BREAst Cancer Personalised NuTrition (BREACPNT): dietary intervention in breast cancer survivors treated with endocrine therapy - a protocol for a randomised clinical trial.

INTRODUCTION: Breast cancer survivors treated with adjuvant endocrine therapy commonly experience weight gain, which has been associated with low adherence to therapy and worse breast cancer prognosis. We aim to assess whether a personalised postprandial glucose targeting diet will be beneficial for weight management as compared with the recommended Mediterranean diet in this patient population METHODS AND ANALYSIS: The BREAst Cancer Personalised NuTrition study is a phase-2 randomised trial in hormone receptor positive patients with breast cancer, treated with adjuvant endocrine therapy. The study objective is to assess whether dietary intervention intended to improve postprandial glycaemic response to meals results in better weight and glycaemic control in this population as compared with the standard recommended Mediterranean diet. Consenting participants will be assigned in a single blinded fashion to either of two dietary arms (Mediterranean diet or an algorithm-based personalised diet). They will be asked to provide a stool sample for microbiome analysis and will undergo continuous glucose monitoring for 2 weeks, at the initiation and termination of the intervention period. Microbiome composition data will be used to tailor personal dietary recommendations. After randomisation and provision of dietary recommendations, participants will be asked to continuously log their diet and lifestyle activities on a designated smartphone application during the 6-month intervention period, during which they will be monthly monitored by a certified dietitian. Participants' clinical records will be followed twice yearly for 5 years for treatment adherence, disease-free survival and recurrence. ETHICS AND DISSEMINATION: The study has been approved by the ethics committee in the Sheba medical centre (file 5725-18-SMC, Ramat Gan, Israel) and the Weizmann Institutional Review Board (file 693-2, Rehovot, Israel). The findings of this study will be published in a peer reviewed publication. TRIAL REGISTRATION NUMBER: NCT04079270.

Pan-cancer analyses reveal cancer-type-specific fungal ecologies and bacteriome interactions.

Cancer-microbe associations have been explored for centuries, but cancer-associated fungi have rarely been examined. Here, we comprehensively characterize the cancer mycobiome within 17,401 patient tissue, blood, and plasma samples across 35 cancer types in four independent cohorts. We report fungal DNA and cells at low abundances across many major human cancers, with differences in community compositions that differ among cancer types, even when accounting for technical background. Fungal histological staining of tissue microarrays supported intratumoral presence and frequent spatial association with cancer cells and macrophages. Comparing intratumoral fungal communities with matched bacteriomes and immunomes revealed co-occurring bi-domain ecologies, often with permissive, rather than competitive, microenvironments and distinct immune responses. Clinically focused assessments suggested prognostic and diagnostic capacities of the tissue and plasma mycobiomes, even in stage I cancers, and synergistic predictive performance with bacteriomes.

Divergence of mutational signatures in association with breast cancer subtype.

Abnormal molecular processes occurring throughout the genome leave distinct somatic mutational patterns termed mutational signatures. Exploring the associations between mutational signatures and clinicopathological features can unravel potential mechanisms driving tumorigenic processes. We analyzed whole genome sequencing (WGS) data of tumor and peripheral blood samples from 37 primary breast cancer (BC) patients receiving neoadjuvant chemotherapy. Comprehensive clinico-pathologic features were correlated with genomic profiles and mutational signatures. Somatic mutational landscapes were highly concordant with known BC data sets. Remarkably, we observed a divergence of dominant mutational signatures in association with BC subtype. Signature 5 was overrepresented in hormone receptor positive (HR+) patients, whereas triple-negative tumors mostly lacked Signature 5, but expectedly overrepresented Signature 3. We validated these findings in a large WGS data set of BC, demonstrating dominance of Signature 5 in HR+ patients, mostly in luminal A subtype. We further investigated the association between Signature 5 and gene expression signatures, and identified potential networks, likely related to estrogen regulation. Our results suggest that the yet elusive Signature 5 represents an alternative mechanism for mutation accumulation in HR+ BC, independent of the homologous recombination repair machinery related to Signature 3. This study provides theoretical basis for further elucidating the processes promoting hormonal breast carcinogenesis.

ICAM-1 on Breast Cancer Cells Suppresses Lung Metastasis but Is Dispensable for Tumor Growth and Killing by Cytotoxic T Cells.

Breast tumors and their derived circulating cancer cells express the leukocyte ß2 integrin ligand Intercellular adhesion molecule 1 (ICAM-1). We found that elevated ICAM-1 expression in breast cancer cells results in a favorable outcome and prolonged survival of breast cancer patients. We therefore assessed the direct in vivo contribution of ICAM-1 expressed by breast cancer cells to breast tumorigenesis and lung metastasis in syngeneic immunocompetent mice hosts using spontaneous and experimental models of the lung metastasis of the C57BL/6-derived E0771 cell line, a luminal B breast cancer subtype. Notably, the presence of ICAM-1 on E0771 did not alter tumor growth or the leukocyte composition in the tumor microenvironment. Interestingly, the elimination of Tregs led to the rapid killing of primary tumor cells independently of tumor ICAM-1 expression. The in vivo elimination of a primary E0771 tumor expressing the ovalbumin (OVA) model neoantigen by the OVA-specific OVA-tcr-I mice (OT-I) transgenic cytotoxic T lymphocytes (CTLs) also took place normally in the absence of ICAM-1 expression by E0771 breast cancer target cells. The whole lung imaging of these cells by light sheet microscopy (LSM) revealed that both Wild type (WT)- and ICAM-1-deficient E0771 cells were equally disseminated from resected tumors and accumulated inside the lung vasculature at similar magnitudes. ICAM-1-deficient breast cancer cells developed, however, much larger metastatic lesions than their control counterparts. Strikingly, the vast majority of these cells gave rise to intravascular tumor colonies both in spontaneous and experimental metastasis models. In the latter model, ICAM-1 expressing E0771- but not their ICAM-1-deficient counterparts were highly susceptible to elimination by neutrophils adoptively transferred from E0771 tumor-bearing donor mice. Ex vivo, neutrophils derived from tumor-bearing mice also killed cultured E0771 cells via ICAM-1-dependent interactions. Collectively, our results are a first indication that ICAM-1 expressed by metastatic breast cancer cells that expand inside the lung vasculature is involved in innate rather than in adaptive cancer cell killing. This is also a first indication that the breast tumor expression of ICAM-1 is not required for CTL-mediated killing but can function as a suppressor of intravascular breast cancer metastasis to lungs.

Chemotherapy Shifts the Balance in Favor of CD8+ TNFR2+ TILs in Triple-Negative Breast Tumors.

Triple-negative breast cancer (TNBC) is primarily treated via chemotherapy; in parallel, efforts are made to introduce immunotherapies into TNBC treatment. CD4+ TNFR2+ lymphocytes were reported as Tregs that contribute to tumor progression. However, our published study indicated that TNFR2+ tumor-infiltrating lymphocytes (TNFR2+ TILs) were associated with improved survival in TNBC patient tumors. Based on our analyses of the contents of CD4+ and CD8+ TILs in TNBC patient tumors, in the current study, we determined the impact of chemotherapy on CD4+ and CD8+ TIL subsets in TNBC mouse tumors. We found that chemotherapy led to (1) a reduction in CD4+ TNFR2+ FOXP3+ TILs, indicating that chemotherapy decreased the content of CD4+ TNFR2+ Tregs, and (2) an elevation in CD8+ TNFR2+ and CD8+ TNFR2+ PD-1+ TILs; high levels of these two subsets were significantly associated with reduced tumor growth. In spleens of tumor-bearing mice, chemotherapy down-regulated CD4+ TNFR2+ FOXP3+ cells but the subset of CD8+ TNFR2+ PD-1+ was not present prior to chemotherapy and was not increased by the treatment. Thus, our data suggest that chemotherapy promotes the proportion of protective CD8+ TNFR2+ TILs and that, unlike other cancer types, therapeutic strategies directed against TNFR2 may be detrimental in TNBC.

Proteomic patterns associated with response to breast cancer neoadjuvant treatment.

Tumor relapse as a consequence of chemotherapy resistance is a major clinical challenge in advanced stage breast tumors. To identify processes associated with poor clinical outcome, we took a mass spectrometry-based proteomic approach and analyzed a breast cancer cohort of 113 formalin-fixed paraffin-embedded samples. Proteomic profiling of matched tumors before and after chemotherapy, and tumor-adjacent normal tissue, all from the same patients, allowed us to define eight patterns of protein level changes, two of which correlate to better chemotherapy response. Supervised analysis identified two proteins of proline biosynthesis pathway, PYCR1 and ALDH18A1, that were significantly associated with resistance to treatment based on pattern dominance. Weighted gene correlation network analysis of post-treatment samples revealed that these proteins are associated with tumor relapse and affect patient survival. Functional analysis showed that knockdown of PYCR1 reduced invasion and migration capabilities of breast cancer cell lines. PYCR1 knockout significantly reduced tumor burden and increased drug sensitivity of orthotopically injected ER-positive tumor in vivo, thus emphasizing the role of PYCR1 in resistance to chemotherapy.

The human tumor microbiome is composed of tumor type-specific intracellular bacteria.

Bacteria were first detected in human tumors more than 100 years ago, but the characterization of the tumor microbiome has remained challenging because of its low biomass. We undertook a comprehensive analysis of the tumor microbiome, studying 1526 tumors and their adjacent normal tissues across seven cancer types, including breast, lung, ovary, pancreas, melanoma, bone, and brain tumors. We found that each tumor type has a distinct microbiome composition and that breast cancer has a particularly rich and diverse microbiome. The intratumor bacteria are mostly intracellular and are present in both cancer and immune cells. We also noted correlations between intratumor bacteria or their predicted functions with tumor types and subtypes, patients' smoking status, and the response to immunotherapy.

TNFR2+ TILs are significantly associated with improved survival in triple-negative breast cancer patients.

In view of the relatively limited efficacy of immunotherapies targeting the PD-1-PD-L1 axis in triple-negative breast cancer (TNBC) and of published reports on tumor-promoting roles of TNFR2+ tumor-infiltrating lymphocytes (TNFR2+ TILs), we determined the incidence of TNFR2+ TILs in TNBC patient tumors, their association with disease outcome and relations with PD-1+ TILs. Using a cohort of treatment-naïve TNBC patients with long follow-up (n = 70), we determined the presence of TNFR2+ TILs and PD-1+ TILs by immunohistochemistry. TILs (≥ 1% of cellular mass) and TNFR2+ TILs (≥ 1% of total TILs) were detected in 96% and 74% of tumors, respectively. The presence of TILs at > 5% of tumor cell mass ("Positive TILs"), as well as of positive TNFR2+ TILs (> 5%), was independently associated with good prognosis, and combination of both parameters demonstrated superior outcome relative to their lower levels. PD1+ TILs (> 5/hot spot) were detected in 63% of patients. High levels of PD-1+ TILs (> 20/hot spot) showed an unfavorable disease outcome, and in their presence, the favorable outcome of positive TNFR2+ TILs was ablated. Thus, TNFR2+ TILs are strongly connected to improved prognosis in TNBC; these findings suggest that TNFR2+ TILs have favorable effects in TNBC patients, unlike the tumor-promoting roles attributed to them in other cancer systems. Overall, our observations propose that the TNFR2+ TIL subset should not be targeted in the course of TNBC therapy; rather, its beneficial impacts may become into power when anti-PD-1 regimens-that may potentiate immune activities-are administered to TNBC patients.

Correction to: ESR1 mutations are frequent in newly diagnosed metastatic and loco-regional recurrence of endocrine-treated breast cancer and carry worse prognosis.

After the publication of the original article [1], we were notified the upper panel of the Fig. 1, where the patients' codes are listed, was cropped by mistake so the patients 1-8 are repeated.

ESR1 mutations are frequent in newly diagnosed metastatic and loco-regional recurrence of endocrine-treated breast cancer and carry worse prognosis.

BACKGROUND: Emerging mutations in the ESR1 gene that encodes for the estrogen receptor (ER) are associated with resistance to endocrine therapy. ESR1 mutations rarely exist in primary tumors (~ 1%) but are relatively common (10-50%) in metastatic, endocrine therapy-resistant cancers and are associated with a shorter progression-free survival. Little is known about the incidence and clinical implication of these mutations in early recurrence events, such as local recurrences or newly diagnosed metastatic disease. METHODS: We collected 130 archival tumor samples from 103 breast cancer patients treated with endocrine therapy prior to their local/metastatic recurrence. The cohort consisted of 41 patients having at least 1 sample from local/loco-regional recurrence and 62 patients with metastatic disease (of whom 41 newly diagnosed and 28 with advanced disease). The 5 most common ESR1 hotspot mutations (D538G, L536R, Y537S/N/C) were analyzed either by targeted sequencing or by droplet digital PCR. Progression-free survival (PFS), disease-free survival (DFS), and distant recurrence-free survival (DRFS) were statistically tested by Kaplan-Meier analysis. RESULTS: The prevalence of ESR1 mutations was 5/41 (12%) in newly diagnosed metastatic patients and 5/28 (18%) for advanced metastases, detected at allele frequency > 1%. All mutations in advanced metastases were detected in patients previously treated with both tamoxifen (TAM) and aromatase inhibitors (AI). However, in newly diagnosed metastatic patients, 4/5 mutations occurred in patients treated with TAM alone. PFS on AI treatment in metastatic patients was significantly shorter for ESR1 mutation carriers (p = 0.017). In the local recurrence cohort, ESR1 mutations were identified in 15/41 (36%) patients but only 4/41 (10%) were detected at allele frequency > 1%. Again, most mutations (3/4) were detected under TAM monotherapy. Notably, 1 patient developed ESR1 mutation while on neoadjuvant endocrine therapy. DFS and DRFS were significantly shorter (p = 0.04 and p = 0.017, respectively) in patients that had ESR1 mutations (> 1%) in their loco-regional recurrence tumor. CONCLUSIONS: Clinically relevant ESR1 mutations are prevalent in newly diagnosed metastatic and local recurrence of endocrine-treated breast cancer. Since local recurrences are amenable to curative therapy, these mutations may inform the selection of subsequent endocrine therapies.

Cancer-associated fibroblast compositions change with breast cancer progression linking the ratio of S100A4+ and PDPN+ CAFs to clinical outcome.

Tumors are supported by cancer-associated fibroblasts (CAFs). CAFs are heterogeneous and carry out distinct cancer-associated functions. Understanding the full repertoire of CAFs and their dynamic changes as tumors evolve could improve the precision of cancer treatment. Here we comprehensively analyze CAFs using index and transcriptional single-cell sorting at several time points along breast tumor progression in mice, uncovering distinct subpopulations. Notably, the transcriptional programs of these subpopulations change over time and in metastases, transitioning from an immunoregulatory program to wound-healing and antigen-presentation programs, indicating that CAFs and their functions are dynamic. Two main CAF subpopulations are also found in human breast tumors, where their ratio is associated with disease outcome across subtypes and is particularly correlated with BRCA mutations in triple-negative breast cancer. These findings indicate that the repertoire of CAF changes over time in breast cancer progression, with direct clinical implications.

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

BACKGROUND: The 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. RESULTS: In 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 100 ng, 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. CONCLUSIONS: Using 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.

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 68 % compared with 37 % among non-carriers, p = 0.01. 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 p = 0.25), whereas in the non-carrier cohort, relapse-free survival was superior for those achieving pathological complete response (Log-rank p < 0.0001). 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.

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.

Estrogen regulation of vascular endothelial growth factor in breast cancer in vitro and in vivo: the role of estrogen receptor alpha 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.

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.

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.

Magnetic resonance imaging of tumor vasculature.

Angiogenic activity and formation of a vascular network facilitate tumor perfusion and play a critical role in tumor growth and metastasis. Tumor vasculature may be visualized by means of parametric imaging of specific morphological and physiological characteristics that collectively describe its properties. In this review, we describe advanced magnetic resonance imaging (MRI) techniques that have been developed in order to image and quantify the distribution of tumor vasculature throughout the tumor and characterize its function. These techniques have been used to monitor changes in the magnetic resonance signal intensity of tissue water hydrogens generated by intrinsic effects, as well as by exogenous contrast agents administered into the blood circulation. We further describe specific applications of magnetic resonance imaging using a contrast agent, gadolinium diethylene triamine penta-acetic acid (GdDTPA), which has long been approved for clinical use. Examples include studies of the vascular properties of breast cancer tumors and metastases in animal models, as well as of breast cancer vasculature in patients. We also discuss the use of MRI to improve breast cancer diagnosis in humans by quantifying the permeability of the tumor vasculature. By maximizing the spatial resolution of the images in both animal and human studies, the capacity of magnetic resonance imaging to enhance our understanding of the processes regulating tumor angiogenesis, and improve the diagnosis of cancer, could be clearly demonstrated.