Rat UCMSCs were prepared from E19 pregnant rats and isolated using the method described previously 15 . Cells were maintained in defined medium, containing a mixture of 56% low glucose Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, CA); 37% MCDB 201 (Sigma; St. Louis, MO); 2% fetal bovine serum (FBS, Atlanta Biologicals Inc, GA); 1x insulin-transferrin-selenium-X (ITS-X, Invitrogen); 1x ALBUMax1 (Invitrogen); 1x penicillin/streptomycin (Pen/Strep, Invitrogen); 10 nM dexamethasone (Sigma); 100 μM ascorbic acid 2-phosphate (Sigma); 10 ng/ml epidermal growth factor (EGF, R&D systems, Minneapolis, MN); and 10 ng/ml platelet derived growth factor-BB (PDGF-BB, R&D systems). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

The LLC cell line was maintained in DMEM (Invitrogen) medium supplemented with 10% FBS and 1x Pen/Strep (Invitrogen). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂.

The MTT assay was performed to study the effect of rat UCMSCs on LLC cell proliferation. In brief, different ratios of rat UCMSCs and LLCs (UCMSCs:LLCs = 1:10, 1:6, and 1:3) in DMEM with 10% FBS were seeded in 96 well plates; cells were allowed to grow for 72 hrs. MTT solution (20 μl of 5 mg/ml) was added 4 hrs before completing 72 hrs of incubation. Formazan crystals formed were dissolved by adding 100 μl solublization buffer (10% SDS containing 0.01N HCl) and incubated overnight at 37°C. The following day, color developed was measured at 550 nm and background absorbance was measured at 630 nm using the Molecular Devices Spectramax 190 plate reader (Global Medical Instrumentation, Inc. Ramsey, MN).

To evaluate cell proliferation from a different angle, a [³H] thymidine uptake assay was carried out. In brief, rat UCMSCs (1 × 10³ or 2 × 10³/well) were mixed with 6 × 10³ LLC cells/well, directly plated in 24-well culture plates, and cultured in a CO₂ incubator for 72 hrs. Radioactivity incorporated into the cells was analyzed using our published protocol 15 .

Direct cell counts via hemocytometer were performed to study the effect of rat UCMSCs on LLC cell growth in culture plates with cell culture inserts (BD Biosciences, San Jose, CA). In brief, LLC cells were seeded in normal growth medium at 1 × 10⁵ cells/well in 6-well plates. After allowing the cancer cells to adhere for 1 hr, 1.67 × 10⁴ and 3.33 × 10⁴ rat UCMSCs were seeded on the cell culture inserts (3.0 μm pore size). The insert pore size of 3 μm is small enough to prevent cells migrating from the inserts to the culture dishes, since UCMSCs do not penetrate this size pores in the insert. After 72 hrs co-culture, cells grown in the bottom culture dish were collected by trypsinization and counted using a hemocytometer.

Various numbers of rat UCMSCs (8.33 × 10² and 1.67 × 10³ cells/well) were grown in a 12 well tissue culture plate. One day after seeding, 0.75 ml 0.8% agar (Sea Plaque agar, Cambrex Bio Science Rockland, Inc. Rockland, ME) in DMEM with 10% FBS was poured into the dish (bottom layer). After the bottom agar layer solidified, LLC cells (5 × 10³ cells) were suspended in 0.5 ml of DMEM containing 10% FBS and 0.5% agar and plated on top of the bottom agar layer. The cells were incubated at 37°C with 5% CO₂ for growth of colonies. On day 16, colony growth was evaluated by an automated phase contrast microscope equipped with Micro Suite Analysis Suite (Olympus CKX41, Center Valley, PA ). Colonies with an area greater than 50000 μm² were counted using Micro Suite Analysis Suite software.

To analyze the effect of rat UCMSC co-culture on LLC cells, cell cycle analysis was carried out using propidium iodide staining. In brief, rat UCMSCs and LLC cells were co-cultured in 6 well Transwell culture dishes as described in the Transwell cell culture study. At the end of incubation LLC cells in the bottom chamber were collected and analyzed for cell cycle using our standard protocol 16 .

Total cellular protein was prepared using lysis buffer (1% TritonX-100, 0.1% SDS, 0.25M sucrose, 1 mM EDTA, 30 mM Tris-HCl (pH 8.0)) supplemented with protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN). Protein samples were separated by a 10% SDS-PAGE gel, electroblotted onto nitrocellulose membrane (GE Healthcare, Uppsala, Sweden) and blocked with 4% nonfat dry milk in 0.1% Tween20 in phosphate buffered saline (PBST) for 1 hr at room temperature. The membranes were washed and incubated with antibodies against cyclin A (1:100, Abcam, Cambridge, MA), cyclin E (1:100, Abcam), and CDK2 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA) with 4% nonfat dry milk in PBST for 1 hr at room temperature and then with a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (GE Healthcare). The protein expression signal was detected with Pierce ECL Western Blotting substrate (Pierce, Rockford, IL). GAPDH was used as the loading control of sample by reprobing with an anti-GAPDH antibody (1:4000, Santa Cruz Biotechnology).

Wild-type female C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All mice were housed in an AAALAC-accredited clean facility and held for 10 days to acclimatize. All animal procedures received prior approval from the Kansas State University Institutional Animal Care and Use Committee (protocol # 2681) and were performed in adherence with all applicable international, federal, state, and local guidelines.

To study the effect of rat UCMSCs on the growth of lung cancer grafts, a syngeneic LLC tumor model was used. In brief, each mouse was injected via the tail vein with 1.5 × 10⁶ LLC cells (for Experimental design I) or 2 × 10⁶ cells (for Experimental design II) suspended in 200 μl of PBS. On day 5 all mice were randomized into two treatment groups: a PBS control group and a rat UCMSC treatment group. To evaluate the trafficking of transplanted UCMSCs, cells were labeled with 10 μg/ml of SP-DiI fluorescent dye (Molecular Probes, CA) for 16 hrs incubation in a 5% CO₂ incubator. After removing excess dye by washing the cells with PBS, cells were incubated with dye-free medium for another 4 hrs. These cells were dispersed by trypsinization and mixed with unlabeled UCMSCs so that 20% of the UCMSCs were labeled with SP-DiI. Although a preliminary cell culture study indicated that SP-DiI does not alter viability of UCMSCs, only 20% of transplanted cells were labeled in order to minimize any potential adverse effects of the dye. Two experimental designs were used for UCMSC injections.

five days after LLC cell injection, these mice were injected intratracheally using a 27 gauge needle with either 35 μl PBS (n = 8) or 35 μl rat UCMSCs suspension (3.5 × 10⁵ cells in PBS, n = 9). The cell suspension in the syringe was released at approximately 5 mm above the dividing point of the bronchi. Our preliminary evaluations have shown that this injection method evenly distributes injected cells into the right and left lobes of the lung. Experimental design II: On day 5 and day 7 after LLC injection, these mice were systemically injected using a 28 gauge needle with either 200 μl PBS (n = 8) or 200 μl UCMSCs suspension (1 × 10⁶ cells in PBS, n = 8) through the tail vein (IV).

All mice were kept in their cages and their physical condition was monitored until sacrifice on day 21 after LLC transplantation. Lungs of mice were collected immediately after sacrifice without inflation treatment, fixed in 10% formalin in saline, and used for histochemical analysis.

Lung tissue fixed in 10% formalin in saline was embedded in paraffin, serially sectioned at 4 μm, and stained with hematoxyline and eosin (H&E) for microscopic examination of morphology of tumors. Unstained serial paraffin sections were counter stained with DAPI for nuclei and observed under epifluorescence microscopy for tracking the rat UCMSCs.

To determine apoptosis in the tumors, the DeadEndTM colorimetric TUNEL system (Promega, Madison, WI) was used according to the manufacturer's protocol with a slight modification. The sections were counterstained with methyl green. To determine the apoptotic index, 10 nodules were selected randomly by light microscopy and the area of TUNEL positive cells in each nodule was calculated using the NIH Image J analysis software. The index was assessed as the percentage of TUNEL-positive area in the tumor. The fold change was calculated by dividing TUNEL-positive area in rat UCMSC treated tumors by those in untreated tumors.

All values are expressed as means ± SE. For all in vitro and in vivo experiments, statistical significance was assessed by Tukey-Kramer Pairwise Comparisons test. All experiments were conducted at least twice with multiple sample determinations. Actual number of experiments repeated and sample numbers/experiments are described in the figure legends. Since all experiments were very closely reproduced, raw values were used for the statistical analysis. Data from replicate experiments as well as replicates within experiments were combined with no for experiment. A value of P < 0.05 was considered significant.

The effect of un-engineered rat UCMSCs on anchorage-dependent growth of LLC cells was evaluated by both direct and indirect co-culture. As shown in Figure 1, direct co-culture of rat UCMSCs with LLC cells (ratio, 1:6 or 1:3) markedly decreased the total LLC cell number as measured by MTT assay. DNA synthesis as determined by thymidine uptake assay was in good agreement with the MTT assay result (Figure 2), although significant growth inhibition of LLC cells by rat UCMSCs was observed with a smaller proportion of UCMSCs in the MTT assay (UCMSC:LLC = 1:6) than in the thymidine uptake assay (UCMSC:LLC = 1:3). In addition to these studies in which both cell types directly contacted each other, an indirect co-culture study was carried out using a Transwell culture system in which rat UCMSCs were cultured in inserts and LLC cells were cultured in the bottom of the well. The effect of this indirect co-culture was assessed by counting the LLC cells after 72 hrs co-culture. The findings of this experiment (Figure 3) also corroborate with the results from the MTT and thymidine uptake assays. Un-engineered rat UCMSCs dose-dependently attenuated the total number of LLC cells. This Transwell experiment suggests that rat UCMSC-dependent cell growth attenuation may be mediated through a diffusible molecule or molecules produced by rat UCMSCs. Another Transwell cell culture in which LLC cells (1 × 10⁵ cells) were cultured in the inserts and rat UCMSCs (1.7 × 10³) were cultured in the bottom of the wells revealed that LLC cells do not alter the growth of rat UCMSCs (data not shown).

Since anchorage-independent growth is a hallmark of tumorigenesis, a soft agar assay was carried out to evaluate the effect of rat UCMSCs on the growth of LLC cell colonies. The soft agar colony assay is an in vitro model to mimic in vivo xenograft models. The results showed that LLC cell colony size and number in soft agar were significantly attenuated when rat UCMSCs were cultured in the bottom of the culture dish (Figure 4). Since LLC cells were separated from rat UCMSCs by a solid agar layer (approximately 1 mm thickness), and the two cell types physically do not contact each other, the colony assay results also suggest that rat UCMSC-dependent growth attenuation was mediated through molecules produced by rat UCMSCs which diffused to LLC cells.

The effect of rat UCMSCs on the cell cycle of LLC cells was evaluated by flow cytometry after the LLC cells were co-cultured with rat UCMSCs in Transwell culture dishes. The result shows that rat UCMSCs caused G0/G1 arrest of LLC cells, thus increasing the G0/G1 population and decreasing the S phase population of LLC cells (Figure 5A). As shown in Figure 5B, the G0/G1 phase cell populations (%) in LLC alone, LLC:UCMSCs (6:1), and LLC:UCMSCs (3:1) were 39.02 ± 0.06, 39.89 ± 0.11, and 45.84 ±0.81, respectively; S phase populations (%, same order as above) were 49.35 ± 0.24, 49.69 ± 0.25, and 41.33 ±0.63, respectively. Furthermore, cyclins and cyclin-dependent kinase were analyzed by Western blot analysis. As shown in Figure 6, co-culture with rat UCMSCs significantly attenuated protein expression levels of cyclin A and CDK2, but not cyclin E. These results also indicate cell cycle arrest in G0/G1 phase.

We tested the growth attenuation ability of un-engineered naïve rat UCMSCs on the lung LLC tumors by local (intratracheal) and systemic (IV) administration. Results indicate that a single intratracheal administration of rat UCMSCs 5 days after LLC transplantation almost completely inhibited tumor growth as measured by tumor weight (Figure 7A). The IV administration of rat UCMSCs also significantly attenuated tumor growth despite the fact that tumor growth was more prominent due to a larger number of the LLC cells transplanted (2 million cells/mouse) (Figure 7B). Histological analysis revealed that lungs of PBS injected mice were occupied by large amounts of cancer tissue, whereas rat UCMSC-treated groups contained almost negligible (intratracheal injection group) or very small amounts of tumor mass (systemic injection group). Fluorescence microscopic observation of serial sections used for H&E staining shows the engraftment of rat UCMSCs in close proximity to or within tumor tissues of tumor bearing mice that received SP-DiI labeled rat UCMSCs either intratracheally or systemically (Figure 8). However, SP-DiI labeled rat UCMSCs were not detected in normal areas of the lung.

To evaluate the effect of the rat UCMSCs on the apoptotic activities of tumor cells in vivo, the percentage of TUNEL-positive cell area in the tumors was determined. The percentage of TUNEL-positive cell area was two times higher in rat UCMSC treated tumors than in PBS treated control tumors (Figure 9). These results indicated that treatment with un-engineered rat UCMSCs increases apoptosis significantly.