Santosh K Bharti1, Louis Dore-Savard1, Aleksander S Popel2, and Zaver M Bhujwalla1
1The Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University, School of Medicine, Division of Cancer Imaging Research, Baltimore, MD, United States, 2Department of Biomedical Engineering, Johns Hopkins University, School of Medicine, Systems Biology Laboratory, Baltimore, MD, United States
Synopsis
Interstitial fluid
(IF) is a key component of the tumor microenvironment (TME) that encompasses
the secretome and holds the key to several of the phenotypic traits of cancer. Modern analytical methods like 1H MR
spectroscopy (MRS) allow for comprehensive metabolic characterization of tissue,
cell, and bio-fluids content to better understand the TME and cancer metabolism.
Here, for the first time, we have metabolically characterized TIF from triple
negative and estrogen receptor (ER)-positive human breast tumor xenografts with
or without VEGF overexpression and detected significant differences between
tumor types and with VEGF overexpression.
Introduction
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.
Methods
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).
Results and Discussion
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.
Acknowledgements
This work was supported by NIH R01CA136576 and P50 CA103175. We thank Mr. Gary Cromwell for valuable
technical assistance.References
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