Korean J Physiol Pharmacol.  2017 Mar;21(2):161-168. 10.4196/kjpp.2017.21.2.161.

Paracrine influence of human perivascular cells on the proliferation of adenocarcinoma alveolar epithelial cells

• Kim E
• Na S
• An B
• Yang SR
• Kim WJ
• Ha KS
• Han ET
• Park WS
• Lee CM
• Lee JY
• Lee SJ
• Hong SH

• KMID: 2371034
• DOI: http://doi.org/10.4196/kjpp.2017.21.2.161

Abstract

Understanding the crosstalk mechanisms between perivascular cells (PVCs) and cancer cells might be beneficial in preventing cancer development and metastasis. In this study, we investigated the paracrine influence of PVCs derived from human umbilical cords on the proliferation of lung adenocarcinoma epithelial cells (A549) and erythroleukemia cells (TF-1α and K562) in vitro using Transwell® co-culture systems. PVCs promoted the proliferation of A549 cells without inducing morphological changes, but had no effect on the proliferation of TF-1α and K562 cells. To identify the factors secreted from PVCs, conditioned media harvested from PVC cultures were analyzed by antibody arrays. We identified a set of cytokines, including persephin (PSPN), a neurotrophic factor, and a key regulator of oral squamous cell carcinoma progression. Supplementation with PSPN significantly increased the proliferation of A549 cells. These results suggested that PVCs produced a differential effect on the proliferation of cancer cells in a cell-type dependent manner. Further, secretome analyses of PVCs and the elucidation of the molecular mechanisms could facilitate the discovery of therapeutic target(s) for lung cancer.

Keyword

Cancer; Paracrine effect; Perivascular cells; Proliferation

MeSH Terms

Adenocarcinoma*
Carcinoma, Squamous Cell
Coculture Techniques
Culture Media, Conditioned
Cytokines
Epithelial Cells*
Humans*
In Vitro Techniques
K562 Cells
Leukemia, Erythroblastic, Acute
Lung
Lung Neoplasms
Neoplasm Metastasis
Umbilical Cord
Culture Media, Conditioned
Cytokines

Figure

• 

  Fig. 1 Isolation and characterization of PVCs derived from HUCs.(A) The morphology of the PVCs in culture. Fibroblast-like colonies were subcultured and expanded. Bars=100 µm. (B) Representative flow cytometry histograms for phenotypes of PVCs (passage 2) are shown. The pink histogram indicates the isotype control. (C) Evaluation of multilineage differentiation potentials of the PVCs. Representative images of Oil Red O staining for adipocytes, Alizarin Red S staining for osteocytes, and Alcian Blue staining for chondrocytes are shown. Bars=100 µm, or 500 µm. (D) Quantification of the dye (Oil Red O and Alizarin Red S) content using spectrophotometry. PVCs, perivascular cells; HUCs, human umbilical cords; CON, control cultures; EXP, experimental cultures with differentiation conditions.

• 

  Fig. 2 PVCs significantly increase the proliferation of human lung adenocarcinoma cells.(A) Indirect co-culturing of PVCs with human erythroleukemia cell lines (TF-1α and K562) and lung adenocarcinoma cell line (A549). The error bars indicate the standard deviation. **p<0.01. (B) The morphology of the cancer cells co-cultured with PVCs for 48 hrs. Bars=50 µm. (C) The diameter of the cancer cells was measured after co-culturing with PVCs for 48 hrs. The error bars indicate the standard deviation. CON, control group; PVCs, perivascular cells.

• 

  Fig. 3 PSPN contributes to the proliferation of human lung adenocarcinoma cells.(A) Secretome analyses of PVC-CM using antibody arrays. A list of selected cytokines that had 1.5 fold or more expression relative to the control sample (non-CM) is shown. (B) A549 cells were plated at a density of 2×105 cells and treated with a variety of concentrations of PSPN (10, 20, and 40 ng/ml) for 48 hrs. The error bars indicate standard deviation. *p<0.05, **p<0.01. (C) Representative images of A549 cells cultured in the presence and absence of PSPN for 48 hrs. Bars=50 µm. CON, control group; PSPN, Persephin; PCV-CM, perivascular cell-conditioned medium; non-CM, non-conditioned control medium.

• 

  Fig. 4 Neutralization of PSPN inhibits proliferation of human lung adenocarcinoma cells.(A) qPCR analysis of relative GFRα4 expression in A549, TF-1α and K562 cells. (B) PSPN neutralizing antibody (500 ng/mL) suppressed the increased proliferation of A549 cells by PSPN (40 ng/mL) treatment. The error bars indicate the standard deviation. **p<0.01, a, p<0.01. (control vs PSPN), b, p<0.01. (PSPN vs PSPN+Ab).

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