The Angiogenic Secretome in VEGF overexpressing Breast Cancer Xenografts

The plasticity of cancer cells and the fluidity of the tumor microenvironment continue to present major challenges in the comprehensive understanding of cancer that is essential to design

effective treatments. The tumor interstitial fluid (TIF) encompasses the secretome and holds the key to several of the phenotypic characteristics of cancer. Difficulties in sampling this

fluid have resulted in limited characterization of its components. Here we have sampled TIF from triple negative and estrogen receptor (ER)-positive human breast tumor xenografts with or

without VEGF overexpression. Angiogenesis-related factors were characterized in the TIF and plasma, to understand the relationship between the TIF and plasma secretomes. Clear differences

were observed between the TIF and plasma angiogenic secretomes in triple negative MDA-MB-231 breast cancer xenografts compared to ER-positive MCF-7 xenografts with or without VEGF

overexpression that provide new insights into TIF components and the role of VEGF in modifying the angiogenic secretome.

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 secretome1. 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.

Some of the earliest work in sampling TIF using a collection chamber was performed by Gullino and colleagues2. Despite the limited analytical methods that were available at the time,

important insights into the protein content, urea, free amino acids, glucose and lactate in TIF were gained2. During the last decade, newer TIF isolation methods, and physiological and

molecular characterization have been applied to unravel the role of the TIF in cancer3,4,5. Methodologies such as the incubation of human tumor samples in a buffer and subsequent

concentration of the samples6 have allowed antibody-based analyses of TIF that identified the presence of several cytokines and other proteins. Microdialysis has also been used to collect

and analyze tumor-secreted proteins in xenograft models using quantitative high-performance liquid chromatography7. This method provided important information about TIF content including

overexpression of proteins such as vascular endothelial growth factor (VEGF)8,9. Factors secreted by breast cancers and their roles in distal metastasis were also recently

investigated4,5,10,11.

Interpretation of TIF content and its influence on the pathophysiological properties of the tumor, such as angiogenesis, invasiveness and metastasis is limited. A few studies have

extensively screened the protein content in TIF12,13,14, but these have primarily been proof-of-principle studies to demonstrate the feasibility of proteomic analysis of the interstitial

fluid.

Angiogenesis, one of the major hallmarks of cancer, is essential for cancers to establish vasculature for growth and hematogenous metastasis15. Cytokines and growth factors secreted by

cancer cells and stromal cells stimulate endothelial cell proliferation16. Among all known angiogenic factors, vascular endothelial growth factor is closely associated with increased

aggressiveness and metastasis17, and is known to be upregulated under hypoxia18. Preclinical and clinical studies have demonstrated that VEGF can be targeted to reduce angiogenesis and VEGF

blockage has been shown to promote survival – especially when combined with chemotherapies – and slow tumor growth in ovarian, cervical, colorectal, renal, lung and breast cancer19.

Additionally, levels of VEGF in plasma may be a biomarker of response to anti-angiogenic therapy20. Despite recent setbacks in the use of anti-angiogenic therapy in breast cancer, the

importance of VEGF in tumor progression, and its potential targeting for treatment remains undeniable21,22,23,24.

To further understand the TIF and plasma secretome in breast cancer, and the role of VEGF in modifying these secretomes, we characterized angiogenesis-related cytokines, small proteins and

peptides in TIF from triple negative MDA-MB-231 and ER-positive MCF-7 human breast cancer xenografts with and without VEGF overexpression. Triple negative breast cancer is characterized by

increased aggressiveness, high recurrence and mortality, and resistance to treatment25 compared to ER-positive breast cancers26. TIF was obtained using a collection chamber modified for

mouse tumors, based on a technique initially described by Gullino and colleagues2. Secretome factors in TIF were compared to those in plasma to understand the role of TIF in modifying the

plasma factors that may impact metastatic dissemination and the formation of the pre-metastatic niche, and also potentially provide plasma-derived biomarkers.

To evaluate the angiogenic secretome, a Proteome Profiler Antibody Array Kit for Human Angiogenesis (R&D systems, Minnesota, USA) was used to determine 55 angiogenesis-related factors in TIF

and plasma samples (Figure S1). As shown in Fig. 1A and B, from a panel of 55, only 4 factors (serpin E1, TIMP metallopeptidase inhibitor 1 (TIMP-1), urokinase plasminogen activator (uPA),

and VEGF) were detectable in TIF derived from MCF-7_WT and MCF-7_VEGF tumors and the plasma from these tumor-bearing mice. VEGF overexpression in MCF-7 tumors resulted in a significant

increase of VEGF and TIMP-1 and a trend towards an increase of serpin E1 and uPA in TIF (Fig. 1A). TIMP-1 was the only factor within the highest 50% range of the assay. Plasma samples from

mice with MCF-7_WT tumors only contained detectable levels of serpin E1 and TIMP-1, but not uPA or VEGF. Plasma samples from mice with MCF-7_VEGF tumors contained detectable uPA and VEGF,

and a trend towards higher serpin E1 (Fig. 1B). A comparison of cytokines between TIF and plasma in the MCF-7_WT tumor group (Fig. 1C) showed that although uPA and VEGF were detected in TIF,

these were not detected in plasma, and there was a trend of lower serpin E1 and higher TIMP-1 in plasma compared to TIF. With VEGF overexpression, even though TIMP-1 increased significantly

in TIF, this was not reflected as a detectable increase of plasma TIMP-1 levels, although increases of uPA and VEGF in TIF were also reflected in plasma (Fig. 1D).

(A) Angiogenic cytokines detected in the TIF of MCF-7_WT (n = 7) and MCF-7_VEGF (n = 13) tumors. (B) Angiogenic cytokines detected in the plasma of MCF-7_WT and MCF-7_VEGF tumors. (C)

Comparison of angiogenic cytokines in TIF and plasma in the MCF-7_WT group. (D) Comparison of angiogenic cytokines in TIF and plasma in the MCF-7_VEGF group. Values represent Mean + SEM. *P