Maya Dadiani

Real-time Imaging of Lymphogenic Metastasis in Orthotopic Human Breast Cancer

Metastatic spread to regional lymph nodes is one of the earliest events of tumor cell dissemination and presents a most significant prognostic factor for predicting survival of cancer patients. Real-time in vivo imaging of the spread of tumor cells through the lymphatic system can enhance our understanding of the metastatic process. Herein, we describe the use of in vivo fluorescence microscopy imaging to monitor the progression of lymph node metastasis as well as the course of spontaneous metastasis through the lymphatic system of orthotopic MDA-MB-231 human breast cancer tumors in severe combined immunodeficient mice. High-resolution noninvasive visualization of metastasizing cancer cells in the inguinal lymph nodes was achieved using cells expressing high levels of red fluorescent protein. Sequential imaging of these lymph nodes revealed the initial invasion of the tumor cells through the lymphatic system into the subcapsular sinuses followed by intrusion into the parenchyma of the nodes. FITC-dextran injected i.d. in the tumor area enabled simultaneous tracking of lymphatic vessels, labeled in green, and disseminated red cancer cells within these vessels. Fast snapshots of spontaneously metastasizing cells in the lymphatic vessels monitored the movement of a few tumor cells and the development of clumps clustered at lymphatic vessel junctions. Quantification of high interstitial fluid pressure (IFP) in the tumors and fast drainage rates of the FITC-dextran into the peritumoral lymphatic vessels suggested an IFP-induced intravasation into the lymphatic system. This work presents unprecedented live fluorescence images that may help to clarify the steps occurring in the course of spontaneous lymphogenic metastasis.

Two DNA-encoded strategies for increasing expression with opposing effects on promoter dynamics and transcriptional noise

Individual cells from a genetically identical population exhibit substantial variation in gene expression. A significant part of this variation is due to noise in the process of transcription that is intrinsic to each gene, and is determined by factors such as the rate with which the promoter transitions between transcriptionally active and inactive states, and the number of transcripts produced during the active state. However, we have a limited understanding of how the DNA sequence affects such promoter dynamics. Here, we used single-cell time-lapse microscopy to compare the effect on transcriptional dynamics of two distinct types of sequence changes in the promoter that can each increase the mean expression of a cell population by similar amounts but through different mechanisms. We show that increasing expression by strengthening a transcription factor binding site results in slower promoter dynamics and higher noise as compared with increasing expression by adding nucleosome-disfavoring sequences. Our results suggest that when achieving the same mean expression, the strategy of using stronger binding sites results in a larger number of transcripts produced from the active state, whereas the strategy of adding nucleosome-disfavoring sequences results in a higher frequency of promoter transitions between active and inactive states. In the latter strategy, this increased sampling of the active state likely reduces the expression variability of the cell population. Our study thus demonstrates the effect of cis-regulatory elements on expression variability and points to concrete types of sequence changes that may allow partial decoupling of expression level and noise.

High-resolution magnetic resonance imaging of disparities in the transcapillary transfer rates in orthotopically inoculated invasive breast tumors

In vivo mapping of the transcapillary fluxes in tumors can help predict the efficacy of delivery of blood-borne anticancer drugs. These fluxes are primarily affected by the vascular permeability and the pressure gradients across the blood vessels walls. We describe herein high-resolution dynamic contrast-enhanced magnetic resonance imaging of the influx and outflux transcapillary transfer rates in vivo in invasive MDA-MB-231 tumors orthotopically inoculated in severe combined immunodeficient mice. The tumors were noted for rapid growth, impaired drainage of fluid, and subsequent formation of cysts. Consequently, the time evolution of the contrast enhancement, induced by i.v. injection of Gadolinium diethylene-triamine-penta-acetate, exhibited two distinct patterns: transcapillary transfer in the cellular regions and simple diffusion in the cyst fluid. Both processes were analyzed at pixel resolution applying to each a physiological model and a corresponding algorithm. In the cellular region, the influx and outflux transcapillary transfer rates decreased during tumor growth; however, an increased disparity between the transfer constants was observed, with the outflux rate exceeding the influx rate. This quantitative spatial and temporal mapping of this disparity can provide a means to assess the physiological barriers to tracer delivery. It is hypothesized that both the increased disparity in transcapillary transfer rates and impaired fluid drainage in these tumors could arise from the development of interstitial hypertension.

Estrogen regulation of vascular endothelial growth factor in breast cancer in vitro and in vivo: the role of estrogen receptor α and c-Myc

The role of c-Myc in estrogen regulation of vascular endothelial growth factor (VEGF) and of the vasculature function has been investigated in breast cancer cells and tumors. The studies were performed on MCF7 wild-type cells and MCF7-35im clone, stably transfected with an inducible c-Myc gene. In vitro and ex vivo methods for investigating molecular events were integrated with in vivo magnetic resonance imaging of the vascular function. The results showed that the c-Myc upregulation by estrogen is necessary for the transient induction of VEGF transcription; however, overexpression of c-Myc alone is not sufficient for this induction. Furthermore, both c-Myc and the activated estrogen receptor alpha (ERalpha) were shown to co-bind the VEGF promoter in close proximity, indicating a novel mechanism for estrogen regulation of VEGF. Studies of long-term estrogen treatment and overexpression of c-Myc alone demonstrated regulation of stable VEGF expression levels in vitro and in vivo, maintaining steady vascular permeability in tumors. However, withdrawal of estrogen from the tumors resulted in increased VEGF and elevated vascular permeability, presumably due to hypoxic conditions that were found to dominate VEGF overexpression in cultured cells. This work revealed a cooperative role for ERalpha and c-Myc in estrogen regulation of VEGF and the ability of c-Myc to partially mimic estrogen regulation of angiogenesis. It also illuminated the differences in estrogen regulation of VEGF during transient and long-term sustained treatments and under different microenvironmental conditions, providing a complementary picture of the in vitro and in vivo results.

Neo-adjuvant doxorubicin and cyclophosphamide followed by paclitaxel in triple-negative breast cancer among BRCA1 mutation carriers and non-carriers

The purpose of this study was to assess pathological complete response and whether it serves a surrogate for survival among patients receiving neo-adjuvant doxorubicin–cyclophosphamide followed by paclitaxel for triple-negative breast cancer with respect to BRCA1 mutation status. From a neo-adjuvant systemic therapy database of 588 breast cancer cases, 80 triple-negative cases who had undergone BRCA genotyping were identified. Logistic regression model was fitted to examine the association between BRCA1 status and pathological complete response. Survival outcomes were evaluated using Kaplan–Meier method, differences between study groups calculated by log-rank test. Thirty-four BRCA1 carriers and 43 non-carriers were identified. The BRCA1 carriers had pathological complete response rate of 68xa0% compared with 37xa0% among non-carriers, pxa0=xa00.01. Yet this did not translate into superior survival for BRCA1 carriers compared with non-carriers. No difference in relapse-free survival were noted among those with or without pathological complete response in BRCA1 carriers regardless of pathological complete response status (Log-rank pxa0=xa00.25), whereas in the non-carrier cohort, relapse-free survival was superior for those achieving pathological complete response (Log-rank pxa0<xa00.0001). Response to neo-adjuvant systemic therapy differed in BRCA1-associated triple-negative breast cancer compared with triple-negative non-carriers, with a higher rate of pathological complete response. However, compared with non-carrier triple-negative breast cancer, pathological complete response was not a surrogate for superior relapse-free survival in BRCA1 patients. Future studies using specific chemotherapy regimens may provide further improvements in outcomes.

Tumor Evolution Inferred by Patterns of microRNA Expression through the Course of Disease, Therapy, and Recurrence in Breast Cancer

Purpose: Molecular evolution of tumors during progression, therapy, and metastasis is a major clinical challenge and the main reason for resistance to therapy. We hypothesized that microRNAs (miRNAs) that exhibit similar variation of expression through the course of disease in several patients have a significant function in the tumorigenic process. Experimental design: Exploration of evolving disease by profiling 800 miRNA expression from serial samples of individual breast cancer patients at several time points: pretreatment, posttreatment, lymph nodes, and recurrence sites when available (58 unique samples from 19 patients). Using a dynamic approach for analysis, we identified expression modulation patterns and classified varying miRNAs into one of the eight possible temporal expression patterns. Results: The various patterns were found to be associated with different tumorigenic pathways. The dominant pattern identified an miRNA set that significantly differentiated between disease stages, and its pattern in each patient was also associated with response to therapy. These miRNAs were related to tumor proliferation and to the cell-cycle pathway, and their mRNA targets showed anticorrelated expression. Interestingly, the level of these miRNAs was lowest in matched recurrent samples from distant metastasis, indicating a gradual increase in proliferative potential through the course of disease. Finally, the average expression level of these miRNAs in the pretreatment biopsy was significantly different comparing patients experiencing recurrence to recurrence-free patients. Conclusions: Serial tumor sampling combined with analysis of temporal expression patterns enabled to pinpoint significant signatures characterizing breast cancer progression, associated with response to therapy and with risk of recurrence. Clin Cancer Res; 22(14); 3651–62. ©2016 AACR.

mRNA-seq whole transcriptome profiling of fresh frozen versus archived fixed tissues

BackgroundThe main bottleneck for genomic studies of tumors is the limited availability of fresh frozen (FF) samples collected from patients, coupled with comprehensive long-term clinical follow-up. This shortage could be alleviated by using existing large archives of routinely obtained and stored Formalin-Fixed Paraffin-Embedded (FFPE) tissues. However, since these samples are partially degraded, their RNA sequencing is technically challenging.ResultsIn an effort to establish a reliable and practical procedure, we compared three protocols for RNA sequencing using pairs of FF and FFPE samples, both taken from the same breast tumor. In contrast to previous studies, we compared the expression profiles obtained from the two matched sample types, using the same protocol for both. Three protocols were tested on low initial amounts of RNA, as little as 100xa0ng, to represent the possibly limited availability of clinical samples. For two of the three protocols tested, poly(A) selection (mRNA-seq) and ribosomal-depletion, the total gene expression profiles of matched FF and FFPE pairs were highly correlated. For both protocols, differential gene expression between two FFPE samples was in agreement with their matched FF samples. Notably, although expression levels of FFPE samples by mRNA-seq were mainly represented by the 3′-end of the transcript, they yielded very similar results to those obtained by ribosomal-depletion protocol, which produces uniform coverage across the transcript. Further, focusing on clinically relevant genes, we showed that the high correlation between expression levels persists at higher resolutions.ConclusionsUsing the poly(A) protocol for FFPE exhibited, unexpectedly, similar efficiency to the ribosomal-depletion protocol, with the latter requiring much higher (2–3 fold) sequencing depth to compensate for the relative low fraction of reads mapped to the transcriptome. The results indicate that standard poly(A)-based RNA sequencing of archived FFPE samples is a reliable and cost-effective alternative for measuring mRNA-seq on FF samples. Expression profiling of FFPE samples by mRNA-seq can facilitate much needed extensive retrospective clinical genomic studies.

PO-312 Longitudinal transcriptomics reveals heterogeneous dynamics through the course of disease and therapy in breast cancer

Introduction Tumours are continuously evolving through their course of progression and treatment, a major process contributing to resistance to therapy. Increasingly, it becomes clear that unravelling the dynamics of gene expression over time, during stages of cancer progression and therapy is fundamental to interpretation of tumour evolution and resistance mechanisms. We postulate that in order to understand the emergence of resistance it is important to immensely profile matched biopsies from individual patients accompanied with their long-term clinical follow-up. Material and methods We collected triplets of archived samples from 33 individual patients that underwent neo-adjuvant (preoperative) treatment. Matched biopsies of tumour pre-treatment, post-treatment and adjacent normal epithelium as well as normal breast tissues from 6 healthy individuals were included. Full transcriptome analysis was performed by mRNA sequencing, after optimising this method for archived samples. Comprehensive clinical and pathological information was collected. A dedicated longitudinal pattern analysis method was developed to follow dynamic expression fluctuations of individual patients. Pathifier was used to calculate pathway deregulation scores. Results and discussions Principle component analysis showed clustering of the samples according to their type. Dynamic fluctuations across the 3 time-points were classified into 8 theoretical patterns, each representing a different scenario through the tumour progression and treatment stages. Genes were divided into two main types: 1. Sharing a common temporal expression pattern across most patients. These genes were associated with tumour progression pathways. 2. Genes that were divided into two or three dominant patterns and this division showed correlation with pathological response score. The dynamic pattern classification enabled to pinpoint genes associated with response that otherwise were difficult to identify using single-time point or two-time points datasets. Furthermore, the dynamics of pathway deregulation scores enabled to detect pathways that were correlated with response to therapy. Conclusion The longitudinal approach of serial sampling and analysis reveals heterogeneous dynamic behaviour across patients through the course of disease. This individual dynamics has higher sensitivity than single-time point measurements in detecting clinically relevant genes that are associated with resistance to therapy and with tumour progression.

Assessment of Interstitial Fluid Pressure in solid tumors via image processing of DCE-MRI

Interstitial Fluid Pressure (IFP) is a major obstacle to intra-tumoral drug delivery. A routine non-invasive assessment of IFP will serve as a valuable predictor for the response to neoadjuvant treatment in breast cancer. It will also be the basis for a rationalized and personalized therapy by improving the efficacy of anti-cancer therapeutics via reduction of tumor IFP. We developed an automated robust algorithm for assessing IFP based on image analysis of dynamic contrast-enhanced MRI (DCE-MRI) examinations. This method is easy to implement on any MRI dataset of breast cancer and does not require any input from the radiologist besides delineating the tumor region.

Abstract 4011: Tumor evolution inferred by patterns of miRNA expression through the course of disease, therapy and recurrence in breast cancer

Tumor heterogeneity frequently develops through the course of disease, therapy and metastasis. This molecular evolution is a major challenge in the clinical setting and is the main reason for resistance to therapy. Although metastatic disease is the cause of death in breast cancer, most studies involve primary tumors compared to normal breast. An alternative approach of serial assessments can add an informative value and highlight important molecular players that may be otherwise underestimated. We sought to explore alterations through the course of disease by profiling serial samples from individual breast cancer cases followed from diagnosis to recurrence. We have assembled a unique cohort of patients having matched samples from pre-treatment, post-treatment and recurrent events. We collected paraffin-embedded samples and profiled miRNA expression across all samples for each patient using NanoString analysis. We hypothesized that expression modulations in individual patients through their course of disease can target miRNAs sets that differentially function at the various stages of the disease. We therefore used an innovative approach for analysis, identifying modulation patterns as opposed to differential absolute expression. We defined all the 8 possible patterns of expression modulations in 3 time points across the course of disease. For each pattern, we identified a set of miRNAs that are shared between patients by limiting the search algorithm to find modulations of at least 1.5 fold in at least 3 patients. Each miRNA set was examined for enrichments in known miRNAs datasets (METABRIC), for target predictions and for literature evidences. We found an agreement between the predicted pattern of each assigned miRNAs and the observed trend. For example, miRNAs assigned to a pattern of an increased expression post-treatment and a decrease at recurrence were found to be down-regulated in breast cancer and negatively correlated with the cell cycle pathway. Interestingly, we found that this miRNAs set can differentiate between individual patients, based on their response to therapy. All patients sharing the same pattern for its assigned set of miRNAs had a partial response to therapy. In contrast, patients that showed the opposite pattern demonstrated a minimal response to therapy. Finally, although this miRNA set were identified by the pattern dynamics, we found a significant difference in its average absolute expression level between patients having recurrence and patients that are recurrence free, suggesting that the identified miRNA set may have a prognostic value. In summary, examining serial assessments enabled us to identify significant molecular signatures characterizing breast cancer progression based on expression patterns through the course of disease. This longitudinal approach of profiling individual patients may have important consequences for personalized medicine. Citation Format: Maya Dadiani, Noa Bossel, Shani Paluch-Shimon, Gili Peri, Anya Pavlovski, Smadar Kahana-Edwin, Nora Balint, Adi Yosepovich, Adi Zundelevich, Einav Gal-Yam, Raanan Berger, Iris Barshack, Eytan Domany, Bella Kaufman. Tumor evolution inferred by patterns of miRNA expression through the course of disease, therapy and recurrence in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4011. doi:10.1158/1538-7445.AM2015-4011