During the metastatic process, cancer cells interact with vascular adventitial fibroblasts (VAF), which are the main components of the outermost connective tissue layer of blood vessels. This activity suggests the presence of a specific tumor microenvironment in the perivascular area. The s.c. coinjection of human lung adenocarcinoma cell lines (A549, PC-14, and CRL-5807) and human VAF (hVAF) resulted in a high rate of tumor formation, compared with the coinjection of these cell lines and human lung tissue-derived fibroblasts (hLF). A cDNA microarray analysis revealed a higher expression level of podoplanin in hVAFs than in hLFs (4.7-fold). Flow cytometry analysis also showed a higher expression level of podoplanin in hVAFs (43% ± 17.5%) than in hLFs (16% ± 10.3%). Sorted podoplanin-positive hVAFs displayed enhanced tumor formation, lymph node metastasis, and lung metastasis of A549 compared to sorted podoplanin-negative hVAFs. Knockdown of podoplanin in hVAFs decreased the augmenting effect of tumor formation and in vitro colony formation. The overexpression of podoplanin in hVAFs hastened the tumor formation of A549, compared with control hVAFs. Furthermore, the analysis of small-sized human lung adenocarcinoma (n = 112) revealed that patients with podoplanin-positive cancer-associated fibroblasts had a significantly higher rate of lymph node metastasis and a high risk of recurrence. These results indicate a promotive effect of hVAFs mediated by podoplanin on cancer progression and suggest that the perivascular environment may constitute a specific niche for tumor progression. Cancer Res; 71(14); 4769–79. ©2011 AACR.
The cancer tissue is composed of different types of stromal cells forming microenvironments (1–3). It has been hypothesized that these stromal components are functionally organized to promote the survival of cancer cells in the host and to generate a favorable microenvironment for cancer cells in both primary and metastatic sites (3–7). The contribution of stromal fibroblasts to the development of a variety of tumors has been supported by extensive clinical evidence and the use of experimental mouse models (8). Accumulating evidence suggests that stromal fibroblasts may promote tumor growth by several mechanisms such as by inducing angiogenesis, recruiting bone marrow–derived endothelial progenitor cells, and remodeling the extracellular matrix (9–11). However, although direct physical interaction between fibroblasts and cancer cells has recently been proposed as a mechanism of cancer cell migration (12), the role of the direct biological interaction between fibroblasts and cancer cells in augmenting tumor progression is not fully understood. It is also increasingly apparent that cancer stromal fibroblasts represent a diverse cell population with different characteristics according to their various origins, such as resident fibroblasts, vascular smooth muscle cells, pericytes (vascular adventitia), and bone marrow–derived cells (13–17).
The vascular adventitia is the outermost connective tissue layer of blood vessels and contains fibroblasts. Animal models have showed that changes in the functions of vascular adventitial fibroblast (VAF) phenotype, such as to a myofibroblast phenotype, contribute to the pathologies of a variety of diseases (18–22). During vascular invasion, cancer cells interact with the VAFs of the existing blood vessels involved with the cancer and migrate into the vascular lumen. Nakayama and colleagues (23) reported that in gastric cancer, blood vessels display structural abnormalities, such as the lack of a vascular adventitia, suggesting that VAFs are absorbed and become part of the stromal fibroblasts. We previously reported the in vitro characteristics of VAFs as fibroblasts containing mesenchymal progenitor/stem cells (MSC; ref. 24), which are reportedly associated with the progression of metastasis in breast cancer (25). The anatomic location and phenotype of VAFs suggest that VAFs may represent one of the origins of the cancer stromal fibroblasts that create a specific microenvironment conductive for cancer progression.
To investigate whether VAFs constitute a specific microenvironment conducive for cancer progression, to determine which compartment is responsible for progression, and to identify the mechanism of progression, we performed in vivo experiments using human VAF (hVAF) and human lung tissue-derived fibroblasts (hLF) in conjunction with the human lung adenocarcinoma cell line A549. We report here that the hVAFs enhanced the tumor formation ability of A549, compared with the hLFs. Moreover, podoplanin was highly expressed in the hVAFs, and podoplanin is the functional protein that is responsible for the enhancement of the tumor formation ability.
Materials and Methods
The hVAFs and hLFs were cultured as described before (24). The study was approved by the Institutional Review Board of the National Cancer Center. The human lung adenocarcinoma cell lines A549 and PC-14 were obtained from the RIKEN BioResource Center. A549 was cultured in DMEM (Sigma) and PC-14 was cultured in RPMI 1640 (Invitrogen). CRL-5807 was obtained from the American Type Culture Collection (ATCC) and was cultured in RPMI 1640. All the cell lines were cultured in a medium containing 10% FBS (Sigma), and 1% penicillin and streptomycin (Sigma) and were incubated at 37 in an atmosphere containing 5% CO2. All the cell lines were used within 2 months after resuscitation of frozen aliquots. Cell lines were authenticated on the basis of viability, growth, and morphology. Passages lower than 10 were used. Primary cells were authenticated using the experiments described before (24) including flow cytometric verification of surface marker expression (CD3, CD14, CD20, CD29, CD34, CD44, CD45, CD68, CD105, CD117, and CD133).
Flow cytometry and cell sorting
The cells were incubated with anti-podoplanin (gp36, clone 18H5, Abcam) antibody and excess antibody was removed by washing with PBS (containing 1% FBS). Polyclonal rabbit anti-mouse immunoglobulins/PE (Dakocytomation) were added as a secondary antibody. The cells were then rinsed with PBS and a FACS scan was performed using FACSCalibur (BD Biosciences). Sorting was performed using FACSAria (BD Biosciences).
Western blot analysis
The cells were lysed in whole-cell extraction buffer (20 mmol/L HEPES-NaOH, 0.5% NP-40, 15% glycerol) containing Complete, a protease inhibitor cocktail tablet (Roche Diagnostics). The proteins were separated on a 12% SDS-polyacrylamide gel and transferred to an Immobilon-P, PVDF membrane (Millipore). The blots were incubated overnight at 4°C with anti-human mouse monoclonal podoplanin antibody (D2-40, Signet) or polyclonal goat actin antibody (Santa Cruz). After washing in TBS-T, the membranes were incubated with HRP-rabbit anti-mouse IgG or HRP-rabbit anti-goat IgG (Zymed). ECL Western Blotting Detection Reagents (GE Healthcare) were used to develop the high-performance chemiluminescence film (GE Healthcare).
Cell growth in soft agar
The anchorage-independent growth of A549 with or without fibroblasts was examined using a colony formation assay in soft agar (Difco Agar Noble). A total of 5 × 103 cells/35 mm dish were suspended in culture medium containing 0.4% agar (1.5 mL) and immediately overlaid onto 0.5% bottom agar in the culture medium (3 mL). The cells were then incubated at 37°C. Two weeks later, colonies with a diameter of >200 μm were counted.
Immunohistochemistry and GFP-positive area analysis
Sections were deparaffinized and heated in citric acid buffer solution at 95°C for 20 minutes. The endogenous peroxidases were quenched with 0.3% H2O2 in PBS. The sections were incubated overnight at 4°C using anti-GFP antibody (Invitrogen). The sections were then washed and incubated using the Envision+ system (Dako Cytomation) at room temperature. The color reaction was developed in 2% 3,3′-diaminobenzidine in 50 mmol/L Tris-buffer (pH 7.6) containing 0.3% H2O2. The sections were counterstained with Meyer's hematoxylin, dehydrated and mounted. The tumor area and GFP-positive areas of the xenograft sections were analyzed using Axio Vision 4.7.1 software (Zeiss).
Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and PCR
Cells were washed with PBS and suspended in 1 mL of TRIzol (Invitrogen), then stored at −80. Total RNA was purified from thawed samples using standard techniques, and cDNA was synthesized using the PrimeScript RT reagent Kit (TaKaRa), according to the manufacturer's instructions. RT-PCR was performed in a Smart Cycler System (TaKaTa) using SYBR Premix Ex Taq (TaKaRa) according to the manufacturer's instructions.
Human WT-podoplanin lentivirus vector was generated by subcloning pcDNA3/human-WT-podoplanin vector (kindly provided by Dr. N. Fujita, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research). The lentiviruses were produced using 293T cells transfected with PCAG-HIV, pCMV-VSV-G-RSV-Rev, and either a podoplanin shRNA vector (CS-H1-shRNA-EG; RIKEN BioResource Center) or a human-WT-podoplanin vector. Transfection was achieved using LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturers' instructions. Vector-containing medium was filtered through a 0.45‐μm filter and 8 μg/mL of polybrene (Sigma) was added for target cell transduction.
The correlation between podoplanin expression in stromal fibroblasts and the clinicopathologic factors were evaluated using the χ2 test. Overall survival was measured from the date of surgery until the date of death from any cause or the date the patient was last known to be alive. The survival curves were estimated using the Kaplan–Meier method, and the differences in survival between the 2 groups were compared using the log-rank test. Statistical analysis software (SPSS, version 11.0) was used to perform the analysis.
A549 human lung adenocarcinoma cells alone, or with either hVAFs or hLFs were injected into the s.c. tissue of SCID mice (8–12 weeks of age; CLEA). Tumor volume was calculated as the product of a scaling factor of 0.52 and the tumor length, width, and height. After some procedures such as FACS sorting and transfection with overexpression lentivirus, injection of at least 5 × 104 cells were necessary to have a tumor. For metastatic analysis, at 12 weeks after injection mice were killed and their organs were removed and fixed in 10% formalin. All 5 lobes of lungs were histologically examined for metastasis.
Gene expression analysis using a microarray
We used GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix), containing 54,675 probe sets, to analyze the mRNA expression levels of approximately 47,000 transcripts and variants from 38,500 well-characterized human genes. Target cRNA was generated from 100 ng of total RNA from each sample using a 3′ IVT Express Kit (Affymetrix). The procedures for target hybridization, washing, and staining with signal amplification were conducted according to the supplier's protocols. The arrays were scanned with a GeneChip Scanner 3000 (Affymetrix), and the intensity of each feature of the array was calculated using GeneChip Operating Software, version 1.1.1 (Affymetrix). The average intensity was standardized to the target intensity, which was set equal to 1,000, to reliably compare various multiple arrays. The values were log transformed and median centered. The programs GeneSpring (Agilent Technologies) and Excel (Microsoft) were used to perform the numerical analysis to permit gene selection. The GEO accession number for microarray data is GSE26146.
All the surgical specimens were fixed with 10% formalin or methanol and embedded in paraffin. Serial 4-μm sections were stained with hematoxylin and eosin and the Verhoeff-van-Gieson method to visualize elastic fibers. Lymphatic invasion was evaluated in sections stained with hematoxylin and eosin. Vascular invasion was evaluated using the Verhoeff-van-Gieson method. Podoplanin staining for human surgical specimens were done using anti-human mouse monoclonal podoplanin antibody (D2-40, Signet). According to the definition described in the previous study (26), the positive stained spindle-shaped cells in the cancer stroma were identified as fibroblasts.
hVAFs enhance tumor formation and colony formation
A549 human lung cancer cells (1 × 106) were s.c. injected alone or with either hVAFs or hLFs into SCID mice. At 3 weeks after the injection, tumors had formed in all the mice injected with A549 alone and with either type of fibroblast (Fig. 1A). Notably, although the rate of tumor formation in mice injected with A549 alone or coinjected with A549 and hLFs decreased as the number of injected cells was gradually diluted, the rate of tumor formation remained 100% in mice coinjected with A549 and hVAFs even when the number of injected cells was diluted to 5 × 104 cells. When 1 × 104 cells were injected, no tumors were detected in mice injected with A549 alone or coinjected with A549 and hLFs, but tumors were identified in 25% (2/8) of the mice that were coinjected with A549 and hVAFs (Fig. 1A). By 1 × 104 cells injection, while a tumor was identified at week 4 in 1 of the 8 mice coinjected with A549 and hLFs, most of the mice coinjected with A549 and hVAFs already exhibited tumors (7/8) at this time point (Fig. 1B). However, the growth kinetics of the hVAFs-containing tumors and the hLFs-containing tumors were similar once the tumors were detected (Fig. 1C). Importantly, none of the animals injected with 1 × 104 of A549 cells alone exhibited tumor formation at any time point until week 8 (Fig. 1B).
When tumors coinjected with A549 and GFP-labeled fibroblasts were analyzed, the area of GFP-positive fibroblasts in the tumor tissue at 5 days after injection was similar in the hVAFs and hLFs groups (Fig. 1D). To verify that the capacity of hVAFs to enhance tumor formation is not restricted to the A549 cell line, we performed an in vivo tumor formation assay using 2 other human lung adenocarcinoma cell lines, CRL-5807 and PC-14. Three weeks after the coinjection of these cell lines and hVAFs (5 × 104 cells each), both cell lines produced tumors in all 4 cases, whereas the tumor formation rate after coinjection with hLFs was 50% (2/4; Fig. 1E). We next performed in vitro A549 colony assay. Colonies with a diameter of 200 μm or larger were counted, and the number of colonies was significantly larger when the cells were seeded with hVAFs (Fig. 1F). In contrast, seeding the A549 cells with hLFs produced only a slight enhancement in colony formation (Fig. 1F).
Podoplanin is overexpressed in hVAFs, and podoplanin-high hVAFs and hLFs enhance A549 tumor formation
We performed a DNA microarray analysis of hVAFs and hLFs (Table 1). Among the genes that were upregulated in hVAFs, we focused on podoplanin, a glycoprotein that is expressed in cancer stromal fibroblasts of human adenocarcinoma in a manner that is correlated with the severity of the disease (26). A flow cytometry analysis revealed that podoplanin was expressed in a higher percentage of hVAFs than hLFs in 10 of the 11 cases (Fig. 2A). The average percentage of podoplanin-high cells in the hVAFs was 43% (SD ± 17.5) and the average percentage of podoplanin-high cells in the hLFs was 16% (SD ± 10.3). To determine whether the podoplanin-high cells were responsible for the enhancement of A549 tumor formation by hVAFs, we coinjected mice with sorted podoplanin-high hVAFs or podoplanin-low hVAFs and A549 s.c. Notably, at week 2, tumors produced by 5 × 104 cells of podoplanin-high hVAFs and A549 were detectable in 70% (7/10) mice, whereas tumors were produced in only 30% (3/10) of the mice coinjected with podoplanin-low hVAFs and A549 (Fig. 2B). We next performed the same assay using hLFs (5 × 104 cells injection). Tumors were detected (at week 2) in 89% (8/9) of the mice coinjected with podoplanin-high hLFs and A549, whereas tumors were detected in only 30% (3/10) of the mice coinjected with podoplanin-low hLFs and A549 (Fig. 2C). The growth kinetics of tumors produced by the coinjection of podoplanin-high or podoplanin-low hVAFs and A549 had comparable slopes (Fig. 2D). We also observed that the numbers of lymph node metastases and lung metastases were much larger in animals coinjected with podoplanin-high hVAFs or podoplanin-high hLFs (Fig. 2E). Among the mice coinjected with podoplanin-high fibroblasts, 92% (11/12) had lymph node metastases; meanwhile 50% (6/12) of the mice coinjected with podoplanin-low fibroblasts exhibited lymph node metastases. In addition, lung metastases were seen in 67% (4/6) of the mice coinjected with podoplanin-high fibroblasts and 33% (2/6) of the mice coinjected with podoplanin-low fibroblasts (Fig. 2E). Bone metastases were not visible in histopathologic investigation.
Podoplanin as a functional protein for A549 tumor formation
Short hairpin (sh) RNA for podoplanin or luciferase were transduced into hVAFs for the stable reduction of podoplanin expression by >80% or as a control, respectively. The expression level of podoplanin in the transduced cells was confirmed using a Western blot analysis and RT-PCR (Fig. 3A and B). Knockdown of podoplanin did not have an effect on hVAFs cell growth on day 3 (data not shown). An in vitro colony assay revealed a 4-fold difference in colony formation between A549 cells seeded with luciferase-shRNA hVAFs and those seeded with podoplanin-shRNA hVAFs (Fig. 3C). Podoplanin-shRNA hVAFs or luciferase-shRNA hVAFs and A549 were then co-injected into mice s.c. (1 × 104 cells each). Fewer tumors were detected in mice co-injected with podoplanin-shRNA hVAFs than in mice coinjected with control hVAFs at all-time points (Fig. 3D). Similar results were seen using another podoplanin-shRNA (Fig. 3D). We further examined the dependence of tumor formation on podoplanin using the enforced expression of podoplanin in hVAFs (Fig. 3E). Podoplanin-overexpressed hVAFs with A549 cells (5 × 104 cells injection each) resulted in a tumor formation rate of 75% at week 2 (3/4), whereas tumor formation was only observed in 25% (1/4) of the animals coinjected with control hVAFs (Fig. 3F).
Podoplanin-positive stromal fibroblasts in human lung adenocarcinoma
We examined the presence of podoplanin positive cancer-associated fibroblasts (CAF) in human lung adenocarcinomas with a tumor size of 3 cm or less (Fig. 4F). Of the 112 specimens, 32 were positive for podoplanin immunostaining. Podoplanin-positive CAF cases displayed significantly more vascular invasion (P < 0.0001) and lymph node metastasis (P < 0.005; Fig. 4B). Furthermore, a Kaplan–Meier analysis showed a significant difference in both the disease-free interval (P < 0.001) and the overall survival (P < 0.001) between 2 groups (Fig. 4C and D).
In this study, we present for the first time the finding that fibroblasts with 2 different origins but from the same organ specimen had distinct effects on the enhancement of tumor formation. We used 2 human-derived primary fibroblasts, hVAFs and hLFs. Both fibroblast types enhanced the tumor formation abilities of A549, CRL-5807 and PC-14, but this ability was especially augmented by hVAFs. Of the genes differentially expressed in hVAFs compared to hLFs in our microarray analysis, we focused on podoplanin because a previous study proposed that the expression of podoplanin in stromal fibroblasts in adenocarcinoma patients was correlated with a poor prognosis and a high rate of recurrence at all stages (26). Podoplanin is known as a lymphatic endothelial marker. It is a mucin-type sialoglycoprotein reported to be expressed in several cancer cells (27–30) and that binds to CLEC-2 on platelets to form platelet-aggregations, enhancing metastasis (31). After sorting podoplanin-high cells from both hVAFs and hLFs, we found that regardless of the fibroblast origin, podoplanin-high fibroblasts were capable of enhancing the tumor formation rate, compared with podoplanin-low cells (Fig. 2B and C). This finding suggests that differences in podoplanin expression could be responsible for the superiority of hVAFs in promoting tumor formation, compared with hLFs.
To determine whether podoplanin-low fibroblasts can become podoplanin-high fibroblasts, we tested the effect of A549 conditioned media on hVAFs in vitro. We observed that less than 10% of podoplanin-low fibroblasts exposed to A549 conditioned media upregulated podoplanin expression by flow cytometry analysis (data not shown). We next investigated whether coinjection of either hVAFs or hLFs (1 × 105 cells) with A549 could lead to an increase in the frequency of podoplanin-expressing cells within the tumor in vivo, 1-week postinjection. However, both hVAFs and hLFs localized mainly in the necrotic area of the tumor, thus hindering accurate quantification and comparison of the percentage of podoplanin-expressing cells in this experiment. Furthermore, there was no correlation between these observations and the frequency of podoplanin-high cells in the injected hVAF or hLF populations (data not shown).
The shRNA knockdown of podoplanin expression not only supported the data that podoplanin-high fibroblasts enhance the tumor formation ability, it also revealed that podoplanin is a functional protein in tumor formation enhancement. This conclusion was also supported by the results of an in vitro colony assay. Tumor progression is thought to occur in several steps, such as colonization, proliferation, inflammation and angiogenesis, infiltration, and metastasis to the new environment (32, 33). Based on the in vivo survival rate of GFP-positive hVAFs and hLFs in the s.c. tumors and the in vivo tumor volume growth rate after tumor formation, podoplanin-high fibroblasts are predicted to function during the early phase of tumorigenesis. Furthermore, the in vitro colony assay suggested that podoplanin in fibroblasts promotes an environment conducive to cancer cell anchorage independence, where neither angiogenesis nor inflammation occurs. Taken together, these phenomena suggest that podoplanin in fibroblasts functions as a tumor formation enhancer during the initiation of tumorigenesis, such as during the pre-angiogenic phase.
How podoplanin-high fibroblasts interact with cancer cells to promote tumor progression remains unclear. One possibility is direct extracellular binding of fibroblasts to cancer cells, promoting cancer progression. In vitro coculturing of A549 and hVAFs showed no difference in A549 cell number even in direct contact with hVAFs (Supplementary Fig. 1A). Furthermore, seeding A549-GFP on either hVAFs or hLFs did not lead to differences in tumor cell proliferation (Supplementary Fig. 1B). These in vitro experiments suggest that direct contact between fibroblasts and lung cancer cells is not sufficient to influence cancer cell proliferation and/or apoptosis. Moreover, the human cancer cell lines used in this study were negative for CLEC-2 or podoplanin expression when examined using quantitative RT-PCR (data not shown), suggesting that the podoplanin-expressing fibroblasts did not bind directly to cancer cells, at least not through CLEC-2, the only receptor for podoplanin described so far. Still, the podoplanin produced in fibroblasts may bind to cancer cells via process mediated by some other proteins. Extracellular binding proteins that interact with podoplanin should be further analyzed to determine the possibility of a direct interaction with cancer cells. Another possibility is stimulation of a signaling pathway within podoplanin-high fibroblasts leading or mediating tumor development. However, although the addition of Luciferase-shRNA hVAFs or podoplanin-shRNA hVAFs to soft agar containing A549 cells revealed the significant ability of podoplanin-expressing hVAFs to enhance colony formation (Fig. 3C), the addition of their culture media did not enhance tumor formation (Supplementary Fig. 2A). Furthermore, the addition of recombinant podoplanin into soft agar did not influence the colony formation rate of A549 (Supplementary Fig. 2B), suggesting that an interaction between podoplanin-high fibroblasts and cancer cells is responsible for enhancing the tumor formation ability. In addition, the fibroblasts alone did not form colonies in either the in vivo or in vitro assays, supporting the concept that the fibroblasts provide a favorable environment in which cancer cells may form colonies but that the fibroblasts themselves do not form or produce proliferative colonies. So far, the ERM protein family is known to bind to the intracellular domain of podoplanin to stimulate the RhoA activity of the cells, inducing cell migration (34). Given that podoplanin expression in fibroblasts contributes to tumorigenesis, coordination of this signaling response involving cell motility and/or direct crosstalk through an extracellular domain might act as molecular cues that trigger tumor progression and promote a poor clinical outcome. Thus, further analysis is needed to determine how podoplanin-high fibroblasts enhance tumor formation progression.
It is interesting that podoplanin-high cells are more common among hVAFs than among hLFs. This may indicate that during vascular invasion, the metastatic ability of cancer cells may be enhanced by interaction with VAFs. Alternatively, circulating cancer cells that encounter podoplanin-low fibroblasts and/or podoplanin-high inducible fibroblasts may have a better chance of colonizing the new environment. Our in vivo mouse model also revealed a difference in the rates of metastasis to the lymph nodes and lungs (Fig. 2C). Consistent with this data, the results of our clinical evaluation of adenocarcinomas with a size of 3 cm or smaller further showed the relevance of podoplanin as a prognostic factor. Although the tumor size at the time of resection was limited to 3 cm or less, the rates of vascular invasion and lymph node metastasis were significantly higher among the patients with podoplanin-positive stromal fibroblasts (Fig. 4B). Moreover, the disease-free interval was significantly lower among patients with podoplanin-positive fibroblasts (Fig. 4C). The current clinic-pathological data also suggest that podoplanin-positive fibroblasts enhance tumor progression.
In colorectal cancer, the presence of podoplanin-positive stromal fibroblasts is associated with a better prognosis, compared with the presence of podoplanin-negative stromal fibroblasts (35). We suspect that this difference can be explained by a difference in how cancer cells react to podoplanin-expressing fibroblasts and/or a difference in the function of podoplanin-expressing fibroblasts in different organs. Therefore, further studies should focus on evaluating in which types of cancer podoplanin-positive fibroblasts contribute to tumor progression and what are the mechanisms that mediate this contribution. Elucidating how different cancer cell types elicit distinct responses from the same fibroblasts may further reveal the function of podoplanin in organ-specific fibroblasts.
Previously, we reported that hVAFs, but not hLFs, contain MSCs (24). It has been reported that bone marrow–derived MSCs can promote breast cancer metastasis in vivo (25). To elucidate whether the MSCs within the hVAFs are responsible for the enhancement of A549 tumor formation, we induced their differentiation into either the adipogenic or an osteogenic lineage. We found that culturing hVAFs with induction medium did not change the ability of hVAFs to enhance tumor formation in the A549 cell line (Supplementary Fig. 3A–D). Because the identification of MSCs in hVAFs has not been clarified, we cannot exclude MSCs as the population responsible for tumor formation. However, we have shown that neither osteogenic nor adipogenic differentiation of hVAFs does not change the potential of hVAFs to augment the tumor formation ability of A549 cells.
In conclusion, we have identified that hVAFs enhance the tumor formation of lung adenocarcinoma cells, compared with hLFs, and that this activity was at least partly dependent on podoplanin, a functional protein expressed on fibroblasts. Podoplanin-positive cancer cells are known to metastasize through the formation of podoplanin–platelet complex. Therefore, the promotive effect of podoplanin-positive stromal fibroblasts on cancer progression suggests that not only tumor cells, but also the fibroblasts that provide a supportive microenvironment (e.g., perivascular stroma cells) can be targeted by cancer therapy. Moreover, further evaluation of the roles that this protein plays in cancer development may reveal the mechanism of cancer–fibroblast interactions and how the microenvironment supports tumor progression.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
This work was supported by the Grant-in-Aid for Cancer Research (19-10) from the Ministry of Health, Labor, and Welfare Programs; the Foundation for the Promotion of Cancer Research, 3rd-Term Comprehensive 10-Year Strategy for Cancer Control; Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation; and JSPS KAKENHI (20590417, 215981). A. Hoshino was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Naoya Fujita (The Cancer Chemotherapy Center of Japanese Foundation for Cancer Research) for kindly providing pcDNA3/human-WT-podoplanin vector and Hiroko Hashimoto for technical support.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received September 1, 2010.
- Revision received April 27, 2011.
- Accepted May 12, 2011.
- ©2011 American Association for Cancer Research.