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The Adipose Stem Cell as a Novel Metabolic Actor in Adrenocortical Carcinoma Progression: Evidence From an In Vitro Tumor Microenvironment Crosstalk Model

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The Adipose Stem Cell as a Novel Metabolic Actor in Adrenocortical Carcinoma Progression: Evidence From an In Vitro Tumor Microenvironment Crosstalk Model

Roberta Armignacco et al. Cancers (Basel).

Abstract

Metabolic interplay between the tumor microenvironment and cancer cells is a potential target for novel anti-cancer approaches. Among stromal components, adipocytes and adipose precursors have been shown to actively participate in tumor progression in several solid malignancies. In adrenocortical carcinoma (ACC), a rare endocrine neoplasia with a poor prognosis, cancer cells often infiltrate the fat mass surrounding the adrenal organ, enabling possible crosstalk with the adipose cells. Here, by using an in vitro co-culture system, we show that the interaction between adipose-derived stem cells (ASCs) and the adrenocortical cancer cell line H295R leads to metabolic and functional reprogramming of both cell types: cancer cells limit differentiation and increase proliferation of ASCs, which in turn support tumor growth and invasion. This effect associates with a shift from the paracrine cancer-promoting IGF2 axis towards an ASC-associated leptin axis, along with a shift in the SDF-1 axis towards CXCR7 expression in H295R cells. In conclusion, our findings suggest that adipose precursors, as pivotal components of the ACC microenvironment, promote cancer cell reprogramming and invasion, opening new perspectives for the development of more effective therapeutic approaches.

Keywords: adipogenesis; adipose precursors; cancer; cell reprogramming; invasion; leptin.

Conflict of interest statement

The Authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Capsular invasion in advanced ACC. (A) Representative Hematoxylin/Eosin staining of an advanced stage 3-ACC showing disruption of the capsule with pushing a well-circumscribed tumor border (*) into the surrounding adipose tissue. (B) Representative Hematoxylin/Eosin staining of an advanced stage 4-ACC displaying cancer extension beyond the capsule with irregular clusters and cords of tumor cells infiltrating the fat. Arrowheads indicate the remaining adrenal capsule. Scale bars = 300 μm (A) and 400 μm (B).
Figure 2
Figure 2
H295R cells stimulate ASC proliferation and drive ASC differentiation toward a myofibroblast-like phenotype. (A) ASCs alone (ASC) or co-cultured with H295R (ASC+H295R) were assessed for cell proliferation at the indicated time points (2, 3, 7 and 9 days) by direct cell count. The proliferative rate was calculated as fold increase (FI) versus the co-culture starting time (Time point = 0), n = 5. (B) Glucose uptake measurement and western blot analysis of GLUT-1 and GLUT-4 expression (inset, fold increase intensity vs. ASC after normalization on actin band is indicated to the right of the bands) assessed in ASCs after 7-day mono- or co-culture, n = 3. (C) Gene expression of specific mesenchymal stem-related markers revealed by RT-qPCR Taqman assay in 7-day co-cultured ASCs compared with the ASC mono-culture, n = 3. (D) Western blot analysis of α-SMA expression and optical microscopy of ASCs cultured alone or in the presence of H295R cells for 7 days. Original magnification: 10×; zoom in: 2×. For western blot analysis, GAPDH or actin were used as internal loading control. Gene expression and glucose uptake are indicated as fold increase (FI) versus ASCs alone. Data are expressed as the mean ± SE in at least three independent experiments; * p < 0.05; ** p < 0.001. Details of western blot can be viewed at the Supplementary Materials.
Figure 3
Figure 3
ASC ultrastructure shows radical changes after co-culture. ASCs cultured alone (A, zoom in B) or in the presence of H295R (C, zoom in: D,E) for 7 days, n = 3 independent experiment. Representative different fields with the same original magnification were digitally merged to reconstruct a wider cell portion. N = nucleus; M = mitochondria; RER = rough endoplasmic reticulum; Ly = lysosome; G = Golgi cisternae; L = lipid droplet. Scale bars = 2 μm (A,C) and 1 μm (B,D,E).
Figure 4
Figure 4
H295R cells affect ASC ability to efficiently differentiate toward mature white adipocytes. (A) ADIPOQ, FABP4 and HSL gene expression measured by RT-qPCR Taqman assay in undifferentiated ASCs (used as negative control) and adipocytes differentiated in vitro from ASCs alone (ADIPO) or induced to differentiate in co-culture with H295R cells (ADIPO+H295R) for 10 days. Gene expression was expressed as fold increase (FI) versus ASCs. The left Y axis refers to AdipoQ and FABP4 expression, the right one to HSL expression. (B) Adipocyte lipid content in ADIPO and ADIPO+H295R was calculated as fold increase (FI) versus undifferentiated ASCs (reference FI = 1, indicated by the white dashed line). (C) AdipoRed-related fluorescence stain of intracellular lipid droplets (green) in ASCs, ADIPO and ADIPO+H295R. Cell nuclei were counterstained with DAPI (blue). Original magnification is indicated for each row. Quantitative data are expressed as the mean ± SE in n = 4 independent experiments; * p < 0.05; ** p < 0.001.
Figure 5
Figure 5
H295R cell proliferation rate increases in the presence of ASCs. (A) H295R cells alone (H295R) or co-cultured with ASCs (H295R+ASC) were assessed for cell proliferation at the indicated time points (2, 3, 7 and 9 days) by direct cell count. The proliferative rate was indicated as fold increase (FI) versus the co-culture starting time (T0: Time = 0). Glucose uptake measurement (B) and western blot analysis of MEK1, ERK and phospho-ERK expression (C) were compared in H295R cells after mono- or co-culture. For western blot analysis, GAPDH was used as internal loading control. Both glucose up-take and protein expression were calculated as fold increase (FI) versus H295R cells alone. (D) Electron microscopy of H295R cells cultured alone or together with ASCs for 7 days. The black arrow indicates the structures corresponding to lipid droplets. N = nucleus. Scale bars = 5 μm. Data are expressed as the mean ± SE vs. H295R cell alone, n = 3 independent experiments; * p < 0.05; ** p < 0.001. Details of western blot can be viewed at the Supplementary Materials.
Figure 6
Figure 6
H295R cell migration and invasion ability is increased by ASC co-culture. (A) Scratch assay: cell migration at 0, 24 and 48 h post-scratch in H295R cells previously cultured alone (H295R) or co-cultured with ASCs (H295R + ASC) for 7 days, and related representative images of n = 3 independent experiments. Original magnification: 5×. (B) Cell invasion assay: the invasion index was calculated as fold increase (FI) versus H295R cells alone. (C) Trans-endothelial migration assay: the invasion index was calculated as fold increase (FI) versus H295R cells alone. (D) Fluorescence staining of F-actin cytoskeleton (in green) in H295R after 7-day mono- or co-culture. DAPI staining (in blue) was used to visualize cell nuclei. Original magnification: 100×; zoom in: 2×. (E) Western blot analysis of FAK, RhoA and Fascin-1 performed on H295R cells cultured alone or together with ASCs for 7 days. GAPDH was used as internal loading control (fold increase intensity vs. H295R cells after normalization on GAPDH band is indicated to the right of the bands). Quantitative data are expressed as the mean ± SE vs. H295R alone in at least three independent experiments; * p < 0.05; ** p < 0.001. Details of western blot can be viewed at the Supplementary Materials.
Figure 7
Figure 7
The potential molecular crosstalk underlying H295R/ASCs interaction. RT-qPCR Taqman analysis of gene expression in both ASCs and H295R cells after 7-day mono- or co-culture (ASC or H295R cells and ASC + H295R or H295R + ASC, respectively). Leptin and IL-8 (A), and CXCL12 (SDF-1) and DDP4 (C) gene expression was evaluated in ASCs, and the intracellular SDF-1 protein expression was assessed by western blot analysis (C), inset; fold increase intensity vs. ASCs after normalization on GAPDH band is indicated to the right of the band). IGF2, IGF-1R and Ob-R gene expression was evaluated in H295R cells (B). For all genes, the relative expression level was calculated as fold increase (FI) versus the mono-culture. For western blot analysis, GAPDH was used as internal loading control. (D) Extracellular levels of SDF-1 measured by ELISA assay in the conditioned medium of ASCs after 7-day mono- and co-culture. The absolute SDF-1 concentrations were normalized on the relative cell number and expressed as fold increase (FI) versus ASCs. (E) CXCR4 and CXCR7 expression was evaluated in H295R cells alone or in co-culture with ASCs. Data are expressed as the mean ± SE vs. the respective monoculture in at least three independent experiments; * p < 0.05; ** p < 0.001 Details of western blot can be viewed at the Supplementary Materials.
Figure 8
Figure 8
Expression of proliferation- and migration-related proteins in H295R cells co-cultured with mature adipocytes. Protein expression was assessed by western blot analysis in cell samples in H295R cells co-cultured for 10 days with previously in vitro differentiated adipocytes (H295R+mADIPO) compared with H295R mono-culture. Bar charts represent protein expression quantified by densitometric analysis of protein bands normalized on GAPDH, used as internal loading control, and calculated as fold increase vs. H295R cells. Data are expressed as the mean ± SE in n = 3 independent experiments, * p < 0.05, ** p < 0.001 vs. H295R cells alone.
Figure 9
Figure 9
Co-culture of H295R cells with mature adipocytes resulted in de-differentiation of the adipose cells. (A) RT-qPCR Taqman assay was performed on cell samples from undifferentiated ASCs, used as negative control, and in vitro differentiated adipocytes cultured alone (mADIPO) or co-cultured with H295R cells (mADIPO+H295R) for 7 days. Gene expression related to AdipoQ and FABP4 is expressed as fold increase (FI) vs. undifferentiated ASCs used as control. (B) Adipocyte lipid content, assessed by AdipoRed assay, was calculated as fold increase (FI) vs. undifferentiated ASCs, (indicated by the white dashed line as = 1). Data are expressed as the mean ± SE in n = 3 independent experiments, ** p < 0.001 vs. mADIPO alone. Representative images of AdipoRed-related fluorescence staining intracellular lipid droplets (green) in samples from undifferentiated ASCs (negative control, (C)), mADIPO (D) and mADIPO+H295R (E). Cell nuclei were counterstained with DAPI (blue). Original magnification: 40×.

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