br Funding Research grant support was provided by Guardant H
Funding Research grant support was provided by Guardant Health Inc. for plasma-based comprehensive genomic testing, grant number C16/12/00442.
Introduction A variety of culture systems have been developed previously to examine cancer biology, among which two-dimensional (2D) systems are the most frequently used models. However, it is well known that these systems do not reflect the complexity, heterogeneity, and plasticity of the human tumor microenvironment. Cancer tissues consist of various cell components with highly complex three-dimensional (3D) microstructures. Recent studies have indicated that 3D culture systems, such as spheroid and/or organoid cultures, can provide better insight on the physiological characteristics of cancer BYL-719 in comparison to 2D culture systems [, , ]. Fibroblasts in cancer tissues, also known as cancer-associated fibroblasts (CAFs), are one of the crucial components of stromal cells. Growing evidence has shown that the proliferation, invasion, and metastasis of cancer cells are influenced by their interaction with CAFs [, , , ]. Together, CAFs and other stromal cells can create specific microenvironments for tumor progression. However, CAFs exhibit heterogeneous phenotypes and functions within the same tumor [, , , ]. Furthermore, the properties of CAFs vary widely from case to case. Thus, an analysis of CAF subpopulations is required in order to gain a functional assessment of the tumor microenvironment. Human podoplanin is a transmembrane glycoprotein that contributes to cancer progression and is upregulated on cancer cells, CAFs, and inflammatory macrophages. Recruitment of podoplanin (+) CAFs is considered as a prognostic indicator of lung adenocarcinoma, lung squamous cell carcinoma, and breast carcinoma [, , , ]. Moreover, in vivo studies have revealed that podoplanin (+) CAFs promoted lung adenocarcinoma cell engraftment into SCID mice . Using an in vitro model, we demonstrated that podoplanin (+) CAFs enhanced fibroblast-dependent cancer cell invasion. These studies suggested that podoplanin (+) CAFs are a special subpopulation of CAFs with tumor-promoting functions . Generally, the high proliferative capacity of cancer cells is associated with their high malignant potential. It is important to elucidate whether podoplanin (+) CAFs are capable of enhancing the proliferative activity of cancer cells to further clarify their biological functions. In the current study, we examined the effect of podoplanin (+) CAFs on the proliferation of cancer cells using a 3D organoid model containing both CAFs and cancer cells, instead of common 2D culture systems. In order to confirm the results obtained from 3D culture, we examined specimens of surgically resected lung adenocarcinoma and evaluated the MIB-1 index of cancer cells in cases involving podoplanin (+) and podoplanin (−) CAF.
Materials and methods
Discussion Recent studies have indicated that 3D culture systems, such as organoid cultures, could reflect the physiological complexity, heterogeneity, and plasticity of human tumor microenvironments better than conventional 2D culture systems [, , ]. However, most studies involving 3D systems use organoid models that contain only cancer cells. As CAFs are a major component of cancer stromal cells, 3D culture systems containing both cancer cells and CAFs can be considered as a better model for cancer research. In the current study, we first generated hybrid cancer organoids containing both cancer cells and podoplanin (+) CAFs, a subpopulation of tumor-promoting CAFs. Using this system, we analyzed the influence of podoplanin (+) CAFs on cancer cell proliferation. In our previous study, we found that in CAFs, podoplanin enhanced tumor formation in lung adenocarcinoma cell lines . Furthermore, during fibroblast-dependent cancer cell invasion, podoplanin (+) CAFs enhanced local invasion of cancer cells . In this study, we clarified that podoplanin (+) CAFs increased cancer cell proliferation using a novel hybrid cancer organoid model and surgically resected samples. The molecular mechanism explaining how podoplanin-overexpressing CAFs enhance the proliferative capacity of PC-9 cells remains unclear. One possibility is that podoplanin in CAFs interacts directly with PC-9 cells through C-type lectin-like receptor 2 (CLEC-2), the known receptor for podoplanin . However, PC-9 cells were negative for CLEC-2 when examined using quantitative reverse transcription polymerase chain reaction, suggesting that podoplanin-positive CAFs did not bind directly to cancer cells through CLEC-2 . Identifying extracellular binding proteins that can interact with podoplanin will be an important topic of research in the future.