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- Materials and Methods
- Literature Cited
- Supporting Information
The aim of this study was to assess the feasibility of applying the single cell network profiling (SCNP) assay to the examination of signaling networks in epithelial cancer cells, using bladder washings from 29 bladder cancer (BC) and 15 nonbladder cancer (NC) subjects. This report describes the methods we developed to detect rare epithelial cells (within the cells we collected from bladder washings), distinguish cancer cells from normal epithelial cells, and reproducibly quantify signaling within these low frequency cancer cells. Specifically, antibodies against CD45, cytokeratin, EpCAM, and cleaved-PARP (cPARP) were used to differentiate nonapoptotic epithelial cells from leukocytes, while measurements of DNA content to determine aneuploidy (DAPI stain) allowed for distinction between tumor and normal epithelial cells. Signaling activity in the PI3K and MAPK pathways was assessed by measuring intracellular levels of p-AKT and p-ERK at baseline and in response to pathway modulation; 66% (N = 19) of BC samples and 27% (N = 4) of NC samples met the “evaluable” criteria, i.e., at least 400,000 total cells available upon sample receipt with >2% of cells showing an epithelial phenotype. The majority of epithelial cells detected in BC samples were nonapoptotic and all signaling data were generated from identified cPARP negative cells. In four of 19 BC samples but in none of the NC specimens, SCNP assay identified epithelial cancer cells with a quantifiable increase in epidermal growth factor-induced p-AKT and p-ERK levels. Furthermore, preincubation with the PI3K inhibitor GDC-0941 reduced or completely inhibited basal and epidermal growth factor-induced p-AKT but, as expected, had no effect on p-ERK levels. This study demonstrates the feasibility of applying SCNP assay using multiparametric flow cytometry to the functional characterization of rare, bladder cancer cells collected from bladder washing. Following assay standardization, this method could potentially serve as a tool for disease characterization and drug development in bladder cancer and other solid tumors. © 2013 International Society for Advancement of Cytometry
Cancer displays biologic and clinical heterogeneity due to a complex range of cytogenetic and molecular aberrations that result in downstream effects on gene expression, protein function, and cell signal transduction pathways, ultimately affecting proliferation, survival, and cellular differentiation (1, 2). Elucidation of the relationship between malignancy and its underlying molecular mechanisms are essential to improve understanding of the tumorigenesis and the mechanisms of drug efficacy and resistance. Since chromosomal, genetic, epigenetic, and other molecular alterations converge at the level of protein function and cell signaling pathways, it is anticipated that tools assessing the activity of these pathways will be highly predictive of the natural history of the disease and the response to therapy.
Single cell network profiling (SCNP) assay is a multiparametric flow cytometry-based assay that simultaneously provides measurements, at the single cell level, of extracellular surface markers and quantitative changes in the activation levels of intracellular signaling proteins in response to extracellular modulators (3–7). This approach interrogates the physiology of signaling pathways and networks by measuring properties beyond those detected in resting cells, which reveals otherwise unseen functional heterogeneity in apparently morphologically and molecularly homogeneous disease groups.
Studies in hematologic malignancies have shown the value of quantitatively measuring single cell signaling networks under modulated conditions as a basis for the development of prognostic and predictive tests. Although the utility of SCNP assay has been well demonstrated in hematologic malignancies (5, 8, 9), to date, its application to solid tumors has not been widely studied, with the exception of a limited number of studies focused on lung cancer (10). The primary reason for this discrepancy is essentially preanalytic. Specifically, in hematologic malignancies, disease samples are represented primarily by aliquots of peripheral blood and/or bone marrow containing discrete numbers of cells that are live, functional, and in suspension, all critical parameters in the application of SCNP analysis by flow cytometry, which are usually absent in the standard collection of solid tumor samples. In particular, SCNP by flow cytometry requires cells in suspension and any assay performed on solid tumors would require some form of mechanical or enzymatic dissociation of the tumor into a single cell suspension. It is still unclear how this manipulation of the solid tumor would affect downstream signaling cascades. Ultimately, performing SCNP analysis on isolated circulating epithelial tumor cells from peripheral blood or bodily fluids, such as bladder washes, could provide an alternative relevant sampling source for generating functional data using SCNP in solid tumors. These data would then be applicable to understanding the biology of the disease and eventually aid in guiding clinical treatments.
The overall aim of this study was to assess the feasibility of applying the SCNP assay to the examination of signaling networks (and their modulation by activation or inhibition) in low numbers of epithelial cells present in otherwise cellularly heterogeneous samples. These data demonstrate that the SCNP assay can be applied reproducibly even on nonconventional samples such as bladder washings, allowing for the identification and characterization of epithelial cells and their response to targeted signaling modulators and inhibitors.
- Top of page
- Materials and Methods
- Literature Cited
- Supporting Information
While the application of SCNP assay in hematologic malignancies has been shown in multiple studies to be feasible, robust, reproducible, and prognostically informative, the application of the same technology to solid tumors has yet to be realized. This is due, at least in part, to the preanalytical challenges associated with solid tumor sampling, which are intrinsically not permissive for a functional, flow cytometry-based analysis. Theoretically, if this limitation can be overcome, then the application of SCNP assay to solid tumors should be straightforward due to the largely overlapping nature of the intracellular pathways found in hematologic and solid tumor malignancies. Hematologic malignancies naturally occur for the most part as single cell suspensions, and tissues such as peripheral blood and bone marrow provide a rich source of live tumor cells for flow-based analysis. Solid tumor samples are rarely collected in live form, since rapid tissue fixation to preserve tissue structure alterations is usually needed for pathology analysis. Additionally, some of the accepted methodologies for studying solid tumors such as immunohistochemistry or genomic sequencing do not allow for functional analysis at the single cell level. While it is possible to quantitatively assess protein expression by immunohistochemistry or identify gene mutations by sequencing, these methodologies do not necessarily reflect biochemical pathway alterations and in the case of genomic sequencing do not take into account possible epigenetic factors such as DNA methylation or histone modification. Furthermore, as targeted therapies become more specific, it is critical to understand the distinct protein and biochemical pathway alterations and functional changes related to a patient's disease in order to guide a clinician in delivering the most effective treatment possible. SCNP is ideally suited to providing this type of information and allows potential testing of drugs on the tumor sample not possible with the other methodologies.
An alternative to solid tumor sampling that is more suitable to SCNP assays would be the use of samples from fluids, which contains tumor cells in suspensions such as pleural effusions and other pathologic fluids or peripheral blood [the latter also known as circulating tumor cells (CTCs)]. The utility of enumerating CTCs as a clinical prognostic tool has been demonstrated in several studies in breast, prostate, and colon cancer; yet, the current methodology does not provide any functional information regarding the biology of the identified tumor cell (19–23). This inability to evaluate relevant functional biology within CTCs is primarily due to the sample collection methodology that requires cell fixation and to the very low tumor cell recovery within the peripheral blood cell repertoire. An assay platform such as SCNP that could be used postcell enrichment to functionally evaluate identified rare CTCs is crucial for further understanding the biology and clinical relevance of CTCs. While peripheral blood is the most common source of CTCs studied to date, other pathological fluids such as pleural effusions or bladder washes may be adaptable to this platform and could be used for studying the biology of those tumor cells.
In this feasibility study, we demonstrated the applicability of the SCNP technology to solid tumors using bladder washes from bladder cancer patients as sample source. Patients with bladder cancer who have completed a cystoscopic procedure have usually low frequency of epithelial cells in the residual bladder wash fluid. Thus, using this sample source allowed us to establish methods for identification of nonapoptotic and aneuploid epithelial cells, measure basal and induced signaling (simultaneously in two parallel pathways, i.e., PI3K and MAPK pathways) in these rare cells, and detect and quantify specific signaling inhibition using targeted inhibitors. To the best of our knowledge, this is the first application of a flow cytometric-based technology platform for functional, quantitative measurement of basal and modulated signaling in bladder washings.
A novel component of this study is the simultaneous use of static and functional measurements consisting of a combination of cell lineage markers, apoptosis, DNA content, and signaling readouts to both define and functionally characterize the biology of malignant epithelial cells in the urine. Importantly, our studies to date demonstrate the ability to detect a consistent and reproducible response across a wide range of cells even at very low numbers. For practical purposes, these experiments were performed by spiking cell lines into surrogate specimens, but the robustness of the assay indicates that a similar approach should be applicable to other tumor types. We have also utilized DNA content analysis to determine if the identified epithelial cell is of tumor origin. While this approach does identify aneuploid cells as being of tumor origin, it does not allow the identification of diploid cells as normal or cancerous. Additional tumor-specific markers will need to be developed to clearly identify tumor from nontumor cells in pathological fluids such as pleural effusions or bladder washes where healthy epithelial cells may be present.
One area for assay enhancement is identification and utilization of markers for more accurately capturing epithelial cells in pathologic fluids as well as peripheral blood. Since it has been reported that a significant number of those tumor cells, in particular CTCs, do not express EpCAM and thus would be excluded from analysis when using EpCAM as a positive selection marker, alternative methodologies are necessary(24–26). One approach that has already shown success in peripheral blood is to enrich for CTCs by removing normal cells rather than positive cell selection using anti-EpCAM or anticytokeratin antibodies (27–29). This approach prevents loss of CTCs that no longer express the prototypical epithelial cell markers cytokeratin and EpCAM possibly due to epithelial to mesenchymal transition or EMT (30–32) To quantify these cells and evaluate signaling pathways via SCNP, additional phenotypic markers are required. Inclusion of mesenchymal cell markers such as vimentin and N-cadherin in conjunction with epithelial cell markers has already shown promise in identifying CTCs in multiple tumor types such as breast, prostate, and head and neck cancers when compared to positive selection of EpCAM alone (28, 33, 34). Combined use of these mesenchymal markers in the described SCNP assay could greatly enhance the cellular detection level (∼30% of samples in this pilot study were not analyzed due to low cell numbers) and allow for the quantification of distinct signaling profiles in phenotypically heterogeneous subsets of tumor cells, information with potential prognostic and predictive value (i.e., use of targeted therapies such as tyrosine kinase inhibitors, etc.).
Although significant progress has been made in the characterization of the molecular pathophysiology of bladder cancer tumorigenesis, these advancements have not resulted in the development of better drug treatments or prediction of tumor behavior. As a functional assay of tumor behavior, this technology has far reaching potential in the treatment of bladder cancer. The ability to measure basal (noninduced) and potentiated signaling levels with SCNP assay may be able to predict platinum sensitivity in patients considering neoadjuvant systemic therapy. Identification of specific pathway activation may lead to incorporation of personalized molecular targeted therapy into treatment regimens. Furthermore, improved disease characterization could aid in the identification of which patients will respond to available therapies, i.e., patient stratification. The assay development concepts shown here could also be informative with other body fluids including the potential identification and characterization of CTCs.
In conclusion, our results demonstrate the initial feasibility of applying SCNP assay to the functional characterization of bladder cancer epithelial cells. Furthermore, our study supports the potential of applying functional pathway analysis using SCNP to other solid tumors. Future development of this technology may allow for the correlation of in vitro network signaling profiles with clinical outcomes, enabling not only a prognostic tool but also furthering progress toward individualized treatment strategies.