Abstract
Simple SummaryHuman breast cancers are not fully autonomous. They are dependent on nutrients and growth-promoting signals provided by stromal cells. In order to instruct the surrounding cells to provide essential growth factors, cancer cells co-opt normal signaling molecules and mechanisms. To inhibit or potentially reverse tumor growth, our goal is to emulate this signaling and reprogram the microenvironment. For example, in a healthy mammary gland, fibroblasts (FBs) overexpress CD36; and the downregulation of CD36 is one of the hallmarks of cancer-associated FBs. Therefore, in this project, we hypothesized that signaling from CD36+ FBs could cause growth suppression in a subset of breast cancer cell lines. We then designed a series of experiments to validate this growth suppression and identified responsible secreted factors by the CD36+ FBs. These experiments suggested that three protein ligands are primarily responsible for growth suppression in a subset of breast cancer cell lines.Reprogramming the tumor stroma is an emerging approach to circumventing the challenges of conventional cancer therapies. This strategy, however, is hampered by the lack of a specific molecular target. We previously reported that stromal fibroblasts (FBs) with high expression of CD36 could be utilized for this purpose. These studies are now expanded to identify the secreted factors responsible for tumor suppression. Methodologies included 3D colonies, fluorescent microscopy coupled with quantitative techniques, proteomics profiling, and bioinformatics analysis. The results indicated that the conditioned medium (CM) of the CD36+ FBs caused growth suppression via apoptosis in the triple-negative cell lines of MDA-MB-231, BT549, and Hs578T, but not in the ERBB2+ SKBR3. Following the proteomics and bioinformatic analysis of the CM of CD36+ versus CD36− FBs, we determined KLF10 as one of the transcription factors responsible for growth suppression. We also identified FBLN1, SLIT3, and PENK as active ligands, where their minimum effective concentrations were determined. Finally, in MDA-MB-231, we showed that a mixture of FBLN1, SLIT3, and PENK could induce an amount of growth suppression similar to the CM of CD36+ FBs. In conclusion, our findings suggest that these ligands, secreted by CD36+ FBs, can be targeted for breast cancer treatment.
Highlights
The tumor microenvironment [1], including fibroblasts (FBs) and immune and endothelial cells, influences human breast cancer progression and malignancy [2]
In a previous work [19], we showed that activin A secreted from tumor epithelial cells decreases CD36 expression in FBs in a coculture assay and that CD36 expression was inversely correlated to the concentration of activin A in a dose-dependent manner
Primary FBs were treated with a nominal concentration of activin A [28] in tumors, at a minimal concentration of 2.25 ng/mL and as high as 20 ng/mL, and the CD36 expression in FBs was quantified under three experimental conditions: (i) three days of treatment followed by fixation, (ii) five days of treatment followed by fixation, and (iii) three days of treatment followed by replacing activin A-containing medium with the normal culture medium, followed by two days of incubation before fixation
Summary
The tumor microenvironment [1], including fibroblasts (FBs) and immune and endothelial cells, influences human breast cancer progression and malignancy [2]. Cancer-associated fibroblasts (CAFs) have emerged as potential targets for reprogramming tumor microenvironment and optimizing therapeutic strategies [4,5]. CD36 is a scavenger receptor that mediates lipid uptake, is expressed in multiple cancer types [7], and accelerates tumor growth [8]. This receptor is downregulated in tumor-associated FBs and has been suggested as a CAF marker [9]. Understanding the normal functioning of FBs could lead to new therapies
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