One of the least examined, and yet critically important factors of the tumor microenvironment (TME) is the tumor interstitial fluid (TIF) that contains the tumor secretome. This fluid surrounds cancer and stromal cells and contains various cytokines, and nutritional and molecular factors that shape the outcome of nearly all aspects of tumor angiogenesis, growth, metastasis, and response to treatment. As mining of targets to treat cancer expands into the TME, the TIF also represents an important source of identifying new targets in cancer treatment. Angiogenesis, one of the major hallmarks of cancer, is essential for cancers to establish vasculature for growth and hematogenous metastasis. To further understand the TIF and plasma secretome in breast cancer, and the role of VEGF in modifying these secretomes, here we have used 1H MRS for characterization of metabolites in TIF collected from the triple negative MDA-MB-231 breast cancer xenograft and the ER-positive MCF-7 breast cancer xenograft models with and without VEGF overexpression.We created a collection chamber to collect IF from mouse tumors (Figure 1) (1, 2). TIF collection was performed from ER-positive MCF-7 and triple negative MDA-MB-231 breast cancer tumors with and without VEGF expression in SCID mice. The chamber was implanted with small tumor pieces in the subcutaneous space until the tumor encompassed the chamber (4 to 12 weeks depending on cell types). The tumor was then removed and the tumor IF and blood plasma were collected. We also collected normal subcutaneous IF (SCIF) using the same chamber in healthy mice. All surgical procedures and animal handling were performed in accordance with protocols approved by the Johns Hopkins University Institutional Animal Care and Use Committee, and conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH. For 1H MRS acquisition, 50μL of IF supernatant was diluted in 550μL of D2O saline buffer. High-resolution 1H MRS was performed on an Avance III 750 MHz Bruker Spectrometer equipped with a 5 mm broad-band inverse probe. The 1H MR spectra with water suppression were acquired using one-dimensional Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with the following parameters: spectral width of 15495.8 Hz, time domain data points of 64K, effective 90° flip angle, relaxation delay 10s, acquisition time of 2.1s, 128 number of scan with 8 dummy scan, a receiver gain of 1030, echo time 25ms. All spectra were processed using line broadening for exponential window function of 0.3 Hz prior to Fourier transformation, they were manually phased and automatically baseline corrected using TOPSPIN 2.1. Characterization of the metabolites was carried out on the basis of chemical shift, coupling constant, and splitting pattern of metabolites, as reported in literature and by comparison with standard MR spectra of metabolites reported by the Biological Magnetic Resonance Bank (BMRB, www.bmrb.wisc.edu) (3, 4). 1H spectra showed several difference in metabolites in TIF compared to SCIF, between MDA-MB-231 and MCF-7 TIF, and with VEGF overexpression (Figure 2). We observed a consumption of amino acids in the TME with decreases ranging from 17% (Glycine) to 77% (Methionine) compared to normal SCIF. Lipids, especially polyunsaturated fatty acids, were markedly increased by VEGF overexpression in both MCF-7 and MDA-MB-231 TIF compared to the wild type TIF (Figure 3). Choline metabolism was also modified by VEGF overexpression and we measured increases of 69% and 20% increases in free choline in MDA-MB-231_VEGF and MCF-_VEGF respectively, compared to their wild type counterpart. A stable glucose concentration was associated with a decrease in lactate (30%) and pyruvate (44%) in MDA-MB-231 tumors while an opposite pattern was observed in MDA-MB-231_VEGF TIF with decreased glucose (55%) but increased lactate (109%). Ketonic metabolism was also modified in those tumors. Beta-hydroxybutyrate and acetoacetate were higher in MDA-MB-231 TIF while acetone was increased in both VEGF-overexpressing TIF. The TIF represents an importance source of information to understand mechanisms that drive aggressiveness, and identify new targets for diagnosis and therapy of cancer.