Lung cancer-associated mesenchymal stem cells promote tumor metastasis and tumorigenesis by induction of epithelial–mesenchymal transition and stem-like reprogram

Characteristics of MSCs derived from primary lung cancer tissues

We firstly identified the cells isolated from primary tumor and tumor-free tissues. A few of adherent cells crawled out from both tumor and tumor-free tissues of all patients after 5 days of initial plating. Multiple fibroblast-like cells distributed spirally around or beside the tissue blocks two weeks later (Figure 1A). Flow cytometry analysis showed the cells isolated from tumor tissues were all positive for CD73, CD90 and CD166, and negative for hematopoietic markers of CD14, CD19, CD34, CD45 and HLA-DR. 55-85% cells expressed CD105 (Figure 1C). When cultured in adipogenic, osteogenic or chondrogenic induction medium, these cells differentiated into adipose cells, osteocytes and chondrocytes, individually (Figure 1B). The cells isolated from tumor or tumor-free tissues showed no significant difference in morphology, phenotype and multidifferential potency except that the percentage of CD105 expression was in a wider range (15%-80%) in the cells derived tumor-free tissues. These results indicated that we isolated MSCs form primary lung cancer (LC-MSCs) and adjacent tumor-free tissues (TF-MSCs) successfully.

Figure 1. Identification of lung cancer-associated mesenchymal stem cells. (A) Tissue block culturing method was used to isolate MSCs from matched primary lung cancer and adjacent tumor-free tissues. Arrow, lung cancer tissue. (B) Adipogenic, osteogenic and chondrogenic differentiation was induced in MSCs in vitro. (C) The expression of surface markers of MSCs was tested by cytometry flow. (A–C) were representative results from LC-MSCs of one patient. LC-MSCs, lung cancer-associated mesenchymal stem cells.

Increased invasion in tumor cells by LC-MSCs in vitro

To investigate how LC-MSCs influenced lung cancer cells, we co-cultured tumor cells with LC-MSCs in vitro and examined the invasion of tumor cells firstly. A549 and H1299 cells expressed CopGFP fluorescence stably by lentivirus transduction. Transwell assay showed the number of migrated tumor cells dramatically increased to twice to six times when tumor cells were co-cultured with LC-MSCs at the cell ratio of 1:1 as well as 1:10 (Figure 2A–2D). We also co-cultured tumor cells with TF-MSCs. Although TF-MSCs also increased tumor invasion, they were not as effective as LC-MSCs (Figure 2A–2D). Additionally, the supernatants of LC-MSC showed similar effect on migration in tumor cells (Supplementary Figure 1). These results indicated that LC-MSCs increased the invasion ability of tumor cells.

Figure 2. Tumor invasion was induced by LC-MSCs in vitro. A549.CopGFP and H1299.CopGFP cells were transduced by lentiviral particles to express CopGFP stably firstly. Then the invasion ability of these cells was tested by transwell invasion assay after they were co-incubated with LC-MSCs or not for 24 hours. (A, B) A549 cells were co-incubated with LC-MSCs at the cell ratio 1:1 and 1:10. (C, D) H1299 cells were co-incubated with LC-MSCs at the cell ratio 1:1 and 1:10. Color bar chart, triplicate data for each patient. Dark bar chart, statistical analysis for all patients. Fluorescent pictures illustrated tumor cells that had migrated through the membrane of transwell inserts. TF-MSCs, tumor-free mesenchymal stem cells, were used as comparison from normal lung tissues. **, P < 0.05 were considered to be statistically significant.

Changes of migration in tumor cells by LC-MSCs in vitro

Next, we used scratch wound healing assay to evaluate the migration ability of tumor cells in co-culture system. We firstly analyzed the results according to each patient. As shown in Figure 3A, 3C, at the cell ratio of 1:1, the migration rate increased significantly in three patient-derived LC-MSC co-culture systems and decreased in one system when compared with that when tumor cells were cultured alone. While, at the cell ratio of 1:10, it did not elevate and even ran down to statistic differences in three co-culture systems (Figure 3A, 3C). TF-MSCs had a similar effect on the migration of tumor cells as LC-MSCs (Figure 3A, 3C). Secondly, we analyzed the migration rate between co-culture and tumor culture alone system by pooling all the data from individual patients, but did not find significant differences (Figure 3B, 3D). Then, we used transwell assay to test the migration of tumor cells to LC-MSC-derived condition medium. The result revealed a great many of tumor cells migrated to the condition medium of LC-MSCs in a concentration-dependent manner. Contrastively, few tumor cells migrated to serum-free medium (Figure 3E). These results suggested tumor cells had tropism to LC-MSCs.

Figure 3. Tumor migration influenced by LC-MSCs in vitro. (A–D) A549.CopGFP and H1299.CopGFP cells were co-incubated with LC-MSCs at the cell ratio 1:1 and 1:10 to 80% confluence before wound healing assay was performed. The migrated ratio was calculated as (the first scratch distance - the scratch distance 48 hours later)/ the first scratch distance×100%. Color bar chart, triplicate data for each patient. Dark bar chart, statistical analysis for all patients. TF-MSCs, comparison from normal lung tissues. (E) The migratory capacity of A549 cells in response to conditioned medium of LC-MSCs was determined using transwell migration assay. Control, serum-free medium without LC/TF-MSCs. *, P < 0.05 were considered to be statistically significant.

Induced EMT-associated gene expression in tumor cells by LC-MSCs

To investigate the mechanism of enhanced invasion of lung cancer cells, we then tested the expression of EMT-associated genes in transcriptional as well as protein levels. A549 cells that expressed CopGFP florescence were firstly sorted from the co-culture systems by cytometry flow. Realtime PCR analysis showed the expression of EMT-associated transcription factors snail and slug elevated remarkably in A549 cells when co-cultured with LC-MSCs derived from all patients and the change of slug expression was the most significant (snail, 1.4 to 4.3 times; slug, 6.5 to 98 times) (Figure 4A). E-cadherin, N-cadherin and β-catenin expression also change obviously in the co-culture systems (Figure 4A). Furthermore, to determine whether LC-MSC-induced EMT in the tumor cells was cell-contact dependence, we cultured tumor cells and LC-MSCs separately by using transwell inserts. A same trend but relative slight changes of the EMT-associated gene expression was found in this indirectly contacted condition (Figure 4B). Western blot results confirmed ascent expression of snail and slug in A549 cells in co-culture system (Figure 4C). Vimentin expression increased. But E-cadherin, β-catenin and N-cadherin did not exhibit much changes in A549 cells (Figure 4C). TF-MSCs increased the EMT-associated gene expression as well but in a lower level than LC-MSCs (Figure 4A–4C). Additionally, we found much higher level of TGF-β secreted from LC-MSCs compared with that from TF-MSCs (Figure 4D). And blockade of TGF-β downstream signaling by galunisertib decreased the levels of snail and slug significantly in A549 cells (Figure 5E). These results indicated that TF-MSCs might promote EMT by TGF-β signaling pathway.

Figure 4. EMT-associated gene expression induced by LC-MSCs. (A) Equal number of A549.CopGFP and LC-MSCs were co-cultured directly for 48 hours and then sorted by cytometry flow to test the expression EMT-associated genes by realtime PCR. (B, C) Equal number of A549.CopGFP and LC-MSCs were co-incubated separately by transwell inserts for 48 hours. The EMT-associated gene expression in A549 cells was evaluated by realtime PCR (B) and western blot (C), individually. Representative results of three patients. TF-MSCs, comparison from normal lung tissues. (D), The level of TGF-β secreted by MSCs that derived from different tissues was tested by ELISA assay. (E), The induced snail and slug expression was blocked by TGF-β signaling inhibitor galunisertib. A549 cells were pre-incubated with galunisertib (5 μM) for 30 min, then treated by the supernatants of LC/NC-MSCs from 3 patients. *, P < 0.05 were considered to be statistically significant. P1-3, three patients. 1, Tumor cells alone. 2, Tumor cells + LC-MSCs. 2, Tumor cells + TF-MSCs. Data from triplicate experiments.

Induced stem-like characteristics of tumor cells by LC-MSCs

As EMT program have a close association with acquisition of stem-like traits in tumor cells, we next assessed the stem-like characteristics in tumor cells in both cell-cell contact (Figure 5A) and noncontact culture condition (Figure 5B). When tumor cells were co-cultured with LC-MSCs directly, we found the level of CD44 but not CD133 in tumor cells increased in all 3 patients (Figure 5A). Enhanced expression of Oct-4 with either Sox2 or Nanog was found in patient 1 and 2. While none of them increase in patient 3 (Figure 5A). LC-MSCs did not show superior capacity to promote stem gene expression in tumor cells compared with TF-MSCs in the cell-cell direct contact system (Figure 5A). However, all the tested genes increased when tumor cells were incubated with LC-MSCs by transwell insert in patient 1 and 2 (Figure 5B). CD44 and Oct-4 increased in patient 3 (Figure 5B). And the level of stem genes increased more significantly induced by LC-MSCs compared with that by TF-MSCs in the cell-cell indirect culture system (Figure 5B). Furthermore, we tested spheroid formation in 3D cell culture system. A549 cells and MSCs were labeled with CopGFP and DsRed florescence, individually. As shown in Figure 5C, on the second day after plating, A549 cells tiled loosely in the bottom of cell culture well where they were plated alone. On the contrary, the cells aggregated in the wells in co-culture condition. The degree of compaction depended on the absolute cell number of LC-MSCs and its ratio to A549 cells plated in each well. The most perfect spheroids were found when cells were co-incubated at the ratio of 1:1. With the time of culturing, the spheroids turned more compacted. MSCs accumulated in the center of the spheroid and A549 cells located in the outer part of the spheroid (Figure 5D). Spheroid also formed when A549 cells were co-incubated with TF-MSCs. But they were not as compacted and round as that when A549 cells were co-incubated with LC-MSCs (Supplementary Figure 2).

Figure 5. Stem-like characteristics induced by LC-MSCs. (A) Equal number of A549.CopGFP and LC-MSCs were co-cultured directly for 48 hours and then sorted by cytometry flow to test the stem-related gene expression by realtime PCR. (B) Equal number of A549.CopGFP and LC-MSCs were co-incubated separately by transwell inserts for 48 hours. The expression of stem-related genes in A549 cells was evaluated by realtime PCR. (C) Different cell ratio of A549.CopGFP and LC-MSCs. DsRed were co-incubated in 96-well 3D cell culture plates. The spheroid formation was observed under inverted fluorescence microscope on the second day of plating cells. (D) Spheroid grew observed at indicated time. *, P < 0.05 were considered to be statistically significant. P1-3, three patients. Data from triplicate experiments.

Enhanced tumor growth and metastasis by LC-MSCs in vivo

Subsequently, we examined the effect of LC-MSCs on tumor progression in vivo by live animal imaging. 10⁶ each of A549.Lu cells and LC/TF-MSCs were co-implanted subcutaneously in mice. The BLI revealed that tumor grew faster in both A549+LC-MSC and A549+TF-MSC group than A549 group. And there was no significant difference of tumor size between the two co-implanted groups (Figure 6A, 6B). Tumors in A549 group gradually grew slowly at the late feed period. When dissecting the sacrificed mice at the end of study, we found the tumor parenchyma was usually occupied by cystic cavities in A549 group, but it was not found in A549+LC-MSC group. H.E staining indicated that the tumor cells in the co-implanted groups presented more derangement distribution which was disordered by fibroblast-like cells than that in A549 group, (Supplementary Figure 3). Interestingly, bioluminescence signaling was detected in sentinel lymph nodes in all mice in A549+LC-MSC group, but not in the other two groups (Figure 6C). Immunohistochemistry result showed a higher level of ki67 staining in the co-implanted group (Figure 6D).

Figure 6. Tumor growth and metastasis promoted by LC-MSCs. 10⁶ each of A549.Luc cells and LC-MSCs or TF-MSCs were co-implanted subcutaneously into female Balb/c nude mice. Tumor growth was monitored by bioluminescence imaging. (A) Bioluminescence images at the indicated time. (B) Total bioluminescence of tumors at indicated time. (C) Bioluminescence images of sentinel lymph nodes from sacrificed mice at the end of experiment. (D) Immunohistochemistry test for Ki67 in tumor tissues. *, P < 0.05 were considered to be statistically significant.

Increased tumorigenesis and metastasis-initiating ability by LC-MSCs in vivo

As LC-MSCs induced stem-like characteristics in tumor cells, we decreased the number of implanted tumor cells to investigate the pro-tumorigenesis of LC-MSCs in vivo. As shown in Figure 7A, 7B, tumor grew in seven out of twelve and three out of thirteen mice when 2×10⁴ or 10³ A549 cells were co-implanted with LC-MSCs. However, no tumor developed when 2×10⁴ A549 cells were implanted alone. We also tested the effect of LC-MSCs that derived from another patient and got the similar result (Supplementary Figure 4 and Table 1). Furthermore, we investigated whether LC-MSCs promoted the metastasis-initiating ability and colonization of circulating tumor cells by intravenous injection of tumor cells in mice model. The result revealed that three out of five mice in A549+LC-MSC group developed lung cancer. Contrastively, there was only one mouse developed lung cancer in A549 group. Meanwhile, the bioluminescent signaling from it was much weaker than that in A549+LC-MSC group and just detected in the last imaging (Figure 7C, 7D).

Figure 7. Increased tumorigenesis and metastasis-initiating ability by LC-MSCs. (A, B) Low number of A549.Luc cells and LC-MSCs were co-implanted subcutaneously into female Balb/c nude mice at different cell ratio. Bioluminescence images (A) and total bioluminescence of tumors (B) at the indicated time. (C, D) A549.Luc cells and LC-MSCs were mixed well and injected intravenously into female Balb/c nude mice. Bioluminescence images (A) and total bioluminescence of tumors (D) at the indicated time.

Ethics approval and consent to participate

The informed consent documents were approved by the participating local institution’s review boards, and all patients were informed in the study which was undertaken in accordance with Declaration of Helsinki. Animal research was performed in compliance with the Guide for the Care and Use of Animal Ethics Committee of Tianjin Medical University (Tianjin, PR China).

MSCs preparation

MSCs were isolated from five pairs of matched primary lung cancer and adjacent tumor-free tissues from the patients (Supplementary Table 1) who newly diagnosed as lung cancer and received lung surgery in Tianjin Medical University Cancer Institute and Hospital (Tianjin, China). Tissue culture method was used to isolate MSCs. Briefly, after lung surgical resection, tissues were immersed immediately in phosphate-buffered saline (PBS) supplemented with 100 units/mL penicillin and 100 μg/mL streptomycin (Gibco, USA) at 4° C till use within 4 hours. Tissues were rinsed with ice-cold PBS to remove the blood and cut into blocks with the size of 8 mm³ with scalpels. During cutting, remove the vessels completely. Tissue blocks were plated on the T-75cm² cell culture flasks (Corning, USA) which had been moistened with 2 mL culture medium including DF-12 (Gibco, USA) supplemented with 2 mmol/L L-glutamine (Gibco, USA) and 10% FSC (Gibco, USA) and incubated upside down at 37° C in a humidified atmosphere containing 5% CO₂ overnight. The flasks were turned over inversely in the second morning. 1 mL culture medium was added into the flasks for the first three days continually and 3 mL culture medium was added on the fourth day. Then culture medium was changed half every three days. MSCs were passaged firstly after two weeks of plating. Cells were subcultured at a density of 4000 cells/cm² in culture medium. MSCs at passages 1 to 3 were cryopreserved with cryprotectant consisted of 45% DF-12, 50% FCS and 5% DMSO (Gibco, USA) in liquid nitrogen for long term storage. MSCs at passages 4 to 6 were used for the following experiments.

Cell line culture

The human lung cancer cell line A549 and H1299 (bought from Cell Resource Center of Shanghai Institute of Life Sciences, Chinese Academy of Sciences, Shanghai, China) were cultured in RPIM1640 (Gibco, USA) supplemented with 2 mmol/L L-glutamine, 100 units/mL penicillin, 100 ug/mL streptomycin and 10% FCS. The human embryonic kidney cell derived 293 T cell line (kindly provided by Professor Dongsheng Xiong, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, PUMC, Tianjin, China) was maintained in DMEM (Gibco, USA) supplemented with 2 mmol/L L-glutamine, 100 units/mL penicillin, 100 ug/mL streptomycin and 10% FCS. Cells were incubated at 37° C in a humidified atmosphere containing 5% CO₂.

Cell phenotype identification

To identify the phenotype of isolated cells from primary lung cancer and adjacent tumor-free tissues, flow cytometry was used to detect the surface markers on these cells. When growing to the confluence of 70%, cells were enzymed and rinsed with ice-cold PBS twice. Cells were suspended at the density of 10⁷/mL with staining buffer and aliquoted to 100 μL. Fluorescein-conjugated mouse anti-human CD73, CD90, CD105, CD166, CD14, CD19, CD34, CD45, and HLA-DR antibodies (BD, USA) were added to the aliquots and incubated on ice for 30 min. After incubation, cells were washed twice, suspended with staining buffer and detected by flow cytometry immediately.

Multi-differentiation of MSCs

To test the multi-differentiation ability of isolated cells, adipogenic, osteogenic and chondrogenic differentiation was induced, individually. All differentiation-induced media were prepared freshly. For adipogenic and osteogenic differentiation, MSCs at passage 2–6 were seeded in a six-well plate (3×10⁵/well) and cultured overnight. On the next day, the cells were washed with PBS twice and induction medium (Cyagen, China) once firstly, and then maintained in induction medium for adipogenic or osteogenic differentiation. The medium was changed every 3 days for 3 weeks. Then, the cells were stained with oil red O for adipogenicity and alizarin red S for osteogenesis, respectively, and observed under an inverted phase contrast microscope (Olympus, Japan). For chondrogenic differentiation, 2 × 10⁶ MSCs at passage 4–6 were harvested in a 15 mL centrifuge tube and washed with PBS twice and induction medium (Cyagen, China) once. After the last wash, supernatant was sucked carefully and thoroughly. 1 mL induction medium were added gently along the pipe wall to avoid disturbing the pellet. The tube with a loosened cap on it was placed in the cell culture incubator, which was humidified at 37° C with 5 % CO₂. Medium was changed every 3 days by using a pipet. After 28 days, the chondrocyte pellet was fixed with neutral buffered formalin, embedded in paraffin, cut into 5-μm sections onto slides, stained with 1 % toluidine blue/1 % sodium borate, and observed under an inverted phase contrast microscope.

Production of lentivirus

To label tumor cells and MSCs in co-culture system in vitro or animal study in vivo, we constructed lentiviral expression vectors encoding label genes firstly. The firefly luciferase (fLuc) sequence which had been amplified from pGL3 basic plasmid (Promega, USA) was cloned into the lentiviral expression vector (System Biosciences, SBI, USA) with a tag gene CopGFP. The DsRed sequence which had been amplified from plasmid DsRed-rab9 WT (Addgene, USA) was cloned into the lentiviral expression vector in place of the tag gene CopGFP. Secondly, the lentiviral particles were produced by transient transfection of 293T cells according to SBI’s protocol. The supernatant of cell culture medium was collected after 48 hours of post-transfection. The lentivirus containing medium was spun at 500g for 5 min, filtered through a 0.45 μm pore size filter (Millipore), and used to transduce tumor cells or MSCs immediately or stored at −80° C.

Transduction of lentiviral particles

A549, H1299 and MSCs were plated at a density of 2×10⁵ per well in T-25cm² cell culture flasks and incubated overnight at 37° C, individually. On the next day, culture medium was removed and 3 ml of fresh medium containing the corresponding lentiviral supernatant at MOI 8 and 8 mg/ml of polybrene (Sigma, USA) was added. And then the flasks were centrifuged at the speed of 1000 rpm at 25° C for 1.5 hours before taken back into the cell incubator. The medium was replaced by culture medium 8 hours later. Cells were incubated for the indicated time, and targeted gene expression (luciferase and CopGFP fluorescence for A549 and H1299; DsRed fluorescence for MSCs) was evaluated by luciferase assay (Promega, USA) or fluorescence microscope (Nikon, Japan).

Transwell assay

To investigate the influence of LC-MSCs on invasion of tumor cells, we co-cultured tumor cells and LC-MSCs directly in vitro. Briefly, the transwell inserts were coated with matrigel (BD, USA), put in 24-well tissue culture plate, and then incubated in 5% CO₂ incubator at 37° C for 5 hours. Excess matrigel that did not freeze was aspired before cell plating. A549 and H1299 cells that stably expressed CopGFP fluorescence and LC-MSCs were collected, washed twice with PBS and suspended in serum-free medium, respectively. Adjust the density of cells with serum-free medium. Add 20% FSC medium into the lower base of inserts. Then tumor cells and LC-MSCs were mixed well in 150μL serum-free medium (2.5×10⁴ cells for each; or 5×10³ tumor cells and 5×10⁴ MSCs) and plated in the upper base of the inserts. Incubate cultures for 24 hours in 5% CO₂ incubator at 37° C. At the end of culture, erase the matrigel on the upper face pf the transwell membrane, and then count the number of tumor cells that migrated through the transwell membrane in five random fields under fluorescence microscope. Tumor cells plated alone were used as control. TF-MSCs were also co-incubated with tumor cells to compare the different effect between tumor and tumor-free derived MSCs. Experiments were repeated in triplicate. We also added the supernatants of LC/TF-MSCs in the culture media of tumor cells in transwell assay to evaluate the changes of invasion ability of tumor cells.

Additionally, we used transwell assay to the tropism of tumor cells induced by LC-MSCs. Briefly, 10⁶, 5×10⁵ and 10⁵ LC-MSCs in 2mL serum-free medium were incubated in 5% CO2 incubator at 37° C for 48 hours respectively. Then the culture medium from LC-MSCs was used as conditioned medium. Serum-free culture medium was used as control. Add the conditioned medium into the lower base of transwell inserts. 2×10⁴ tumor cells that was suspended in serum-free culture medium containing 0.2% BSA were plated in the upper base of transwell inserts. Put the transwell system in cell incubator for 5 hours. Then take out of the inserts. Slightly remove the cells that did not migrate through transwell membrane. Then stain the cells on the lower side of transwell membrane with 0.1% crystal violet. The number of cells that had migrated to the lower side of the membrane was counted under a light microscope with five high-power fields. Experiments were done in triplicate.

Wound healing assay

To test tumor cell migration in vitro, we used wound healing assay in co-culture condition. Firstly, A549 and H1299 cells labeled with CopGFP fluorescence and LC-MSCs were collected, respectively. Then tumor cells and LC-MSCs at the ratio of 1:1 (2.5×10⁵ for each) or 1:10 (5×10⁴ tumor cells and 5×10⁵ LC-MSCs) were mixed well in 2 mL 10% FSC culture medium and incubated in 6-well tissue culture plate in 5% CO2 incubator at 37° C overnight. On the second day, cells should reach 80% confluence as monolayer. Gently and slowly scratch the monolayer with a new 1 mL pipette tip across the center of the well. Scratch another two straight lines parallel to the first one. Gently wash the wells twice with medium to remove the detached cells. Replenish the well with fresh medium. Take photos for the scratched monolayer for the first time. Grow the cells for additional 48 hours. Take photos for the scratched monolayer for the second time. The configurations of the microscope should be the same as the first time. The migrated ratio was calculated as (the first distance - the second distance)/ the first distance×100%.

Quantitative real-time PCR

To evaluate the EMT and stem-like associated gene expression in transcriptional levels, we applied real-time PCR in the study. Total RNA was extracted from tumor cells with TRIzol (Gibco, USA), and cDNA was synthesized using PrimeScript™ RT Master Mix (TaKaRa, Japan) according to the manufacturer’s instructions. Real-time PCR amplification was performed with the 7500 real time PCR system (Applied Biosystems, USA) using SYBR Green qPCR kit (TaKaRa, Japan). The PCR conditions included 40 cycles of 95° C for 5 s and 60° C for 34 s. The primer pairs used for real-time PCR were described in the supplementary data (Supplementary Table 2). The relative expression levels of the target gene were evaluated by the 2^−ΔΔCT method, and β-actin gene expression was used for normalization of each sample.

Western blotting

To evaluate the expression of EMT associated gene expression in protein levels, we used western blot in the study. Briefly, cells were lysed and protein was extracted using M-PER (Pierce, Rockford, IL, USA) plus protease inhibitor cocktail (Halt; Pierce). Ultrasonic the cell lysis in ice bath (300W for 4 seconds and rest for 8 seconds. Repeat three times.) was Protein concentrations were determined using BCA assay (Pierce). Aliquots of protein lysates were separated on SDS−polyacrylamide gels and transferred onto PVDF membrane, which was blocked with 5% blotting grade milk (Bio-Rad, USA) in PBST (0.1% Tween 20 in PBS). The membrane was then hybridized with the indicated primary antibodies to human E-cadherin, N-cadherin, β-catenin, vimentin, snail, slug and β-actin (Cell Signaling Technology, USA) followed by corresponding secondary antibodies conjugated with horseradish peroxidase, and then detected using a chemiluminescence assay (Millipore, USA). Membranes were exposed to X-ray film to visualize the bands. The band of β-actin was used as endogenous control.

Spheroid culture

To investigate the stem-like characteristics of tumor cells induced by LC-MSCs or TF-MSCs, we tested the ability of spheroid formation of co-cultured cells by using 3D cell culture system. Briefly, 10⁴ A549 cells that had been labeled with CopGFP florescence and MSCs that had been labeled with DsRed florescence were co-plated at the cell ratio of 1:1, 1:3 (3×10³ MSCs), or 1: 10 (10³ MSCs) in six repeated wells of 96-well black round bottom polystyrene ultra-low attachment microplates (Corning, USA) in a volume of 0.2 mL. Centrifuge the microplates at the speed of 800 rpm for 5min to spin down cells in the bottom of the wells. Observe the sphere formation under an inverted fluorescence microscope at indicated time.

In vivo experiments

To test the effect of MSCs on tumor progression in vivo, animal studies in two mouse models were carried out. Animal research was performed in compliance with the Guide for the Care and Use of Animal Ethics Committee of Tianjin Medical University (Tianjin, PR China). Female Balb/c nude mice aged 5−6 weeks (Beijing Vital River Laboratory Animal Technology Co., Ltd.; Beijing, China) were raised under pathogen-free conditions with irradiated fodder. A459 cells were firstly labeled with firefly luciferase (A459.Lu) by lentivirus transduction. In the subcutaneous mouse model, 10⁶, 2×10⁴ or 10³ A459.Lu cells respectively with or without MSCs (10⁶ or 2×10⁵) were implanted subcutaneously into the right flank of each mouse in a volume of 0.2 mL using 1 mL syringe with 18g 1 1/2” needle tip (BD, USA). In lung cancer metastasis mouse model, 5×10⁵ A459.Lu cells with or without 5×10⁵ MSCs were injected intravenously into the tail vein of each mouse in a volume of 0.1 mL using 1mL syringe with 28G 1/2” needle tip (BD, USA). The growth of tumor was monitored by bioluminescence imaging (BLI) (In Vivo Imaging System-Xenogen 100 system; Caliper Lifesciences, USA). For BLI, mice were administrated intraperitoneally D-luciferin (15 mg/mL in PBS, Promega, USA) at a dosage of 150 mg/kg 10 min prior to imaging. At the end of experiment, tumor and sentinel lymph nodes were taken out from the sacrificed mice. Histopathological test was used to confirm the tumor tissues. Immunohistochemistry test was used to the expression of Ki67 to evaluate the proliferation of tumor cells.

Statistical analyses

Experimental data are presented as the mean and standard deviation (SD) values. Statistical analysis was performed using SPSS22.0 software. Differences between groups were examined for significant differences by ANOVA LSD or Dunnett post hoc procedure. Limiting dilution analyses were performed based on previous report [49], using the limdil function of the “statmod” package (http://bioinf.wehi.edu.au/software/elda/index.html). Lung cancer-initiating cell frequencies were compared using likelihood ratio tests. Values of P < 0.05 were considered to be statistically significant. Data of each patient was assessed in triplicate.