Rong Fan

Yale University, New Haven, Connecticut, United States

Are you Rong Fan?

Claim your profile

Publications (24)183.1 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: It is increasingly recognized that infiltrating immune cells contribute to the pathogenesis of a wide range of solid tumors. The paracrine signaling between the tumor and the immune cells alters the functional state of individual tumor cells and, correspondingly, the anticipated response to radiation or chemotherapies, which is of great importance to clinical oncology. Here we present a high-density microchip platform capable of measuring a panel of paracrine signals associated with heterotypic tumor-immune cell interactions in the single-cell, pair-wise manner. The device features a high-content cell capture array of 5000+ sub-nanoliter microchambers for the isolation of single and multi-cell combinations and a multi-plex antibody "barcode" array for multiplexed protein secretion analysis from each microchamber. In this work, we measured a panel of 16 proteins produced from individual glioma cells, individual macrophage cells and varying heterotypic multi-cell combinations of both on the same device. The results show changes of tumor cell functional phenotypes that cannot be explained by an additive effect from isolated single cells and, presumably, can be attributed to the paracrine signaling between macrophage and glioma cells. The protein correlation analysis reveals the key signaling nodes altered by tumor-macrophage communication. This platform enables the novel pair-wise interrogation of heterotypic cell-cell paracrine signaling at the individual cell level with an in-depth analysis of the changing functional phenotypes for different co-culture cell combinations.
    Lab on a Chip 07/2014; · 5.70 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Nanostructured surfaces emerge as a new class of material for capture and separation of cell populations including primary immune cells and disseminating rare tumor cells, but the underlying mechanism remains elusive. Although it has been speculated that nanoscale topological structures on cell surface are involved in the cell capture process, there are no studies that systematically analyze the relation between cell surface structures and the capture efficiency. Here we report on the first mechanistic study by quantifying the morphological parameters of cell surface nanoprotrusions, including filopodia, lamellipodia, and microvilli in the early stage of cell capture (< 20 min) in correlation to the efficiency of separating primary T lymphocytes. This was conducted by using a set of nanohole arrays (NHAs) with varying hole and pitch sizes. Our results showed that the formation of filopodia (e.g., width of filopodia and the average number of the filopodial filaments per cell) depends on the feature size of the nanostructures and the cell separation efficiency is strongly correlated to the number of filopodial fibers, suggesting a possible role of early stage mechanosensing and cell spreading in determining the efficiency of cell capture. In contrast, the length of filopodial filaments was less significantly correlated to the cell capture efficiency and the nanostructure dimensions of the NHAs. This is the first mechanistic study on nanostructure-based immune cell capture and provides new insights to not only the biology of cell-nanomaterial interaction but also the design of new rare cell capture technologies with improved efficiency and specificity.
    Journal of Biomedical Nanotechnology 06/2014; 10(6):1030-40. · 7.58 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Despite the presence of the blood-brain barrier (BBB) that restricts the entry of immune cells and mediators into the central nervous system (CNS), a small number of peripheral leukocytes can traverse the BBB and infiltrate into the CNS. The cerebrospinal fluid (CSF) is one of the major routes through which trafficking leukocytes migrate into the CNS. Therefore, the number of leukocytes and their phenotypic compositions in the CSF may represent important sources to investigate immune-to-brain interactions or diagnose and monitor neurodegenerative diseases. Due to the paucity of trafficking leucocytes in the CSF, a technology capable of efficient isolation, enumeration, and molecular typing of these cells in the clinical settings has not been achieved. In this study, we report on a biofunctionalized silicon nanowire array chip for highly efficient capture and multiplexed phenotyping of rare trafficking leukocytes in small quantities (50 microliters) of clinical CSF specimens collected from neurodegenerative disease patients. The antibody coated 3D nanostructured materials exhibited vastly improved rare cell capture efficiency due to high-affinity binding and enhanced cell-substrate interactions. Moreover, our platform creates multiple cell capture interfaces, each of which can selectively isolate specific leukocyte phenotypes. A comparison with the traditional immunophenotyping using flow cytometry demonstrated that our novel silicon nanowire-based rare cell analysis platform can perform rapid detection and simultaneous molecular characterization of heterogeneous immune cells. Multiplexed molecular typing of rare leukocytes in CSF samples collected from Alzheimer's disease patients revealed the elevation of white blood cell counts and significant alterations in the distribution of major leukocyte phenotypes. Our technology represents a practical tool for potentially diagnosing and monitoring the pathogenesis of neurodegenerative diseases by allowing an effective hematological analysis of the CSF from patients.
    Nanoscale 04/2014; · 6.23 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We report on the rapid and direct quantification of specific cell captures using a micro-patterned streptavidin (STR)-functionalized silicon nanowire (SiNW) platform, which was prepared by Ag-assisted wet chemical etching and a photo-lithography process. This platform operates by high-affinity cell capture rendered by the combination of antibody-epithelial cell surface-binding, biotin-streptavidin binding, and the topologically enhanced cell-substrate interaction on a 3-dimensional SiNWs array. In this work, we developed a micro-patterned nanowire platform, with which we were able to directly evaluate the performance enhancement due to nanotopography. An excellent capture efficiency of ~96.6±6.7%, which is the highest value achieved thus far for the targeting specific A549 cells on a selective area of patterned SiNWs, is demonstrated. Direct comparison between the nanowire region and the planar region on the same substrate indicates dramatically elevated cell-capture efficiency on nanotopological surface identical surface chemistry (<2% cell-capture efficiency). An excellent linear response was seen for quantifying captured A549 cells with respect to loaded cells. This study suggests that the micro-patterned STR-functionalized SiNWs platform provides additional advantage for detecting rare cells populations in a more quantitative and specific manner.
    Biosensors & bioelectronics 11/2013; 54C:181-188. · 5.43 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Biodegradable metal alloys emerge as a new class of biomaterials for tissue engineering and medical devices such as cardiovascular stents. Deploying biodegradable materials to fabricate stents not only obviates a second surgical intervention for implant removal but also circumvents the long-term foreign body effect of permanent implants. However, these materials for stents suffer from an un-controlled degradation rate, acute toxic responses, and rapid structural failure presumably due to a non-uniform, fast corrosion process. Here we report that highly uniform, nanophasic degradation is achieved in a new Mg alloy with unique interstitial alloying composition as the nominal formula Mg-2.5Nd-0.2Zn-0.4Zr (wt%, hereafter, denoted as JDBM). This material exhibits highly homogeneous nanophasic biodegradation patterns as compared to other biodegradable metal alloy materials. Consequently it has significantly reduced degradation rate determined by electrochemical characterization. The in vitro cytotoxicity test using human vascular endothelial cells indicates excellent biocompatibility and potentially minimal toxic effect on arterial vessel walls. Finally, we fabricated a cardiovascular stent using JDBM and performed in vivo long-term assessment via implantation of this stent in an animal model. The results confirmed the reduced degradation rate in vivo, excellent tissue compatibility and long-term structural and mechanical durability. Thus, this new Mg-alloy with highly uniform nanophasic biodegradation represents a major breakthrough in the field and a promising material for manufacturing the next generation biodegradable vascular stents.
    Nanoscale 08/2013; · 6.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Single-cell functional proteomics assays can connect genomic information to biological function through quantitative and multiplex protein measurements. Tools for single-cell proteomics have developed rapidly over the past 5 years and are providing approaches for directly elucidating phosphoprotein signaling networks in cancer cells or for capturing high-resolution snapshots of immune system function in patients with various disease conditions. We discuss advances in single-cell proteomics platforms, with an emphasis on microchip methods. These methods can provide a direct correlation of morphological, functional and molecular signatures at the single-cell level. We also provide examples of how those platforms are being applied to both fundamental biology and clinical studies, focusing on immune-system monitoring and phosphoprotein signaling networks in cancer.
    Genome Medicine 08/2013; 5(8):75. · 4.94 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: From the lungs to the central nervous system, cilia-driven fluid flow plays a fundamental role in many facets of life. Yet, there are few quantitative methods for analysing the function of ciliated surfaces. Here, we report a novel microfluidic approach for quantifying the performance of a ciliated surface using mixing performance as an integrated readout.
    Lab on a Chip 08/2013; · 5.70 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Secreted proteins dictate a range of cellular functions in human health and disease. Due to the high degree of cellular heterogeneity and, more importantly, polyfunctionality of individual cells, there is an unmet need to simultaneously measure an array of proteins from single cells and to rapidly assay a large number of single cells (more than 1000) in parallel. We describe a simple bioanalytical assay platform consisting of a large array of sub-nanoliter microchambers integrated with high-density antibody barcode microarrays for highly multiplexed protein detection from over a thousand single cells in parallel. This platform has been tested for both cell lines and complex biological samples such as primary cells from patients. We observed distinct heterogeneity among the single cell secretomic signatures that, for the first time, can be directly correlated to the cells' physical behavior such as migration. Compared to the state-of-the-art protein secretion assay such as ELISpot and emerging microtechnology-enabled assays, our approach offers both high throughput and high multiplicity. It also has a number of clinician-friendly features such as ease of operation, low sample consumption and standardized data analysis, representing a potentially transformative tool for informative monitoring of cellular function and immunity in patients.
    Analytical Chemistry 01/2013; · 5.70 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: An accurate measurement of the immune status in patients with immune system disorders is critical in evaluating the stage of diseases and tailoring drug treatments. The functional cellular immunity test is a promising method to establish the diagnosis of immune dysfunctions. The conventional functional cellular immunity test involves measurements of the capacity of peripheral blood mononuclear cells to produce pro-inflammatory cytokines when stimulated ex vivo. However, this "bulk" assay measures the overall reactivity of a population of lymphocytes and monocytes, making it difficult to pinpoint the phenotype or real identity of the reactive immune cells involved. In this research, we develop a large surface micromachined poly-dimethylsiloxane (PDMS) microfiltration membrane (PMM) with high porosity, which is integrated in a microfluidic microfiltration platform. Using the PMM with functionalized microbeads conjugated with antibodies against specific cell surface proteins, we demonstrated rapid, efficient and high-throughput on-chip isolation, enrichment, and stimulation of subpopulations of immune cells from blood specimens. Furthermore, the PMM-integrated microfiltration platform, coupled with a no-wash homogeneous chemiluminescence assay ("AlphaLISA"), enables us to demonstrate rapid and sensitive on-chip immunophenotyping assays for subpopulations of immune cells isolated directly from minute quantities of blood samples.
    Advanced healthcare materials. 01/2013;
  • Source
    Chao Ma, Rong Fan, Meltem Elitas
    [Show abstract] [Hide abstract]
    ABSTRACT: In the past decade, significant progresses have taken place in the field of cancer immunotherapeutics, which are being developed for most human cancers. New immunotherapeutics, such as Ipilimumab (anti-CTLA-4), have been approved for clinical treatment; cell-based immunotherapies such as adoptive cell transfer (ACT) have either passed the final stage of human studies (e.g., Sipuleucel-T) for the treatment of selected neoplastic malignancies or reached the stage of phase II/III clinical trials. Immunotherapetics has become a sophisticated field. Multimodal therapeutic regimens comprising several functional modules (up to five in the case of ACT) have been developed to provide focused therapeutic responses with improved efficacy and reduced side-effects. However, a major challenge remains: the lack of effective and clinically applicable immune assessment methods. Due to the complexity of antitumor immune responses within patients, it is difficult to provide comprehensive assessment of therapeutic efficacy and mechanism. To address this challenge, new technologies have been developed to directly profile the cellular immune functions and the functional heterogeneity. With the goal to measure the functional proteomics of single immune cells, these technologies are informative, sensitive, high-throughput, and highly multiplex. They have been used to uncover new knowledge of cellular immune functions and have been utilized for rapid, informative, and longitudinal monitoring of immune response in clinical anti-cancer treatment. In addition, new computational tools are required to integrate high-dimensional data sets generated from the comprehensive, single cell level measurements of patient's immune responses to guide accurate and definitive diagnostic decision. These single cell immune function assessment tools will likely contribute to new understanding of therapy mechanism, pre-treatment stratification of patients, and ongoing therapeutic monitoring and assessment.
    Frontiers in Oncology 01/2013; 3:133.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Secreted proteins including cytokines, chemokines and growth factors represent important functional regulators mediating a range of cellular behavior and cell-cell paracrine/autocrine signaling, e.g. in the immunological system, tumor microenvironment or stem cell niche. Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically-identical cell population can give rise to diverse phenotypic differences. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this Perspective Article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer.
    Frontiers in Oncology 01/2013; 3(10).
  • Yu Wu, Lana X Garmire, Rong Fan
    [Show abstract] [Hide abstract]
    ABSTRACT: We conducted inter-cellular cytokine correlation and network analysis based upon a stochastic population dynamics model that comprises five cell types and fifteen signaling molecules inter-connected through a large number of cell-cell communication pathways. We observed that the signaling molecules are tightly correlated even at very early stages (e.g. the first month) of human glioma, but such correlation rapidly diminishes when tumor grows to a size that can be clinically detected. Further analysis suggests that paracrine is shown to be the dominant force during tumor initiation and priming, while autocrine supersedes it and supports a robust tumor expansion. In correspondence, the cytokine correlation network evolves through an increasing to decreasing complexity. This study indicates a possible mechanistic transition from the microenvironment-controlled, paracrine-based regulatory mechanism to self-sustained rapid progression to fetal malignancy. It also reveals key nodes that are responsible for such transition and can be potentially harnessed for the design of new anti-cancer therapies.
    Integrative Biology 10/2012; · 4.32 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: High-content cellomic analysis is a powerful tool for rapid screening of cellular responses to extracellular cues and examination of intracellular signal transduction pathways at the single-cell level. In conjunction with microfluidics technology that provides unique advantages in sample processing and precise control of fluid delivery, it holds great potential to transform lab-on-a-chip systems for high-throughput cellular analysis. However, high-content imaging instruments are expensive, sophisticated, and not readily accessible. Herein, we report on a laser scanning cytometry approach that exploits a bench-top microarray scanner as an end-point reader to perform rapid and automated fluorescence imaging of cells cultured on a chip. Using high-content imaging analysis algorithms, we demonstrated multiplexed measurements of morphometric and proteomic parameters from all single cells. Our approach shows the improvement of both sensitivity and dynamic range by two orders of magnitude as compared to conventional epifluorescence microscopy. We applied this technology to high-throughput analysis of mesenchymal stem cells on an extracellular matrix protein array and characterization of heterotypic cell populations. This work demonstrates the feasibility of a laser microarray scanner for high-content cellomic analysis and opens up new opportunities to conduct informative cellular analysis and cell-based screening in the lab-on-a-chip systems.
    Lab on a Chip 09/2012; · 5.70 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We report on the development of a nanowire substrate-enabled laser scanning imaging cytometry for rare cell analysis in order to achieve quantitative, automated, and functional evaluation of circulating tumor cells. Immuno-functionalized nanowire arrays have been demonstrated as a superior material to capture rare cells from heterogeneous cell populations. The laser scanning cytometry method enables large-area, automated quantitation of captured cells and rapid evaluation of functional cellular parameters (e.g., size, shape, and signaling protein) at the single-cell level. This integrated platform was first tested for capture and quantitation of human lung carcinoma cells from a mixture of tumor cells and leukocytes. We further applied it to the analysis of rare tumor cells spiked in fresh human whole blood (several cells per mL) that emulate metastatic cancer patient blood and demonstrated the potential of this technology for analyzing circulating tumor cells in the clinical settings. Using a high-content image analysis algorithm, cellular morphometric parameters and fluorescence intensities can be rapidly quantitated in an automated, unbiased, and standardized manner. Together, this approach enables informative characterization of captured cells in situ and potentially allows for subclassification of circulating tumor cells, a key step toward the identification of true metastasis-initiating cells. Thus, this nanoenabled platform holds great potential for studying the biology of rare tumor cells and for differential diagnosis of cancer progression and metastasis.
    Nano Letters 05/2012; 12(6):2697-704. · 13.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Human embryonic stem cells (hESCs) have great potentials for future cell-based therapeutics. However, their mechanosensitivity to biophysical signals from the cellular microenvironment is not well characterized. Here we introduced an effective microfabrication strategy for accurate control and patterning of nanoroughness on glass surfaces. Our results demonstrated that nanotopography could provide a potent regulatory signal over different hESC behaviors, including cell morphology, adhesion, proliferation, clonal expansion, and self-renewal. Our results indicated that topological sensing of hESCs might include feedback regulation involving mechanosensory integrin-mediated cell-matrix adhesion, myosin II, and E-cadherin. Our results also demonstrated that cellular responses to nanotopography were cell-type specific, and as such, we could generate a spatially segregated coculture system for hESCs and NIH/3T3 fibroblasts using patterned nanorough glass surfaces.
    ACS Nano 04/2012; 6(5):4094-103. · 12.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Tumor cells do not develop in isolation, but co-evolve with stromal cells and tumor-associated immune cells in a tumor microenvironment mediated by an array of soluble factors, forming a complex intercellular signaling network. Herein, we report an unbiased, generic model to integrate prior biochemical data and the constructed brain tumor microenvironment in silico as characterized by an intercellular signaling network comprising 5 types of cells, 15 cytokines, and 69 signaling pathways. The results show that glioma develops through three distinct phases: pre-tumor, rapid expansion, and saturation. We designed a microglia depletion therapy and observed significant benefit for virtual patients treated at the early stages but strikingly no therapeutic efficacy at all when therapy was given at a slightly later stage. Cytokine combination therapy exhibits more focused and enhanced therapeutic response even when microglia depletion therapy already fails. It was further revealed that the optimal combination depends on the molecular profile of individual patients, suggesting the need for patient stratification and personalized treatment. These results, obtained solely by observing the in silico dynamics of the glioma microenvironment with no fitting to experimental/clinical data, reflect many characteristics of human glioma development and imply new venues for treating tumors via selective targeting of microenvironmental components.
    PLoS Computational Biology 02/2012; 8(2):e1002355. · 4.87 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We report the development of a novel quartz nanopillar (QNP) array cell separation system capable of selectively capturing and isolating a single cell population including primary CD4(+) T lymphocytes from the whole pool of splenocytes. Integrated with a photolithographically patterned hemocytometer structure, the streptavidin (STR)-functionalized-QNP (STR-QNP) arrays allow for direct quantitation of captured cells using high content imaging. This technology exhibits an excellent separation yield (efficiency) of ~95.3 ± 1.1% for the CD4(+) T lymphocytes from the mouse splenocyte suspensions and good linear response for quantitating captured CD4(+) T-lymphoblasts, which is comparable to flow cytometry and outperforms any non-nanostructured surface capture techniques, i.e. cell panning. This nanopillar hemocytometer represents a simple, yet efficient cell capture and counting technology and may find immediate applications for diagnosis and immune monitoring in the point-of-care setting.
    Nanoscale 01/2012; 4(7):2500-7. · 6.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We describe a microchip designed to quantify the levels of a dozen cytoplasmic and membrane proteins from single cells. We use the platform to assess protein-protein interactions associated with the EGF-receptor-mediated PI3K signaling pathway. Single-cell sensitivity is achieved by isolating a defined number of cells (n = 0-5) in 2 nL volume chambers, each of which is patterned with two copies of a miniature antibody array. The cells are lysed on-chip, and the levels of released proteins are assayed using the antibody arrays. We investigate three isogenic cell lines representing the cancer glioblastoma multiforme, at the basal level, under EGF stimulation, and under erlotinib inhibition plus EGF stimulation. The measured protein abundances are consistent with previous work, and single-cell analysis uniquely reveals single-cell heterogeneity, and different types and strengths of protein-protein interactions. This platform helps provide a comprehensive picture of altered signal transduction networks in tumor cells and provides insight into the effect of targeted therapies on protein signaling networks.
    Proceedings of the National Academy of Sciences 12/2011; 109(2):419-24. · 9.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cellular immunity has an inherent high level of functional heterogeneity. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. We report a microfluidic platform designed for highly multiplexed (more than ten proteins), reliable, sample-efficient (∼1 × 10(4) cells) and quantitative measurements of secreted proteins from single cells. We validated the platform by assessment of multiple inflammatory cytokines from lipopolysaccharide (LPS)-stimulated human macrophages and comparison to standard immunotechnologies. We applied the platform toward the ex vivo quantification of T cell polyfunctional diversity via the simultaneous measurement of a dozen effector molecules secreted from tumor antigen-specific cytotoxic T lymphocytes (CTLs) that were actively responding to tumor and compared against a cohort of healthy donor controls. We observed profound, yet focused, functional heterogeneity in active tumor antigen-specific CTLs, with the major functional phenotypes quantitatively identified. The platform represents a new and informative tool for immune monitoring and clinical assessment.
    Nature medicine 06/2011; 17(6):738-43. · 27.14 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein signaling networks among cells play critical roles in a host of pathophysiological processes, from inflammation to tumorigenesis. We report on an approach that integrates microfluidic cell handling, in situ protein secretion profiling, and information theory to determine an extracellular protein-signaling network and the role of perturbations. We assayed 12 proteins secreted from human macrophages that were subjected to lipopolysaccharide challenge, which emulates the macrophage-based innate immune responses against Gram-negative bacteria. We characterize the fluctuations in protein secretion of single cells, and of small cell colonies (n = 2, 3,···), as a function of colony size. Measuring the fluctuations permits a validation of the conditions required for the application of a quantitative version of the Le Chatelier's principle, as derived using information theory. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein-protein interaction network.
    Biophysical Journal 05/2011; 100(10):2378-86. · 3.67 Impact Factor

Publication Stats

404 Citations
183.10 Total Impact Points

Institutions

  • 2011–2014
    • Yale University
      • Department of Biomedical Engineering
      New Haven, Connecticut, United States
  • 2013
    • Chung-Ang University
      • Department of Physics
      Sŏul, Seoul, South Korea
    • University of New Haven
      New Haven, Connecticut, United States
  • 2012
    • Yale-New Haven Hospital
      • Anesthesiology Program
      New Haven, Connecticut, United States
  • 2008–2011
    • California Institute of Technology
      • Division of Chemistry and Chemical Engineering
      Pasadena, CA, United States