We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.
Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 µm pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.
Mesenchymal stem cells (MSCs), with the ability of differentiation and contribution to regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and adipose, have been isolated from almost all tissues in the adult body1,2. Other than providing tissue homeostasis by producing resident cells in case of chronic inflammation or an injury, they produce vital cytokines and growth factors to orchestrate angiogenesis, immune system, and tissue remodeling3. The interaction of MSCs with cancer tissue is not well-understood, but accumulating evidence suggests that MSCs might promote tumor initiation, progression, and metastasis4.
The homing ability of MSCs to the injured or chronically inflamed area makes them a valuable candidate for stem cell-based therapies. However, cancer tissues, "never healing wounds", also release inflammatory cytokines, pro-angiogenic molecules, and vital growth factors, which attract MSCs to the cancerogenous area5. While there are limited reports showing inhibitory effects of MSCs on cancer growth6,7, their cancer progression and metastasis promoting effects have been extensively reported8. MSCs directly or indirectly affect carcinogenesis in different ways including suppressing immune cells, secreting growth factors/cytokines that support cancer cell proliferation and migration, enhancing angiogenic activity, and regulating epithelial-mesenchymal transition (EMT)9,10. Tumor environment consists of several cell types including cancer-associated fibroblasts (CAFs) and/or myofibroblasts, endothelial cells, adipocytes, and immune cells11. Of those, CAFs are the most abundant cell type in the tumor area that secrete various chemokines promoting cancer growth and metastasis8. It has been shown that bone marrow-derived MSCs can differentiate into CAFs in the tumor stroma12.
Dental pulp stem cells (DPSCs), characterized as the first dental tissue-derived MSCs by Gronthos et al.13 in 2000 and then widely investigated by others14,15, express pluripotency markers such as Oct4, Sox2, and Nanog16 and can differentiate into various cell linages17. Gene and protein expression analysis proved that DPSCs produce comparable levels of growth factors/cytokines with other MSCs such as vascular endothelial growth factor (VEGF), angiogenin, fibroblast growth factor 2 (FGF2), interleukin-4 (IL-4), IL-6, IL-10, and stem cell factor (SCF), as well as fms-like tyrosine kinase-3 ligand (Flt-3L) that might promote angiogenesis, modulate immune cells, and support cancer cell proliferation and migration18,19,20. While the interactions of MSCs with cancer environment have been well-documented in the literature, the relationship between DPSCs and cancer cells has not been evaluated yet. In the present study, we established co-culture and condition medium treatment strategies for a highly metastatic prostate cancer cell line, PC-3, and DPSCs to propose potential action of mechanism of dental MSCs in cancer progression and metastasis.
Written informed consent of the patients was obtained after the approval from the Institutional Ethics Committee.
1. DPSC Isolation and Culture
2. Characterization of DPSCs
3. Preparation of Condition Medium (CM)
4. Treatment of Cancer Cells with CM
5. Cell Migration by Indirect Contact of Cancer Cells and DPSCs
6. Co-culture Assay and Flow Cytometry Analysis
Figure 1 depicts the general MSC characteristics of DPSCs under culture conditions. DPSCs exert fibroblast-like cell morphology after plating (Figure 1B). MSC surface antigens (CD29, CD73, CD90, CD105, and CD166) are highly expressed while hematopoietic markers (CD34, CD45, and CD14) are negative (Figure 1C). Changes at the morphological and molecular level related to osteo-, chondro-, and adipo-genic differentiation are observed in DPSCs culture followed by differentiation cocktail application (Figure 1D).
To determine the activity of secreted molecules from DPSCs on prostate cancer cell proliferation and migration, we applied CM that is collected from cultured dental stem cells and analyzed cancer cell proliferation and migratory behavior. CM treatment (20% v/v) was selected based on MTS cell viability analyses. PC-3 cells treated with stem cell CM were subjected to the TUNEL assay and qPCR analyses to determine cell death and apoptotic regulation under control and experimental conditions. We concluded that CM treatment increased cell viability and reduced cell death in PC-3 cell culture. Ex vivo cell migration assay (scratch assay) was performed to evaluate whether CM of DPSCs affects PC-3 cancer cell migration. Treatment with the concentrations of 10% and 20% CM (v/v) increased scratch closure significantly in comparison to the control group at 24 h (Figure 2). Similarly, 20% CM (v/v) treatment induced upregulation of extracellular matrix protein gene expressions such as collagen I, fibronectin, and laminin, which play significant roles in cell migration.
We used two different culture techniques to establish direct and indirect co-cultures of PC-3 cells and DPSCs under ex vivo conditions. Trans-well system was selected to create an indirect interaction environment for PC-3 cancer cells. PC-3 cells were seeded onto the bottom of the well plate and inserts carrying DPSCs were placed on the top. Because we aimed to generate an in-direct interaction, trans-well membranes with 0.4 μm pore size were used to prevent physical cell movement of DPSCs from the upper part to bottom part through the membrane. Secreted molecules from DPSCs increased scratch closure of PC-3 cells significantly compared to control cells. PC-3 cells co-cultured with DPSCs demonstrated a 51% scratch closure while control cells had a 38% closure after 24 h (Figure 3A). Direct co-cultures containing 1:1 ratio of DPSCs and PC-3 cells were used to analyze self-organization of stem cells and cancer cells. Cells were stained with red and green membrane dyes to distinguish different cell types under microscope. PC-3 cells stained with red fluorescence dye were surrounded by DPSCs in a tube-like structure after a 24 h incubation period. Co-cultured cells stained with fluorescent dyes were analyzed by flow cytometry based on fluorescence staining, and cell ratios were detected. DPSCs and PC-3 cells created a well-organized structure in which PC-3 cells proliferated rapidly after 48 h (Figure 3B). Although cells were seeded at an equal ratio (1:1) for direct co-culture, 62.22% of PC-3 cells were determined after 48 h incubation, indicating the higher proliferation rate of PC-3 cells.
Figure 1: Characterization of dental pulp stem cells (DPSCs). (A) Pulp tissue obtained from the center of tooth. (B) Fibroblast-like cell morphology of DPSCs. Scale bar: 100 μm. (C) Flow cytometry analyses of DPSCs. CD29, CD73, CD90, CD105, and CD 166 are positive surface markers, while CD14, CD34, and CD45 are negative surface markers. NC: negative control (growth medium treated cells). (D) Differentiation of DPSCs to mesenchymal cell types was confirmed with von Kossa staining, Alcian Blue staining, and lipid droplets (scale bar: 200 μm). Osteocalcin, collagen type II (Col II), and fatty acid binding protein 4 (FABP4) immunostainings showed the osteo-, chondro-, and adipogenic differentiation of DPSCs. Scale bar: 200 μm. This figure is adapted from Dogan et al.34. Please click here to view a larger version of this figure.
Figure 2: Collection of condition medium (CM) from DPSCs for cell viability and scratch analyses. TUNEL positive cells were decreased by 20% CM (v/v) application. Quantitative measurement of scratch closure showed that CM increased PC-3 cell migration. CM: conditioned medium; NC: negative control (growth medium treated cells). *P < 0.05. This figure is adapted from Dogan et al.34. Please click here to view a larger version of this figure.
Figure 3: Methodology for trans-well cell migration assay and co-culture. (A) PC-3 cell scratch closure in the trans-well co-culture system. (B) Interaction pattern of stained DPSCs (green fluorescence) and PC-3 cells (red fluorescence) after 24 h and 48 h of co-culture (1:1 seeding ratio). Higher PC-3 cell number was detected with respect to DPSCs. Scale bar: 200 μm. This figure is adapted from Dogan et al.34. Please click here to view a larger version of this figure.
Contribution of MSCs to tumor environment is regulated by several interactions including hybrid cell generation via cell fusions, entosis or cytokine and chemokine activities between stem cells and cancer cells28. Structural organization, cell-cell interactions, and secreted factors determine cancer cell behavior in terms of tumor promotion, progression, and metastasis to surrounding tissue. Proper ex vivo model systems to investigate the mechanisms behind the interactions of resident cell populations are required to understand cellular communications for cancer progression and metastasis.
We used DPSCs to create a model system for prostate cancer and dental stem cell interactions. The advantage of this type of adult stem cell is the accessibility of tissue source and easy isolation steps. On the other hand, using only DPSCs without comparison with well-known adult stem cell types is a limitation of this protocol. Current co-culture methods are categorized into direct and indirect techniques which include paracrine signaling of soluble secreted molecules and direct culture of different cell populations in the same environment29,30,31. CM of fully characterized DPSCs was used to evaluate paracrine signaling mediated cancer cell growth in culture. CM application is the simplest method for cell interaction studies and allows for observation of soluble mediator activities in the culture system. Although CM is not a fully adequate model system, CM application as a co-culture method is very efficient to observe cell-cell interactions ex vivo. CM of DPSCs increased the cell proliferation and migration of prostate cancer cells, indicating the acquisition of metastatic phenotype due to the factors secreted from DPSCs.
Another model of cell-cell interaction is the establishment of a physical barrier such as a trans-well membrane between cell populations30. Trans-well systems are divided into two types: those which allow cell movement through the pores, and those enabling transfer of secreted factors while hindering cell contact through the membrane as well. We used trans-wells with 0.4 μm pore size to analyze the closure of PC-3 scratches, revealing higher cancer cell migration rate in the DPSC group compared to the control cells.
Although CM and trans-well based systems are advantageous for simply analyzing contributions of a particular cell type32, direct culturing of different subpopulations in the same environment is necessary in parallel with indirect co-culture methods. Seeding ratio can be controlled and structural organization of distinct populations can be easily analyzed by direct co-culture of MSC with cancer cells. We used 1:1 ratio of DPSCs and PC-3 cells stained with green and red fluorescence membrane dyes, respectively. DPSCs encircled PC-3 cells and created a tube-like structure in culture wells. PC-3 cells formed clusters in the form of islets surrounded by channel-like structures generated by DPSCs.
Recently, Brunetti et al. showed that DPSCs secrete TNF-related apoptosis-inducing ligand (TRAIL) during osteogenic differentiation and affect myeloma cancer cell viability, indicating the possible interactions of dental stem cells with cancer cells33. Our study is the first report that evaluates interactions of dental derived MSCs and prostate cancer cells as an ex vivo model. We used three different direct/indirect approaches in our experiments. Proliferation of cancer cells and high migration rates were detected either by CM and trans-well assays or by co-culturing differentially stained cells that allows for multiple interactions.
The authors have nothing to disclose.
This study was supported by Yeditepe University. All data and figures used in this article were previously published34.
DMEM | Invitrogen | 11885084 | For cell culture |
FBS | Invitrogen | 16000044 | For cell culture |
PSA | Lonza | 17-745E | For cell culture |
Trypsin | Invitrogen | 25200056 | For cell dissociation |
PBS | Invitrogen | 10010023 | For washes |
Dexamethasone | Sigma | D4902 | Component of differentiation media |
β-Glycerophosphate | Sigma | G9422 | Component of osteogenic differentiation medium |
Ascorbic acid | Sigma | A4544 | Component of osteo- and chondro-genic differentiation medium |
Insulin-Transferrin-Selenium (ITS −G) | Invitrogen | 41400045 | Component of chondrogenic differentiation medium |
TGF-β | Sigma | SRP3171 | Component of chondrogenic differentiation medium |
Insulin | Sigma | I6634 | Component of adipogenic differentiation medium |
Isobutyl-1-methylxanthine (IBMX) | Sigma | I7018 | Component of adipogenic differentiation medium |
Indomethacin | Sigma | I7378 | Component of adipogenic differentiation medium |
MTS Reagent | Promega | G3582 | Cell viability analyses |
TUNEL Assay | Sigma | 11684795910 | Apoptotic analyses |
24-well plate inserts | Corning | 3396 | For trans-well migration assay |
PKH67 | Sigma | PKH67GL | For co-culture cell staining |
PKH26 | Sigma | PKH26GL | For co-culture cell staining |
Paraformaldehyde | Sigma | P6148 | For cell fixation |
von Kossa Kit | BioOptica | 04-170801.A | For cell staining (differentiation) |
Alcian blue | Sigma | A2899 | For cell staining (differentiation) |