Obesity-associated dysregulation of calpastatin and MMP-15 in adipose-derived stromal cells results in their enhanced invasion

Abstract

Adipose tissue maintains a subpopulation of cells, referred to as adipose-derived stromal/stem cells (ASCs), which have been associated with increased breast cancer tumorigenesis and metastasis. For ASCs to affect breast cancer cells, it is necessary to delineate how they mobilize and home to cancer cells, which requires mobilization and invasion through extracellular matrix barriers. In this study, ASCs were separated into four different categories based on the donor's obesity status and depot site of origin. ASCs isolated from the subcutaneous abdominal adipose tissue of obese patients (Ob(+)Ab(+)) demonstrated increased invasion through Matrigel as well as a chick chorioallantoic membrane, a type I collagen-rich extracellular matrix barrier. Detailed mRNA and protein analyses revealed that calpain-4, calpastatin, and MMP-15 were associated with increased invasion, and the silencing of each protease or protease inhibitor confirmed their role in ASC invasion. Thus, the data indicate that both the donor's obesity status and depot site of origin distinguishes the properties of subcutaneous-derived ASCs with respect to enhanced invasion and this is associated with the dysregulation of calpain-4, calpastatin, and MMP-15.

Figure 1. Characterization of ASCs isolated from donors based on obesity status and deposit site

(A) ASCs in each of the four groups were stained with antibodies against the indicated antigens and analyzed by flow cytometry. Histograms are shown as colored lines and the respective isotype controls are shaded in gray. (B) ASCs were grown until 70% confluent in CCM and then switched to differentiation media. After 21 days, cells were fixed and stained with Alizarin Red for osteogenesis and Oil Red O for adipogenesis. Representative images for each group are shown. Original magnification for osteogenesis is 4x and for adipogenesis is 10x for all panels. (C) CFUs were seeded at low density and incubated in CCM for 14 days. Cells were fixed and stained with crystal violet. Representative image for each group are shown. Bars, ± SD.

Figure 2. Invasion of ASCs towards conditioned media

ASCs were placed onto transwell filters coated with growth factor reduced Matrigel and incubated for 24 hours in serum free or MDA-MB-231 conditioned media (CM) or serum free media (SF). Data was normalized by dividing the values obtained with or without MDA-MB-231 conditioned media as a chemoattractant by the values obtained without conditioned media (set as 1). Values are means of three independent experiments, each performed in triplicate. Bars, ± SD. *P < .05, **P < .01, ***P < .001.

Figure 3. Protease inhibitors limit ASC invasion

Ob⁺Ab⁺ ASCs were pretreated with protease inhibitors for 4 hours and placed onto transwell inserts with 8 μm pore membrane filters pre-coated with growth factor-reduced Matrigel in the presence of protease inhibitors. The bottom chamber was loaded with MDA-MB-231 conditioned media and incubated for 24 hours. (A) ASCs were pretreated with broad-spectrum protease inhibitor Pepstatin A (100 mM), SBTI (100 μg/mL), E64d (1 μM), or GM6001 (25 μM) for 4 hours prior to being placed onto transwell inserts. (B) ASCs were pretreated with cysteine protease inhibitor 1,3-Bis(CBZ-Leu-NH)-2-propanone (10 μM), acetyl-calpastatin (0.2 μM), or Z-VAD-FMK (20 μM) for 4 hours prior to being placed onto transwell inserts. Data was normalized by dividing the values obtained with each treatment group by the values obtained without protease inhibitor treatment and represented as a percentage. Values are means of three independent experiments, each performed in triplicates. Bars, ± SD. *P < .05, **P < .005.

Figure 4. RT-PCR analyses of MMP, TIMP, calpain, and calpastatin from different ASC populations

ASCs were cultured in CCM until 70% confluent and grown in breast cancer conditioned media for 24 hours. Cells were harvested and RNA extraction was conducted with TRIzol and purified with DNase digestion. A total of 200 ng of total RNA was used for cDNA synthesis and PCR analysis. No-template controls (neg ctrl) were run to rule out cross contamination of reagents and surfaces. PCR products were analyzed by gel electrophoresis on 2.0% agarose gels with ethidium bromide stain. β-actin is shown as a standard reference. Primer sets are listed in Supplemental Material 2. Bars, ± SD. *P < .05

Figure 5. Zymographic and Western blot analyses of MMP, TIMP, calpain, and calpastatin in different ASC population

ASCs were cultured in CCM until 70% confluent and grown in breast cancer conditioned media for 24 hours. (A) Zymographic analysis of gelatinase secretion from ASCs. A total of 20 μg of culture supernatants were analyzed. Breast cancer conditioned media was used as a control to demonstrate basal levels of protein content prior to ASC exposure. (B) Western blot detection of ASC cell lysate. Again, 20 μg of protein was separated by SDS-PAGE under reducing conditions, blotted, and probed with indicated antibodies. Bars, ± SD. *P < .05

Figure 6. ASC invasion influenced by shRNA knockdown

ASCs were transfected with an shRNA construct targeting a non-human gene (shRNA Neg Ctrl) or an shRNA construct targeting the gene of interest (shRNA) followed by antibiotic selection. (A) ASCs expressing the shRNA vectors were assessed for their efficiency of gene inhibition by Western blot analysis. About 20 μg of cell lysate isolated from transfected ASCs grown in conditioned media for 24 hours were separated by SDS-PAGE under reducing conditions, blotted, and probed with the indicated antibodies. (B) The shRNA transfected ASCs were seeded in the upper compartment of the transwell inserts with 8-μm pore membrane filters pre-coated with growth factor-reduced Matrigel and incubated for 24 hours. Data was normalized by dividing the values obtained with serum-free media or MDA-MB-231 conditioned media as a chemoattractant by the values obtained for the serum-free media (set as 1). Values are means of three independent experiments, each performed in triplicates. Bars, ± SD. *P < .05, **P < .01.

Figure 7. The role of MMP-15, calpain-4, and calpastatin in ASC invasion in the CAM model

ASCs transfected with an shRNA construct targeting a non-human gene (shRNA Neg Ctrl) or an shRNA construct targeting the gene of interest (shRNA) underwent antibiotic selection. (A) ASCs were labeled with Cell Tracker Green (10μM) and seeded atop the CAM of 10-day-old chick embryos. After 3 days, CAM cross-sections were stained with DAPI and visualized by fluorescent microscopy. Dashed lines outline the upper CAM surface such that cells below the dashed line demarcate invading ASCs. Bar represents 10 μM. (B) CAM invasion is quantified as the number of ASCs that cross the CAM surface and average depth of the leading front of the invading cells. Values are means of three independent fields. Bars, ± SD. *P < .01., **P < .001.