Supplementary Figure 1 from Preparing the “Soil”: The Primary Tumor Induces Vasculature Reorganization in the Sentinel Lymph Node before the Arrival of Metastatic Cancer Cells
ABSTRACT. Metanephroi, the embryonic precursors of the adult kidney, can be induced in vivo to grow and develop. Despite their potential clinical utility for transplantation, the ability of human metanephroi to differentiate after transplantation into functional mature nephrons is mostly unknown. To address this, 70-d human metanephroi were transplanted into NOD/SCID mice; global gene expression patterns that underlie development of human metanephric transplants were analyzed and compared with normal human kidney development. In addition, functionality of the grafts was assessed by dimercaptosuccinic acid radioisotope scans at different times after transplantation. The results of hybridization to cDNA arrays when RNA was derived from normal human kidneys at 8, 12, 16, and 20 wk gestation demonstrated that a subset of 240 genes changed substantially with time. The induced genes can be classified as cell cycle regulators, transcription and growth factors, and signaling, transport, adhesion, and extracellular matrix molecules. Strikingly, clustering analysis of global gene expression at 2, 6, and 10 wk after metanephric transplantation revealed an expression profile that characterizes normal human kidney development. Moreover, maturation of the transplants was accompanied by an increased uptake of dimercaptosuccinic acid. Nevertheless, expression levels of specific genes were mostly found to be suppressed in the transplants compared with the normal kidneys. These data provide insights into human kidney development and support the possibility of the transplantability of human metanephroi. Understanding of the molecular regulation of the transplanted developing metanephroi might lead to the development of strategies aimed at increasing the levels of specific genes, nephron endowment, and graft function.
The tyrosine kinase receptor Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), play an important role in normal developmental processes, as well as in tumorigenicity and metastasis. We constructed a green fluorescent protein (GFP) Met chimeric molecule that functions similarly to the wild-type Met receptor and generated GFP-Met transgenic mice. These mice ubiquitously expressed GFP-Met in specific epithelial and endothelial cells and displayed enhanced GFP-Met fluorescence in sebaceous glands. Thirty-two percent of males spontaneously developed adenomas, adenocarcinomas, and angiosarcomas in their lower abdominal sebaceous glands. Approximately 70% of adenocarcinoma tumors metastasized to the kidneys, lungs, or liver. Quantitative subcellularresolution intravital imaging revealed very high levels of GFP-Met in tumor lesions and in single isolated cells surrounding them, relative to normal sebaceous glands. These single cells preceded the formation of local and distal metastases. Higher GFP-Met levels correlated with earlier tumor onset and aggressiveness, further demonstrating the role of Met-HGF/SF signaling in cellular transformation and acquisition of invasive and metastatic phenotypes. Our novel mouse model and high-resolution intravital molecular imaging create a powerful tool that enables direct realtime molecular imaging of receptor expression and localization during primary events of tumorigenicity and metastasis at single-cell resolution.
Abstract Triple-negative breast cancer (TNBC) is a type of aggressive breast cancer lacking the expression of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor-2 (HER-2). TNBC patients have a high propensity for distant metastasis and limited treatment options. Cancer cell motility and invasion are fundamental steps in metastasis. MET, a tyrosine kinase receptor, and its ligand, Hepatocyte Growth Factor/Scatter Factor (HGF/SF), induce specific signal transduction in tumor cells leading to cell motility and metastasis. MET's induced tumorigenesis and metastatic processes make it an ideal target for anti-cancer therapy. We characterized the motility patterns of m-Cherry labeled TNBC cells (BT-549, MDA-MB-231) and ER-positive cells (MCF7) subjected to time-lapse fluorescence wound healing assay. We also studied the effect of MET inhibition, chemotherapy, and their combined impact on breast cancer motility. Time-lapse images were subjected to commercial packages for segmentation and tracking - allowing us to extract morphokinetic (MK) information at a single-cell resolution. To better understand cell motility patterns, we developed the Tool for Analysis of Single-Cell (TASC) infrastructure, which utilizes unsupervised machine learning methods to demonstrate a high-dimensional feature set at a single-cell resolution. We compared our experimental MK results to simulations of three modified classical physical models: Lévy flight (LF), fractal Brownian motion, and random walk (RW) with or without a back-propagating wave (BPW). TASC analysis demonstrated a fundamental difference between ER-positive and TNBC cells: both cell types contained two subpopulations harboring similar MK characteristics. TNBC cells presented an additional subpopulation characterized by 1) dominantly increasing cumulative kinetics values (mean square displacement - MSD), 2) highest temporal wave values (velocity starting-time), and 3) a decrease in non-cumulative kinetics values. The combined treatment of TNBC cells with MET inhibition and chemotherapy eliminates this high-motility group. Cell motility models and motion simulations were compared using Wasserstein's analysis. Untreated TNBC cells showed high similarities to RW with BPW simulations, while MET-activated TNBC cells transformed into LF without BPW simulations, indicating more aggressive behavior. TASC analysis revealed a previously undefined high motility subpopulation of TNBC that was eradicated by combining MET inhibition and chemotherapy. The primary benefit of TASC is the ability to cluster and characterize similar behaviors across different motility models while still detecting subtle changes within each one. MK analysis combined with TASC can lead to discovering novel targets for precise therapy. Moreover, this infrastructure may determine patient tumor susceptibility to chemotherapy and biological treatments and operate as an analytical tool for personalized, targeted therapy. Citation Format: Ilan Tsarfaty, Or Megides, Ohad Doron, Hagar Alaloof, Ori Moskowitz, Judith Horev. Morphokinetic single-cell analysis and machine learning as a tool to characterize breast cancer cell motility and response to therapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PO-089.
Abstract Metastasis is the major cause for cancer patients’ death and despite all the recent advances in cancer research it is still mostly incurable. Understanding the mechanisms that are involved in the migration of the cells in a complex environment is a key step towards successful anti-metastatic treatment. Using experimental data-based modeling, we focus on the fundamentals of metastatic invasion: motility, invasion, proliferation and metabolism and study how they may be combined to maximize the cancer’s ability to metastasize. The modeled cells’ performance is measured by the number of cells that succeed in migration in a maze, which mimics the extracellular environment. We show that co-existence of different cell clones in the tumor, as often found in experiments, optimizes the invasive ability in a frequently-changing environment. We study the role of metabolism and stimulation by growth factors and show that metabolism plays a crucial role in the metastatic process and should therefore be targeted for successful treatment.
Abstract During the past decade, considerable efforts have been devoted to the identification of agents that selectively kill neoplastic breast cancer cells based on their signaling and metabolic alterations. We hypothesis that combination tyrosine kinase and metabolic inhibitors are potential novel therapies for breast cancer. Aberrant signaling through Met-tyrosine kinase receptor and its ligand hepatocyte growth factor/scatter factor (HGF/SF) has been shown to play a critical role in development of tumors and metastasis in a variety of cancers, including breast cancer. We have previously shown that HGF/SF-induced Met-activation leads to diverse metabolic alteration in the tumor. Activation of Met signaling increases glycolytic flux into lactate and increases glycolysis, oxidative phosporylation, oxygen consumption, and tumor blood volume. The voltage-dependent anion channel 1 (VDAC1), located in the mitochondrial outer membrane, functions as gatekeeper for the entry and exit of mitochondrial metabolites, thereby controlling crosstalk between mitochondria and the rest of the cell. VDAC1 is also a key player in mitochondria-mediated apoptosis. Thus, in addition to regulating the metabolic and energetic functions of mitochondria, VDAC1 appears to act as a convergence point for a variety of cell survival and cell death signals, mediated by its association with various ligands and proteins. Shoshan-Barmatz’s lab developed the retro-Tf-D-LP4 peptide, a cell-penetrating VDAC1-based peptide that acts by inducing apoptosis and impairment of cell energy and metabolism. To study the possible therapeutic effect of Met inhibition and alteration of VDAC1 combined therapy on breast tumor cells, we evaluated cell proliferation and motility in BC cells. Using our newly developed single-cell 35 parameters morphokinetic analysis infrastructure, we demonstrate that after 24 hours, the peptide decreased all cell lines velocity (5µM; MDMDA-MB-231_velocity=8.2%, p=0.024. 15µM; MDMDA-MB-231_velocity=62.4%, p<0.001). The peptide in combination with Met inhibitor further decreased cell velocity and acceleration (MDMDA-MB-231_Velocity=85.9%, p<0.001; MDMDA-MB-231_Acceleration=76%, p<0.001). The combined treatment increased cell coordination but abrogated the back-propagating wave. The morphologic effect of the treatment was dependent on the cell line. Cluster analysis of morphokinetic parameters demonstrated that untreated or low doses of treatment MDA-MB-231 and HB2 are clustered together; MCF7 and T47D have slower motility parameters. Upon high-dose combined treatment MDA-MB-231 cells change are clustered with slow-velocity T47D and MCF7. Combined Met inhibition and VDAC1 alteration dramatically reduced cell motility and increased coordinated motility. In vitro experiments show a slight inhibitory effect of VDAC1 on MDA-MB-231 cell proliferation. However, in vivo experiments demonstrate an inhibitory effect on tumor growth. These results indicate that the combined therapy can reduce cell motility and metastasis in triple-negative breast cancer tumors. Citation Format: Guy Namir, Varda Shoshan-Barmatz, Ilan Tsarfaty. Met and VDAC1 as target for breast cancer therapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B24.
To objectively quantify the expression and prognostic implications of the met protooncogene product (Met) in human breast cancer.One hundred eighty-two cases of primary human breast cancer were collected. Both the normal and tumor portions of the original surgical pathology specimen were immunostained for Met and imaged using laser scanning confocal microscopy. Then the cases were ranked according to relative concentrations of normal and tumor Met expression. Subsequently, they were quantified using image analysis and the results correlated with clinical outcome to determine the prognostic value of relative levels of Met.Using a quantitative index to evaluate the relative levels of Met expression, high levels of Met expression in the tumor as compared with the adjacent normal ducts predicted poor prognosis for overall survival and metastasis-free survival. The risk ratio for elevated Met expression was 3.94 (P = .0009). This new method also allows determination of the clinical relevance of low levels of Met in the tumor. The overall survival between the patient population with higher, lower and unchanged levels of Met in normal tissue as compared to tumor were significantly different (P = .0020).Our studies suggest that in a subpopulation of node-negative breast cancer patients, either high or low levels of Met in tumor tissue relative to normal tissue is an indicator of poor overall survival (P = .0068). Thus, Met expression could be useful for identifying node-negative patients who could benefit from adjuvant therapy.
Multi-cellular segmentation of bright field microscopy images is an essential computational step when quantifying collective migration of cells in vitro. Despite the availability of various tools and algorithms, no publicly available benchmark has been proposed for evaluation and comparison between the different alternatives. A uniform framework is presented to benchmark algorithms for multi-cellular segmentation in bright field microscopy images. A freely available set of 171 manually segmented images from diverse origins was partitioned into 8 datasets and evaluated on three leading designated tools. The presented benchmark resource for evaluating segmentation algorithms of bright field images is the first public annotated dataset for this purpose. This annotated dataset of diverse examples allows fair evaluations and comparisons of future segmentation methods. Scientists are encouraged to assess new algorithms on this benchmark, and to contribute additional annotated datasets.
Fyn kinase is implicated in prostate cancer.We illustrate the role of miR-125a-3p in cellular pathways accounted for motility and migration of prostate cancer cells, probably through its regulation on Fyn expression and Fyn-downstream proteins. Prostate cancer PC3 cellswere transiently transfected with empty miR-Vec (control) or with miR-125a-3p.Overexpression of miR-125a-3p reduced migration of PC3 cells and increased apoptosis.Live cell confocal imaging indicated that overexpression of miR-125a-3p reduced the cells' track speed and length and impaired phenotype.Fyn, FAK and paxillin, displayed reduced activity following miR-125a-3p overexpression.Accordingly, actin rearrangement and cells' protrusion formation were impaired.An inverse correlation between miR-125a-3p and Gleason score was observed in human prostate cancer tissues.Our study demonstrated that miR-125a-3p may regulate migration of prostate cancer cells.
