected in neurite channel. In neurite images, we propose a novel neurite centerline extraction approach using the improved line-pixel detection technique. The proposed neurite tracing method can detect the curvilinear structure more accurately compared with the current existing methods. An interface called NeuriteIQ based on the proposed algorithms is developed finally for better application in high content screening.
doi:10.1016/j.jneumeth.2010.05.012
PMCID: PMC3184395  PMID: 20580743
High content screening; Microscopy image; Nuclei segmentation; Neurite outgrowth; Line-pixel detection; Branch area
16.  Image-Based Chemical Screening Identifies Drug Efflux Inhibitors In Lung Cancer Cells 
Cancer research  2010;70(19):7723-7733.
Cancer cells with active drug-efflux capability are multidrug resistant and pose a significant obstacle for the efficacy of chemotherapy. Moreover, recent evidence suggests that high drug-efflux cancer cells (HDECCs) may be selectively enriched with stem-like cancer cells, which are believed to be the cause for tumor initiation and recurrence. There is a great need for therapeutic reagents that are capable of eliminating HDECCs. We developed an image-based high-content screening (HCS) system to specifically identify and analyze the HDECC population in lung cancer cells. Using the system, we screened 1,280 pharmacologically active compounds which identified twelve potent HDECC inhibitors. It is shown that these inhibitors are able to overcome MDR and sensitize HDECCs to chemotherapeutic drugs, or directly reduce the tumorigenicity of lung cancer cells possibly by affecting stem-like cancer cells. The HCS system we established provides a new approach for identifying therapeutic reagents overcoming MDR. The compounds identified by the screening may potentially be used as potential adjuvant to improve the efficacy of chemotherapeutic drugs.
doi:10.1158/0008-5472.CAN-09-4360
PMCID: PMC2948619  PMID: 20841476
high drug-efflux cancer cells; multidrug resistance; high content screening; image-based assay
17.  A Time-Series Method for Automated Measurement of Changes in Mitotic and Interphase Duration from Time-Lapse Movies 
PLoS ONE  2011;6(9):e25511.
Background
Automated time-lapse microscopy can visualize proliferation of large numbers of individual cells, enabling accurate measurement of the frequency of cell division and the duration of interphase and mitosis. However, extraction of quantitative information by manual inspection of time-lapse movies is too time-consuming to be useful for analysis of large experiments.
Methodology/Principal Findings
Here we present an automated time-series approach that can measure changes in the duration of mitosis and interphase in individual cells expressing fluorescent histone 2B. The approach requires analysis of only 2 features, nuclear area and average intensity. Compared to supervised learning approaches, this method reduces processing time and does not require generation of training data sets. We demonstrate that this method is as sensitive as manual analysis in identifying small changes in interphase or mitotic duration induced by drug or siRNA treatment.
Conclusions/Significance
This approach should facilitate automated analysis of high-throughput time-lapse data sets to identify small molecules or gene products that influence timing of cell division.
doi:10.1371/journal.pone.0025511
PMCID: PMC3180452  PMID: 21966537
18.  Differential diagnosis of breast cancer using quantitative, label-free and molecular vibrational imaging 
Biomedical Optics Express  2011;2(8):2160-2174.
We present a label-free, chemically-selective, quantitative imaging strategy to identify breast cancer and differentiate its subtypes using coherent anti-Stokes Raman scattering (CARS) microscopy. Human normal breast tissue, benign proliferative, as well as in situ and invasive carcinomas, were imaged ex vivo. Simply by visualizing cellular and tissue features appearing on CARS images, cancerous lesions can be readily separated from normal tissue and benign proliferative lesion. To further distinguish cancer subtypes, quantitative disease-related features, describing the geometry and distribution of cancer cell nuclei, were extracted and applied to a computerized classification system. The results show that in situ carcinoma was successfully distinguished from invasive carcinoma, while invasive ductal carcinoma (IDC) and invasive lobular carcinoma were also distinguished from each other. Furthermore, 80% of intermediate-grade IDC and 85% of high-grade IDC were correctly distinguished from each other. The proposed quantitative CARS imaging method has the potential to enable rapid diagnosis of breast cancer.
doi:10.1364/BOE.2.002160
PMCID: PMC3149516  PMID: 21833355
(170.3880) Medical and biological imaging; (170.4580) Optical diagnostics for medicine; (180.4315) Nonlinear microscopy; (300.6230) Spectroscopy, coherent anti-Stokes Raman scattering microscopy
19.  Label-free high-resolution imaging of prostate glands and cavernous nerves using coherent anti-Stokes Raman scattering microscopy 
Biomedical Optics Express  2011;2(4):915-926.
A custom built coherent anti-Stokes Raman scattering (CARS) microscope was used to image prostatic glands and nerve structures from 17 patients undergoing radical prostatectomy. Imaging of glandular and nerve structures showed distinctive cellular features that correlated to histological stains. Segmentation of cell nucleus was performed to establish a cell feature-based model to separate normal glands from cancer glands. In this study, we use a single parameter, average cell neighbor distance based on CARS imaging, to characterize normal and cancerous glandular structures. By combining CARS with our novel classification model, we are able to characterize prostate glandular and nerve structures in a manner that potentially enables real-time, intra-operative assessment of surgical margins and neurovascular bundles. As such, this method could potentially improve outcomes following radical prostatectomy.
doi:10.1364/BOE.2.000915
PMCID: PMC3072130  PMID: 21483613
(180.4315) Nonlinear microscopy; (170.1610) Clinical applications; (170.3880) Medical and biological imaging; (170.4580) Optical diagnostics for medicine
20.  Cell Segmentation Using Front Vector Flow Guided Active Contours 
Phase-contrast microscopy is a common approach for studying the dynamics of cell behaviors, such as cell migration. Cell segmentation is the basis of quantitative analysis of the immense cellular images. However, the complicated cell morphological appearance in phase-contrast microscopy images challenges the existing segmentation methods. This paper proposes a new cell segmentation method for cancer cell migration studies using phase-contrast images. Instead of segmenting cells directly based on commonly used low-level features, e.g. intensity and gradient, we first identify the leading protrusions, a high level feature, of cancer cells. Based on the identified cell leading protrusions, we introduce a front vector flow guided active contour, which guides the initial cell boundaries to the real boundaries. The experimental validation on a set of breast cancer cell images shows that the proposed method demonstrates fast, stable, and accurate segmentation for breast cancer cells with wide range of sizes and shapes.
PMCID: PMC2896022  PMID: 20426162
21.  INTEGRATING MULTI-SCALE BLOB/CURVILINEAR DETECTOR TECHNIQUES AND MULTI-LEVEL SETS FOR AUTOMATED SEGMENTATION OF STEM CELL IMAGES 
Studies of differentiation abilities of stem cells have been attracting a lot of attention over the last years. Microscopy can be used to record details of the differentiation process of stem cells under different perturbations and is an important tool for studying stem cell differentiation. Since it is infeasible to quantitatively analyze a huge amount of image data manually, automated image analysis systems are urgently needed. However, the complicated morphological appearances of stem cells are challenging to the existing segmentation methods. Herein, we propose a new, automated scheme for stem cell segmentation. This scheme first uses the multi-scale blob and curvilinear structure detectors to delineate the skeletons of stem cells quickly and then segment out stem cells by refining the skeletons to the cell boundaries using multi-level sets. The initial experimental results indicate the effectiveness of the proposed scheme.
doi:10.1109/ISBI.2009.5193318
PMCID: PMC2888517  PMID: 20585412
stem cell differentiation; blob detection; curvilinear structure detection; cell segmentation; level set
22.  Analysis of clinical and dosimetric factors associated with severe acute radiation pneumonitis in patients with locally advanced non-small cell lung cancer treated with concurrent chemotherapy and intensity-modulated radiotherapy 
Background
To evaluate the association between the clinical, dosimetric factors and severe acute radiation pneumonitis (SARP) in patients with locally advanced non-small cell lung cancer (LANSCLC) treated with concurrent chemotherapy and intensity-modulated radiotherapy (IMRT).
Methods
We analyzed 94 LANSCLC patients treated with concurrent chemotherapy and IMRT between May 2005 and September 2006. SARP was defined as greater than or equal 3 side effects and graded according to Common Terminology Criteria for Adverse Events (CTCAE) version 3.0.
The clinical and dosimetric factors were analyzed. Univariate and multivariate logistic regression analyses were performed to evaluate the relationship between clinical, dosimetric factors and SARP.
Results
Median follow-up was 10.5 months (range 6.5-24). Of 94 patients, 11 (11.7%) developed SARP. Univariate analyses showed that the normal tissue complication probability (NTCP), mean lung dose (MLD), relative volumes of lung receiving more than a threshold dose of 5-60 Gy at increments of 5 Gy (V5-V60), chronic obstructive pulmonary disease (COPD) and Forced Expiratory Volume in the first second (FEV1) were associated with SARP (p < 0.05). In multivariate analysis, NTCP value (p = 0.001) and V10 (p = 0.015) were the most significant factors associated with SARP. The incidences of SARP in the group with NTCP > 4.2% and NTCP ≤4.2% were 43.5% and 1.4%, respectively (p < 0.01). The incidences of SARP in the group with V10 ≤50% and V10 >50% were 5.7% and 29.2%, respectively (p < 0.01).
Conclusions
NTCP value and V10 are the useful indicators for predicting SARP in NSCLC patients treated with concurrent chemotherapy and IMRT.
doi:10.1186/1748-717X-5-35
PMCID: PMC2883984  PMID: 20462424
23.  Conditional random pattern model for copy number aberration detection 
BMC Bioinformatics  2010;11:200.
Background
DNA copy number aberration (CNA) is very important in the pathogenesis of tumors and other diseases. For example, CNAs may result in suppression of anti-oncogenes and activation of oncogenes, which would cause certain types of cancers. High density single nucleotide polymorphism (SNP) array data is widely used for the CNA detection. However, it is nontrivial to detect the CNA automatically because the signals obtained from high density SNP arrays often have low signal-to-noise ratio (SNR), which might be caused by whole genome amplification, mixtures of normal and tumor cells, experimental noise or other technical limitations. With the reduction in SNR, many false CNA regions are often detected and the true CNA regions are missed. Thus, more sophisticated statistical models are needed to make the CNAs detection, using the low SNR signals, more robust and reliable.
Results
This paper presents a conditional random pattern (CRP) model for CNA detection where much contextual cues are explored to suppress the noise and improve CNA detection accuracy. Both simulated and the real data are used to evaluate the proposed model, and the validation results show that the CRP model is more robust and reliable in the presence of noise for CNA detection using high density SNP array data, compared to a number of widely used software packages.
Conclusions
The proposed conditional random pattern (CRP) model could effectively detect the CNA regions in the presence of noise.
doi:10.1186/1471-2105-11-200
PMCID: PMC2876128  PMID: 20412592
24.  Workflow and Methods of High-Content Time-Lapse Analysis for Quantifying Intracellular Calcium Signals 
Neuroinformatics  2008;6(2):97-108.
Abstract Calcium ions (Ca2+) play a fundamental role in a variety of physiological functions in many cell types by acting as a secondary messenger. Variation of intracellular Ca2+ concentration ([Ca2+]i) is often observed when the cell is stimulated. However, it is a challenging task to automatically quantify intracellular [Ca2+]i in a population of cells. In this study, we present a workflow including specific algorithms for the automated intracellular calcium signal analysis using high-content, time-lapse cellular images. The experimental validations indicate the effectiveness of the proposed workflow and algorithms. We applied the workflow to analyze the intracellular calcium signals induced by different concentrations of H2O2 in the cell lines transfected by presenilin-1 (PS-1) that is known to be closely related to the familial Alzheimer's disease (FAD). The analysis results imply an important role of mutant PS-1, but not normal human PS-1 and mutant human amyloid precursor protein (APP), in enhancing intracellular calcium signaling induced by H2O2.
doi:10.1007/s12021-008-9016-z
PMCID: PMC2853186  PMID: 18506641
High content image analysis; Oxidative stress; Calcium oscillation; Familial Alzheimer's disease; Mutant presenilin 1
25.  Multiple Nuclei Tracking Using Integer Programming for Quantitative Cancer Cell Cycle Analysis 
Automated cell segmentation and tracking are critical for quantitative analysis of cell cycle behavior using time-lapse fluorescence microscopy. However, the complex, dynamic cell cycle behavior poses new challenges to the existing image segmentation and tracking methods. This paper presents a fully automated tracking method for quantitative cell cycle analysis. In the proposed tracking method, we introduce a neighboring graph to characterize the spatial distribution of neighboring nuclei, and a novel dissimilarity measure is designed based on the spatial distribution, nuclei morphological appearance, migration, and intensity information. Then, we employ the integer programming and division matching strategy, together with the novel dissimilarity measure, to track cell nuclei. We applied this new tracking method for the tracking of HeLa cancer cells over several cell cycles, and the validation results showed that the high accuracy for segmentation and tracking at 99.5% and 90.0%, respectively. The tracking method has been implemented in the cell–cycle analysis software package, DCELLIQ, which is freely available.
doi:10.1109/TMI.2009.2027813
PMCID: PMC2846554  PMID: 19643704
Anti-cancer drug screening; cell cycle analysis; segmentation and tracking; time-lapse fluorescence microscopy

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