Individual Tumor Cells Research Articles

Clusters of circulating tumor cells traverse capillary-sized vessels

Multicellular aggregates of circulating tumor cells (CTC clusters) are potent initiators of distant organ metastasis. However, it is currently assumed that CTC clusters are too large to pass through narrow vessels to reach these organs. Here, we present evidence that challenges this assumption through the use of microfluidic devices designed to mimic human capillary constrictions and CTC clusters obtained from patient and cancer cell origins. Over 90% of clusters containing up to 20 cells successfully traversed 5- to 10-μm constrictions even in whole blood. Clusters rapidly and reversibly reorganized into single-file chain-like geometries that substantially reduced their hydrodynamic resistances. Xenotransplantation of human CTC clusters into zebrafish showed similar reorganization and transit through capillary-sized vessels in vivo. Preliminary experiments demonstrated that clusters could be disrupted during transit using drugs that affected cellular interaction energies. These findings suggest that CTC clusters may contribute a greater role to tumor dissemination than previously believed and may point to strategies for combating CTC cluster-initiated metastasis.

Implications of Extracellular Vesicle Transfer on Cellular Heterogeneity in Cancer: What Are the Potential Clinical Ramifications?

The functional and phenotypic heterogeneity of tumor cells represents one of the greatest challenges in the successful treatment of cancer patients, because it increases the risk that certain individual tumor cells possess the ability to, for example, metastasize or to tolerate cytotoxic drugs. This heterogeneity in cellular behavior is driven by genetic and epigenetic changes and environmental differences. Recent studies suggest that an additional layer of complexity of tumor heterogeneity exists, based on the ability of cells to share functional biomolecules through local and systemic transfer of extracellular vesicles (EV), with profound effects on cellular behavior. The transfer of functional biomolecules between various populations of tumor cells and between tumor cells and nontumor cells has large consequences for both the tumor cells and the microenvironment that support the cellular behavior of tumor cells, and therefore for the clinical outcome of cancer. Here, we discuss the latest findings on EV transfer and the potential implications of EV-mediated local and systemic transmission of phenotypic behavior, particularly in the context of tumor heterogeneity, metastatic disease, and treatment response. Cancer Res; 76(8); 2071-5. ©2016 AACR.

New pattern-based personalized risk stratification system for endocervical adenocarcinoma with important clinical implications and surgical outcome

We present a recently introduced three tier pattern-based histopathologic system to stratify endocervical adenocarcinoma (EAC) that better correlates with lymph node (LN) metastases than FIGO staging alone, and has the advantage of safely predicting node-negative disease in a large proportion of EAC patients. The system consists of stratifying EAC into one of three patterns: pattern A tumors characterized by well-demarcated glands frequently forming clusters or groups with relative lobular architecture and lacking destructive stromal invasion or lymphovascular invasion (LVI), pattern B tumors demonstrating localized destructive invasion (small clusters or individual tumor cells within desmoplastic stroma often arising from pattern A glands), and pattern C tumors with diffusely infiltrative glands and associated desmoplastic response. Three hundred and fifty-two cases were included; mean follow-up 52.8months. Seventy-three patients (21%) had pattern A tumors; all were stage I and there were no LN metastases or recurrences. Pattern B was seen in 90 tumors (26%); all were stage I and LVI was seen in 24 cases (26.6%). Nodal disease was found in only 4 (4.4%) pattern B tumors (one IA2, two IB1, one IB not further specified (NOS)), each of which showed LVI. Pattern C was found in 189 cases (54%), 117 had LVI (61.9%) and 17% were stage II or greater. Forty-five (23.8%) patients showed LN metastases (one IA1, 14 IB1, 5 IB2, 5 IB NOS, 11 II, 5 III and 4 IV) and recurrences were recorded in 41 (21.7%) patients. This new risk stratification system identifies a subset of stage I patients with essentially no risk of nodal disease, suggesting that patients with pattern A tumors can be spared lymphadenectomy. Patients with pattern B tumors rarely present with LN metastases, and sentinel LN examination could potentially identify these patients. Surgical treatment with nodal resection is justified in patients with pattern C tumors.

Interobserver Agreement Between Pathologists Assessing Tumor-Infiltrating Lymphocytes (TILs) in Breast Cancer Using Methodology Proposed by the International TILs Working Group.

The presence of tumor-infiltrating lymphocytes (TILs) in breast tumors is prognostic and predictive, suggesting that TILs may be an important biomarker. Recently, an international TILs working group formulated consensus recommendations for TIL evaluation. The current study was performed to determine interobserver agreement using that methodology. Tumor-infiltrating lymphocytes were assessed on a single hematoxylin and eosin (H&E)-stained slide obtained from the core biopsy of 75 triple-negative breast cancers. Four pathologists independently reviewed each slide and evaluated stromal TILs (sTILs) and intratumoral TIL (iTILs). The kappa statistic was used to estimate interobserver agreement for identification of sTILs, and the intraclass correlation coefficient (ICC) was used to estimate the agreement among observers for iTILs. Cases with poor agreement were reviewed to identify pathologic factors that may contribute to the lack of agreement. The kappa statistic for sTIL evaluation was 0.57 (standard error, 0.04). For iTILs, the ICC calculated to determine internal consistency within raters was 0.65 (95% confidence interval [CI] 0.56-0.74; p<0.0001), and the ICC calculated to determine agreement among raters was 0.62 (95% CI 0.50-0.72; p<0.0001). In 10 cases (13%), there was not agreement between three of four pathologists. The pathologic features contributing to difficulty in TIL enumeration included marked individual tumor cell necrosis or apoptosis, the presence of reactive plasma cells mimicking tumor cells, plasmatoid tumor cells, and accurate quantification of TILs in specimens with focal areas of heavy immune infiltrate. Acceptable agreement in TIL enumeration was observed, suggesting that the proposed methodology can be used to facilitate the use of TILs as a biomarker in research and clinical trial settings.

A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues.

Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.

Abstract P6-04-01: Single cell RNA sequencing reveals key expression signatures of primary breast cancer cells and immune infiltrates

Abstract Introduction: Cancers display intratumoral heterogeneity which interferes with the precise analyses of the tumor entity, and which may affect therapeutic outcomes of targeted treatments. The aim of this study was to evaluate the feasibility of single cell RNA sequencing on primary breast cancer cells and to demonstrate the key gene expression signatures and transcriptome heterogeneity of breast cancer subtypes. Methods: We performed RNA sequencing on 246 individual cells from 4 primary breast tumors and 2 metastatic lymph nodes from 4 patients, using C1™Single-Cell Auto Prep System (Fluidigm, South San Francisco, CA). RNA sequencing reads were aligned to the human genome reference (hg19) using the 2-pass default mode of STAR_2.4.0d, and gene expression was quantified by RSEM v1.2.18 as the sum of isoform expression. Results: Pathologic characteristics of the 4 patients is summarized in Table 1. Table 1. Pathologic characteristics of patients BC01BC02BC03BC04pathologic stagepT1N0 (IA)pT2N0 (IIA)pT1N3 (IIIC)pT2N1 (IIB)Immunohistochemistry estrogen receptorpositivenegativenegativepositiveprogesterone receptornegativenegativenegativepositiveHER2 (FISH)2+/3 (negative)3+/3 (positive)1+/3 (negative)3+/3 (positive)No. of single cells tumor (lymph node)214947(50)28(51)HER2, human epidermal growth factor receptor 2; FISH, fluorescence in situ hybridization To distinguish tumor cells, we chose epithelial cell adhesion molecule (EpCAM) as an archetypal epithelial tumor marker and performed gene signature enrichment analysis for the EpCAM-positive cells. A total of 673 genes with enrichment score &amp;gt; |0.5| were selected and used as an "epithelial breast cancer" signature. Consensus clustering with the epithelial breast cancer signature separated 246 cells into 145 epithelial tumor and 101 non-tumor cell groups. Gene expression profiling in the tumor cells revealed many co-regulated genes in the estrogen receptor (ER)-positive tumor at a single cell resolution, which represent previously known ER-associated genes including MYC, BCL-2, and GATA3. Individual tumor cells from the two human epidermal growth factor receptor 2 (HER2)-amplified tumors demonstrated drastically different degree of HER2 signaling pathway activation, indicating the necessity of molecular subtyping for the identification of HER2-activated tumors. TNBC tumor cells showed an overall upregulation of activator protein 1 transcriptional pathway and strong activation of epithelial-mesenchymal transition signatures in a small sub-population. Immune cells comprised all the non-epithelial population, with mostly T lymphocytes in the primary tumor samples and B lymphocytes in the lymph nodes. The tumor-infiltrating T cells expressed an activated phenotype and many cytotoxic components. Altogether, the single cell RNA sequencing revealed the true identity of the tumor cells and the tumor-associated immune cells. Conclusion: Single cell RNA sequencing of breast cancer was used to reveal the true gene expression characteristics of the tumor cells and tumor-associated non-tumor compartments. The results showed key gene expression signatures of specific tumor subtypes and a wide range of transcriptome heterogeneity which is shaped by the tumor and microenvironments. Citation Format: Lee H-B, Eum HH, Chung W, Lee H-O, Lee K-M, Kim K-T, Moon H-G, Noh D-Y, Han W, Park W-Y. Single cell RNA sequencing reveals key expression signatures of primary breast cancer cells and immune infiltrates. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-04-01.

Lipid tethering of breast tumor cells enables real-time imaging of free-floating cell dynamics and drug response

Free-floating tumor cells located in the blood of cancer patients, known as circulating tumor cells (CTCs), have become key targets for studying metastasis. However, effective strategies to study the free-floating behavior of tumor cells in vitro have been a major barrier limiting the understanding of the functional properties of CTCs. Upon extracellular-matrix (ECM) detachment, breast tumor cells form tubulin-based protrusions known as microtentacles (McTNs) that play a role in the aggregation and re-attachment of tumor cells to increase their metastatic efficiency. In this study, we have designed a strategy to spatially immobilize ECM-detached tumor cells while maintaining their free-floating character. We use polyelectrolyte multilayers deposited on microfluidic substrates to prevent tumor cell adhesion and the addition of lipid moieties to tether tumor cells to these surfaces through interactions with the cell membranes. This coating remains optically clear, allowing capture of high-resolution images and videos of McTNs on viable free-floating cells. In addition, we show that tethering allows for the real-time analysis of McTN dynamics on individual tumor cells and in response to tubulin-targeting drugs. The ability to image detached tumor cells can vastly enhance our understanding of CTCs under conditions that better recapitulate the microenvironments they encounter during metastasis.

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

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

Instability in Oncogenesis Directly Implicates Mutator Phenotype Progression through Genetic Lesion Heterogeneity

Theoretical uncertainties regarding the dimensions of mutator mutations are reflected particularly in the parameter uncertainties of the oncogenic pathways that are heterogeneous in individual tumor cells. The key attribute of marked heterogeneity in genomic lesions attests for the essential evolution of the mutator phenotype per se, and allows for the emergence of oncogenesis beyond initial or middle course events in malignant transformation. Although initial or early mutator mutations may prove rare events in oncogenesis, the emergence also of passenger mutations reflect an essential establishment of a mutator phenotype as essential genomic instability and mutability.

Handheld confocal laser endomicroscopic imaging utilizing tumor-specific fluorescent labeling to identify experimental glioma cells in vivo.

Background:We have reported that handheld confocal laser endomicroscopy (CLE) can be used with various nonspecific fluorescent dyes to improve the microscopic identification of brain tumor and its boundaries. Here, we show that CLE can be used experimentally with tumor-specific fluorescent labeling to define glioma margins in vivo.Methods:Thirteen rats underwent craniectomy and in vivo imaging 21 days after implantation with green fluorescent protein (GFP)-labeled U251 (n = 7) cells or epidermal growth factor receptor (EGFR) overexpressing F98 cells (n = 6). Fluorescein isothiocyanate (FITC) conjugated EGFR fluorescent antibody (FITC-EGFR) was applied for contrast in F98 tumors. Confocal images of normal brain, obvious tumor, and peritumoral zones were collected using the CLE system. Bench-top confocal microscopy and hematoxylin and eosin-stained sections were correlated with CLE images.Results:GFP and FITC-EGFR fluorescence of glioma cells were detected by in vivo visible-wavelength fluorescence CLE. CLE of GFP-labeled tumors revealed bright individual satellite tumor cells within peritumoral tissue, a definitive tumor border, and subcellular structures. Imaging with FITC-EGFR labeling provided weaker contrast in F98-EGFR tumors but was able to delineate tumor cells. Imaging with both methods in various tumor regions correlated with standard confocal imaging and clinical histology.Conclusions:These data suggest that in vivo CLE of selectively tagged neoplasms could allow specific interactive identification of tumoral areas. Imaging of GFP and FITC-EGFR provides real-time histologic information precisely related to the site of microscopic imaging of tumor.

Quantitative imaging of magnesium distribution at single-cell resolution in brain tumors and infiltrating tumor cells with secondary ion mass spectrometry (SIMS).

Glioblastoma multiforme (GBM) is one of the deadliest forms of human brain tumors. The infiltrative pattern of growth of these tumors includes the spread of individual and/or clusters of tumor cells at some distance from the main tumor mass in parts of the brain protected by an intact blood-brain-barrier. Pathophysiological studies of GBM could be greatly enhanced by analytical techniques capable of in situ single-cell resolution measurements of infiltrating tumor cells. Magnesium homeostasis is an area of active investigation in high grade gliomas. In the present study, we have used the F98 rat glioma as a model of human GBM and an elemental/isotopic imaging technique of secondary ion mass spectrometry, a CAMECA IMS-3f ion microscope, for studying Mg distribution with single-cell resolution in freeze-dried brain tissue cryosections. Quantitative observations were made on tumor cells in the main tumor mass, contiguous brain tissue, and infiltrating tumor cells in adjacent normal brain. The brain tissue contained a significantly lower total Mg concentration of 4.70±0.93mmol/kg wet weight (mean±SD) in comparison to 11.64±1.96mmol/kg wet weight in tumor cells of the main tumor mass and 10.72±1.76mmol/kg wet weight in infiltrating tumor cells (p<0.05). The nucleus of individual tumor cells contained elevated levels of bound Mg. These observations have established that there was enhanced influx and increased binding of Mg in tumor cells. They provide strong support for further investigation of altered Mg homeostasis and activation of Mg-transporting channels in GBMs as possible therapeutic targets.

THE CYTOTOXIC EFFECT OF CHELIDONIUM MAJUS IN VITRO

The effect of aqueous and ether Chelidonium majus haulms extract on cervical HeLa tumor cells, mammary adenocarcinoma MCF 7 tumor cells and acute lymphoblastic leukemia CEM tumor cells in vitro have been studied. The purpose of this research was to compare the effect of aqueous and ether Chelidonium majus haulms extract on selected tumor cells. Colorimetric MTT assay have been used for the study of the antiproliferative effect of aqueous and ether haulms extract of Chelidonium majus on cell viability in vitro. The results of the experiments have shown the cytotoxic effect of the aqueous and the ether Chelidonium majus haulms extract on the individual tumor cells. The aqueous Chelidonium majus haulms extract was the most effective on CEM cells, it was less effective on MCF 7 cells and it was the least effective on HeLa cells. The ether haulms extract of Chelidonium majus was the most effective at all of studied concentrations on CEM cells and MCF 7 cells in comparison with HeLa cells, where it was significantly effective only at the highest concentration. Aqueous and ether haulms extract of Chelidonium majus tested in vitro indicated their cytotoxic activity. Both haulms extract of Chelidonium majus were more efficient on CEM cells. It is assumed that higher antiproliferative activity of ether haulms extract of Chelidonium majus is the result of higher antiproliferative activity of lipophilic substances. The lipophilic substances pass through membrane and bind to various proteins and change their biological activity.

Single-cell functional analysis of parathyroid adenomas reveals distinct classes of calcium sensing behaviour in primary hyperparathyroidism.

Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder caused by a failure of calcium sensing secondary to tumour development in one or more of the parathyroid glands. Parathyroid adenomas are comprised of distinct cellular subpopulations of variable clonal status that exhibit differing degrees of calcium responsiveness. To gain a clearer understanding of the relationship among cellular identity, tumour composition and clinical biochemistry in PHPT, we developed a novel single cell platform for quantitative evaluation of calcium sensing behaviour in freshly resected human parathyroid tumour cells. Live-cell intracellular calcium flux was visualized through Fluo-4-AM epifluorescence, followed by in situ immunofluorescence detection of the calcium sensing receptor (CASR), a central component in the extracellular calcium signalling pathway. The reactivity of individual parathyroid tumour cells to extracellular calcium stimulus was highly variable, with discrete kinetic response patterns observed both between and among parathyroid tumour samples. CASR abundance was not an obligate determinant of calcium responsiveness. Calcium EC50 values from a series of parathyroid adenomas revealed that the tumours segregated into two distinct categories. One group manifested a mean EC50 of 2.40 mM (95% CI: 2.37-2.41), closely aligned to the established normal range. The second group was less responsive to calcium stimulus, with a mean EC50 of 3.61 mM (95% CI: 3.45-3.95). This binary distribution indicates the existence of a previously unappreciated biochemical sub-classification of PHPT tumours, possibly reflecting distinct etiological mechanisms. Recognition of quantitative differences in calcium sensing could have important implications for the clinical management of PHPT.

The Novel Technique of Genetically Engineered Patient-Derived Xenografts (GEPDX) Reveals That the X-Linked Inhibitor of Apoptosis Protein (XIAP) Plays an Essential Role for Maintenance and Growth of Patients' Acute Lymphoblastic Leukemia In Vivo

Expression profiling and next generation sequencing have enabled a detailed knowledge on alterations present in tumors from individual patients. In contrast, only limited understanding exists on the role that each alteration plays for the existing tumor. Of direct clinical interest, genes are of special interest which harbor an essential function for tumor maintenance and growth as they represent putative targets for anti-cancer therapy.Characterizing gene functions is demanding regarding both techniques and resources. Questions on gene function are often studied in established tumor cell lines, although establishing cell lines from primary tumors is rarely successful in acute leukemias. Primary acute leukemia cells poorly grow in vitro and established acute leukemias cell lines rely on additional mutations enabling in vitro growth, making them doubtful models to study genes with essential function.To bridge the gap, we aimed at studying gene function in the complex environment of individual tumors. As primary acute leukemia cells are unable to grow in vitro, we used the orthotopic model of patient-derived xenograft (PDX) leukemia and amplified cells in mice.We established a novel technique to manipulate distinct signaling proteins in PDX cells using lentiviruses and knockdown. We expressed small hairpin RNA (shRNA) in the background of micro RNA 30 (miR30) under control of a Pol II promoter and 3 prime of dsRED as molecular marker. This approach closely links expression of the shRNA to the fluorochrome and resulted in a potent and stable knockdown. We expressed a control shRNA targeting Renilla luciferase and several shRNA sequences targeting XIAP. In order to discriminate different derivative cell populations within a single mouse, we co-expressed a second fluorochrome from a second plasmid so that green cells harbored a knockdown of XIAP, while blue cells harbored the control construct, thus allowing in vivo outcompete proliferation assays. We called our new approach "genetically engineered PDX (GEPDX)" models in parallel to genetically engineered mouse models (GEMM).We used our new technology to study the role of XIAP, the X-linked inhibitor of apoptosis for acute lymphoblastic leukemia (ALL), the single most frequent tumor in children. XIAP is frequently and highly overexpressed in hematological malignancies and its up-regulation was shown to be associated with inferior prognosis of patients in different tumors. Nevertheless XIAP's role for tumor maintenance remains unclear.In several preB- and T-cell ALL cell lines, potent and stable knockdown of XIAP did not alter cell proliferation in vitro or upon xeno-transplantation in vivo. Thus expression levels of XIAP seem irrelevant for spontaneous proliferation of established acute leukemia cell lines in vitro and in vivo.We next studied PDX ALL cells growing in mice as model closer related to patients. We generated GEPDX cells from two children with relapse of a B precursor ALL with either knockdown of XIAP or control knockdown together with the appropriate molecular color markers. When blue and green GEPDX cells harboring control or XIAP knockdown, respectively, were co-transplanted into mice at a 1:1 ratio in a competitive outcompete assay, control transfected cells significantly overgrew or even eliminated cells with XIAP knockdown in both samples studied. GEPDX cells with knockdown of XIAP showed a significant and dose-dependent growth disadvantage in vivo compared to control cells indicating that XIAP played an essential role for PDX cells growing in vivo.Thus, our novel technique of genetic engineering in PDX cells revealed an essential role of XIAP for tumor maintenance and growth in patients' tumor cells making XIAP an attractive therapeutic target in ALL. As established ALL cell lines were unable to unravel this important role of XIAP, GEPDX might be superior to cell lines for identifying genes with essential function. GEPDX represent a powerful new tool to characterize the complex environment of individual patients' tumor cells in vivo, the function of the many lesions and alterations described by expression profiling and next generation sequencing. DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-B18: Epithelial to mesenchymal transition in human tumor biopsies: Quantitative, histopathological proof of the existence of EMT in vivo by immunofluorescence microscopy

Abstract Epithelial-mesenchymal transition (EMT) is a dynamic process whereby epithelial cells acquire mesenchymal properties. Despite the clinical significance of the acquired mesenchymal properties for metastasis and drug resistance, histopathological evidence of transitional cells in patient samples is lacking and EMT remains an unproven clinical hypothesis. We previously developed and validated a multiplex immunofluorescence assay (IFA) that quantifies the levels of EMT biomarkers (E-Cadherin, Vimentin, β-catenin) in snap-frozen, formalin-fixed, paraffin-embedded (FFPE) tumor tissue (Navas et al, AACR 2013). Building upon that method, we now report a precise, quantitative and unbiased IFA method of tissue analysis (EMT-IFA) using Definiens® software to quantify co-expression of the epithelial marker E-cadherin (E) and mesenchymal marker Vimentin (V) at the cellular level in FFPE clinical biopsies. FFPE human tumor xenografts and cell lines serve as calibrators and reference materials for establishing initial image acquisition parameters for segmented tumor regions of interest. Flanking H&amp;E sections of the clinical biopsies are initially annotated by an anatomic pathologist to identify regions of tumor tissue, non-tumor related areas (including normal or stromal tissues), inflammatory and/or necrotic regions, and freeze artifacts. Adjacent sections are used for the EMT-IFA, and further tumor segmentation from stroma is applied to extracted regions of interests (fields) using the β-catenin layer. Within segmented tumor regions, E-cadherin (E) and Vimentin (V) sub-cellular pixel areas are measured using predetermined thresholds for each biomarker and reported as Log10 (V:E). High resolution images confirmed the co-localization of plasma membrane E-cadherin and cytoplasmic Vimentin to individual transitional tumor cells, and the co-localization of E and V within individual tumor cells of the segmented regions of interest was key for distinguishing EMT from tumor heterogeneity in which adjacent tumor cells can be exclusively E+ or V+. Quantitative analysis of a clinical biopsy series of various histologies revealed all possible phenotypes: epithelial (E+V- colorectal carcinoma), mesenchymal (E-V+ sarcomas), heterogeneous mixtures of E+V- and E-V+ subpopulations, and EMT. Importantly, using the EMT-IFA, we discovered that pharmacological targeting of VEGFR/FGFR/PDGFR signaling with the multi-kinase inhibitor pazopanib stimulated EMT in a preclinical xenograft model of gastric carcinoma (MKN45), as revealed by the significant increase in the V:E ratio (P = 0.0023) coupled with an increase in the EMT phenotype (co-localized V+E+ at the individual cell level) (P = 0.0070) in biopsy specimens. Thus, the EMT-IFA has potential value for investigating EMT in the clinic and its ramifications for drug response and other clinical endpoints. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Tony Navas, Robert J. Kinders, Scott M. Lawrence, Katherine Ferry-Galow, Thomas D. Pfister, Apurva K. Srivastava, Sergio Y. Alcoser, Melinda G. Hollingshead, Lindsay M. Dutko, Brad A. Gouker, Donna O. Butcher, Hala Makhlouf, Rodrigo Chuaqui, Donald P. Bottaro, Shivaani Kummar, Alice Chen, James H. Doroshow, Ralph E. Parchment. Epithelial to mesenchymal transition in human tumor biopsies: Quantitative, histopathological proof of the existence of EMT in vivo by immunofluorescence microscopy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B18.

Targeted Therapies for Non-Small Cell Lung Cancer: An Update on Epidermal Growth Factor Receptor and Anaplastic Lymphoma Kinase Inhibitors.

The development of targeted therapies has revolutionized the treatment of advanced non-small cell lung cancer (NSCLC), with new clinical trials and therapies consistently providing new information. This rapidly changing field mandates ongoing education for nursing professionals whose foremost priority is patient care. This review aims to summarize the history and current status of targeted therapies for NSCLC, focusing on two types of drugs that have had the most impact to date. The safety profiles of first- and second-generation EGFR and ALK inhibitors are described, and strategies for the management of the most commonly experienced adverse events are summarized. Information is also provided to help identify which patients might be eligible for treatment with EGFR or ALK inhibitors in addition to the implications of targeted therapies. Therapies designed to target specific molecular features of individual tumor cells are one of the most important developments in treating NSCLC. The safety profiles of targeted therapies differ greatly from chemotherapy and present unique challenges to nurses. Education of nurses and patients on implementation of effective adverse event management and improvement in patient adherence will maximize the benefits of these drugs.

Potential role of the glycolytic oscillator in acute hypoxia in tumors

Tumor acute hypoxia has a dynamic component that is also, at least partially, coherent. Using blood oxygen level dependent magnetic resonance imaging, we observed coherent oscillations in hemoglobin saturation dynamics in cell line xenograft models of head and neck squamous cell carcinoma. We posit a well-established biochemical nonlinear oscillatory mechanism called the glycolytic oscillator as a potential cause of the coherent oscillations in tumors. These data suggest that metabolic changes within individual tumor cells may affect the local tumor microenvironment including oxygen availability and therefore radiosensitivity. These individual cells can synchronize the oscillations in patches of similar intermediate glucose levels. These alterations have potentially important implications for radiation therapy and are a potential target for optimizing the cancer response to radiation.

P148: CD68 expression in inflammatory cell infiltration of nonspecific invasive breast cancer

Background Tumor-associated macrophages play a main role in tumor progression and dissemination. Taking into account the high heterogeneicity of tumor the different clinical impact of macrophages, infiltrating different sites of tumor, could be expected. The aim was to detect the level of CD68+ cells (macrophages) in the different site of stroma in breast tumor in comparison to clinical course. Materials and methods One thirty-six women with nonspecific invasive breast cancer T1-4N0-3M0, who were treated in General Oncology Department of Tomsk Cancer Research Institute (Tomsk, Russia), were included in the present study. Patients did not receive preoperative treatment. The material was fixed in 10–12% neutral formalin. Preparation of the histological material was carried out according to standard procedures. Morphological examination of the surgical specimens was performed by the standard method using a light microscope “Carl Zeiss Axio Lab.A1” (Germany) and slidescanner “MiraxMidiZeiss” (Germany). Metastatic lesion was detected in regional lymph nodes. Immunohistochemical study was performed according to standard procedures. Cytoplasmic expression of these markers was determined in the inflammatory cell infiltrate of different tumor segments: (1) in areas with soft fibrous stroma; (2) in areas with coarse fibrous stroma; (3) in the areas of the so-called “maximum stromal-and-parenchymal relationship” where the individual tumor cells, short strands and groups of tumor cells arranged in soft fibrous stroma; (4) among parenchymal elements; (5) in gaps of ductal tumor structures. Double-stained immunofluorescence was performed according to standard procedures using Leica TCS SP2 laser-scanning spectral confocal microscope (Germany). The following primary antibodies were used: mouse monoclonal anti-human CD68 (BD Biosciences) and rabbit polyclonal anti-stabilin-1 or RS1 (marker of M2 macrophages). Results The highest expression of CD68 in the inflammatory cell infiltrate was detected more frequently in areas with soft fibrous stroma (54%) or the so-called “maximum stromal-and-parenchymal relationship” (79%) in patients with breast cancer. The lowest expression of CD68 was observed in areas with coarse fiber stroma (23%). The CD68-positive cells of the inflammatory infiltrate were located between parenchymal elements of tumor (88%). Inverse correlation ( R = −0.67; p = 0.02) observed between tumor size and the expression of CD68 in the cells of the inflammatory infiltrate in gaps of tubular tumor structures. The CD68 expression in cells of the inflammatory tumor infiltrate was correlated with the presence of metastatic regional lymph nodes. It was found that in the case of the lymph node metastases the average score of CD68 expression in cells of ductal gaps tumor structures was lower (1.4 ± 0.5) in comparison with the negative lymph nodes case (3.1 ± 1.0; F = 10.9; p = 0.007). Same time no correlation between the CD68 expression in the inflammatory cell tumor infiltrate and the rate of tumor malignancy was found. Using confocal microscopy domination of CD68 + /RS1 + cells were found. Conclusion So, low CD68 expression level in ductal gaps tumor structures is associated with the presence of metastatic regional lymph nodes.

GENO-04REGULATION OF GLIOBLASTOMA BY NOVEL LONG NONCODING RNA (lncRNA)

The human genome produces many thousands of long noncoding RNAs (lncRNAs) – transcripts >200 nucleotides long with little evidence of protein coding potential. It is now clear that lncRNAs can have critical biological functions and roles in human disease including cancer. Because lncRNAs are particularly cell and disease specific, they are attractive as therapeutic targets. However, very few specific lncRNAs have been identified as potential targets for cancer therapy. We have recently identifiedPinky (Pnky) as a novel lncRNA that regulates the production of neurons from neural stem cells (NSCs) in the embryonic and adult brain (Ramos et al., Cell Stem Cell 2013; Ramos et al., Cell Stem Cell 2015). Pnky-deficiency in NSCs increased neuronal differentiation, depleting the NSC population. Moreover, human GBM tumors expressed the human PNKY transcript, and single cell RNA-seq data analysis indicated that PNKYis present in a specific subset of individual GBM tumor cells. In the Ink/Arf-null, EGFRvIII mouse glioma model, Pnky was also found to be highly expressed, and ongoing studies indicate a function for Pnky in this brain tumor model. Upcoming results may reveal the critical importance of Pnky and other specific lncRNAs in the proliferation of GBM. Overall, our work lays important groundwork for the development of lncRNAs as a large new class of therapeutic targets for the treatment of gliomas.

P77: Clonal evolution of breast tumor during neoadjuvant chemotherapy and metastasis

Background There are numerous evidences suggesting that tumor evolution follows the laws of Darwinian evolution, whereby individual tumor cell clones have private genetic aberrations, including chromosomal abnormalities. The combined effect of genetic instability and differential selective pressures of the microenvironment and chemotherapy can result in the creation of new tumor clones (Navin et al., 2011; Ng et al., 2012). The aim of this study is to show breast tumor clonal evolution during neoadjuvant chemotherapy (NAC) using microarray analysis. Material and methods Breast cancer patients ( n = 26) with stage IIA to IIIC (T1-4N0-3M0), were treated with NAC (FAC or CAX regimens). DNA was extracted from 26 samples of tumor tissue derived before or after NAC using QIAamp DNA mini Kit (Qiagen, Germany). Copy Number Aberrations (CNA, deletions and amplifications, or Loss and Gain, respectively) and number of mutant clones were detected in pre- and post-NAC tumor samples using the high density microarray platform Affymetrix (USA) CytoScanTM HD Array. This study was approved by Tomsk Cancer Research Institute review board. Results We have revealed that 19% (5/26) of patients during the NAC showed the decrease in the number of mutant clones and CNA frequency right up to their complete elimination (genetic regression) at one case. In 7 (27%) cases chemotherapy had no any effect on number of mutant clones and the frequency of CNA in tumor. In the tumors of 10 patients the elimination of some mutant clones as well as the formation of new clones with deleted genetic material occurred under the influence of chemotherapy. 6 patients have demonstrated the appearance of new tumor clones with gene amplifications which were associated with the development of metastases in 83% of cases (5/6). All other patients ( n = 21) who has not acquired the new tumor clones with Gain function mutation after NAC did not manifest distant metastasis in 5-year follow-up (Kaplan–Meier, p = 0.00001 Log-rank test). Conclusion The first time evidence is presented that the formation of new tumor clones may occur during the NAC. Metastasis of breast cancer is associated with the appearance of new clones with DNA amplifications. Detection of these clones allow getting new prognostic factor in breast cancer. The study was supported by the Russian Foundation for Basic Research – Russia (Project 15-04-03091).