Supplementary Information for

Synergistic Effects of Co-administration of Suicide Gene Expressing

Mesenchymal Stem Cells and Prodrug-encapsulated Liposome on

Aggressive Lung Melanoma Metastases in Mice

Tian-Yuan Zhang, Bing Huang, Hai-Bin Wu, Jia-He Wu, Li-Ming Li, Yan-Xin Li, Yu-Lan Hu,

Min Han, You-Qing Shen, Yasuhiko Tabata, Jian-Qing Gao*

*Correspondence author. Email: gaojianqing@zju.edu.cn

The supplementary information file includes:

Supplementary Methods.

Supplementary Figure 1. Tumor tropism and permeation of BMSCs into 3D tumor spheroids in

vitro.

Supplementary Figure 2. Fluorescence intensities of CM-Dil labeled BMSCs in vivo after

systemic delivery from Day 1 to Day 5 in pulmonary melanoma metastasis-bearing C57BL6

mice (left) or healthy normal mice (right).

Supplementary Figure 3. Tumor regression effects post suicide gene treatment.

Supplementary Table 1. Formulation of the liposome impacts on the size and entrapment

efficiency.

Supplementary Table 2. Number of the metastatic nodules and weight of the lung.

Supplementary Video 1. Permeability of BMSCs into the 3D tumor spheroid 24 h post

incubation.

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Supplementary Video 2. Permeability of BMSCs into the 3D tumor spheroid 48 h post

incubation.

Supplementary Video 3. Permeability of BMSCs into the 3D tumor spheroid 72 h post

incubation.

1. Supplementary Methods

1.1. Particle size, size distribution and ganciclovir encapsulation efficacy determination

Particle size and polydispersity index (PDI) of ganciclovir (GCV) encapsulated liposomes

were measured by using a Nano-S90 laser particles size analyzer (Zetaszier Nano-S90, Malvern,

UK) at 20 °C. The morphological examination of liposomes was performed via transmission

electron microscopy (TEM, JEM1200, Japan) using 2 % phosphotungstic acid solution negative

staining. Besides, the liposomes were also analyzed with atomic force microscopy (AFM)

(Veeco Dimension Icon, Veeco, USA).

GCV encapsulation efficiency was determined by using a dialysis method (dialysis

membrane with molecular weight (MW) cutoff of 8000–14,000). In brief, liposome suspension

was placed in a dialysis membrane bag and dialyzed against physiologic saline (pH = 7.4) for 4 h

by gentle shaking. After dialysis, the free drug content (W f) was determined by high-

performance liquid chromatography (HPLC). Then the entrapped amount of GCV in liposomes

(We) was determined. Generally, 3 mL methyl ethanol was mixed with 0.5 mL GCV liposomes,

followed by vortex for 5 min to destroy the liposome’s structure. After centrifuging at 13,000

rpm for 5 min, the supernatant was suitably diluted with the mobile phase (methyl ethanol and

water, 5:95, v/v) and analyzed by HPLC. Each analysis was performed in triplicate. The percent

encapsulation efficiency (EE %) was calculated as the ratio between the amount of GCV

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encapsulated (We) and the total amount of drug initially added (Wf + We). The applied formula

was: EE % = We / (Wf + We) × 100 %.

1.2. HPLC analysis of GCV concentration

GCV concentration was determined by reverse-phase HPLC (Agilent series 1200, Agilent

Technologies, USA). The column was packed with Diamonsil® C18 5 μm (150× 4.6 mm,

Dikma, China). The mobile phase consisting of methyl alcohol and water (5:95, v/v) was

prepared daily and filtered through a Millipore membrane filter (0.22 μm) and degassed by

sonication in an ultrasonic bath before use. The flow rate was 1.0 mL/min, and the UV detection

(Agilent 1200 VWD, Agilent Technologies, USA) wavelength was 254 nm. The analyses were

performed at room temperature. The injection volume was 20 μL. Data collection and processing

were performed using Agilent series 1200 Chemstation software (Agilent Technologies, USA).

1.3. GCV release in vitro

In vitro release of GCV from liposomes in physiologic saline (pH 7.4) was monitored using

a dialysis method. In brief, 2 mL of liposomal solution was sealed into dialysis tubes (MW cutoff

8000-14,000), and incubated in 40 ml of physiologic saline at 37 °C in thermal bath on

continuous stirring. Samples were collected at 0.5 h, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h and 72 h

during the dialysis process, and the GCV concentration was analyzed by HPLC. The cumulative

release percentage (%) was indicated by dividing the cumulative amount of GCV recovered in

the dialysis medium with the total amount in the liposomes.

1.4. Isolation and culture of bone marrow-derived mesenchymal stem cells

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Rat bone marrow-derived mesenchymal stem cells (BMSCs) were isolated from the bone

shaft of femurs of three-week-old male Sprague-Dawley (SD) rats. Briefly, cells from both ends

of rat femurs were plated in 100 mm × 25 mm dish (Corning Inc., USA) with 10 ml of a

modified isolation medium included low-sugar Dulbecco’s modified Eagle’s medium (DMEM),

15 % fetal bovine serum (FBS), L-glutamine, penicillin, and streptomycin. After 24 h of

incubation at 37 °C in 5 % CO2, non-adherent cells were removed, adherent cells were washed

with phosphate buffered saline (PBS), and fresh medium was replaced every 2 days. After

incubation for 4 or 5 days, the cells of the primary passage became sub-confluent and were

detached from the dish using 0.25 wt % trypsin and 0.02 wt % ethylene diamine tetraacetic acid.

Cells were collected by centrifuge at 1200 rpm for 5 min and were replanted in modified

isolation medium. Second-passage cells at sub-confluence were used for all experiments up to

the fifth passages.

1.5. Fluorescence labeling for BMSCs

BMSCs were stained with the cell tracker CM-Dil (Invitrogen, USA). In short, BMSCs (1 ×

106 cells) were suspended in 500 μL PBS containing 1 μL CM-Dil (1 mg/mL) and incubated at

37 °C in 5 % CO2 for 5 min. Afterwards, the cells were transferred to 4 °C for 15 min. Finally,

cells were washed twice in PBS and applied in the experiments. The CM-Dil-labeled BMSCs

were examined by fluorescence microscopy (Eclipse Ti-s, Nikon, Japan).

1.6. Estimation of GCV in lung

Male C57BL6 mice (six week) were given GCV solution (GCV-so) or GCV liposomes

(GCV-lipo) by intravenous injection via tail vein at a single dose of 40 mg GCV/kg. Mice were

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anesthetized by ether inhalation 2 h, 6 h and 24 h after the injection and immediately sacrificed

by cervical dislocation. Then the lung was harvested, rinsed in physiologic saline, weighed, and

frozen at -20 °C until measurement. To determine the GCV concentration, lung samples were

firstly cut into small pieces and then homogenized in ice-cold physiologic saline by using an

Ultra-Turrax Homogenizer (IKA T10 basic, IKA Werke GmbH and Co., Germany). The

homogenates were deproteinized with methyl alcohol, vortex for 5 min and centrifuged at 13,000

rpm for 10 min at 4 °C. The supernatants were collected to clean eppendorf tubes and dried using

a stream of nitrogen gas at 60 °C. The sediments then dissolved with 400 μL double-distilled

water, vortex for 5 min and centrifuged at 13,000 rpm for 10 min. GCV concentration in the

supernatant samples was determined by HPLC. The HPLC analysis was similar as mentioned

above, but with several modifications. The composition of mobile phase of methyl alcohol and

water was changed to 4:96 (v/v), and the flow rate was changed to 0.8 mL/min. The analysis was

performed in triplicate.

1.7. Transduction and screening of green fluorescent protein (GFP)-expressing B16F10 cells

B16F10 cells were transduced via pGC FU-GFP-LV (GeneChem, Canada). For lentivirus

transduction, cells were seeded in a 12-well plate at a density of 5 × 10 4 cells per well,

transduced in DMEM culture medium with 10% FBS and 8 μg/mL polybrene (GeneChem,

Canada) at an multiplicity of infection of 100 and left to incubate overnight. The culture medium

was replaced on the next day.

To screen the GFP expressing B16F10 cells (B16F10-GFP+), 600 μg/mL G418 sulfate

(Invitrogene, USA) was added and full change of the medium every 3 days. Fluorescence

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microscopy and flow cytometry were applied to examine the positive rate of B16F10-GFP+. For

maintain culture, 300 μg/mL G418 sulfate was added to the DMEM medium.

2. Supplementary Figures:

Supplementary Fig. 1. Tumor tropism and permeation of BMSCs into 3D tumor spheroids in

vitro. (A) The randomly seeded BMSCs were found can migrate to and permeate into the 3 D

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tumor spheroid. (B) Fluorescence microscopy showed the time course of the migration and

permeation of CM-Dil-labeled BMSCs (red fluorescence) into 3D tumor spheroid. BMSCs were

firstly distributed randomly around the tumor spheroid (see blue arrows), but 24 h after

incubation, almost all BMSCs were observed that had migrated to the tumor spheroid. Moreover,

these migrated BMSCs would further permeate into the tumor spheroid 48 h post incubation

compared with the images that 24 h post incubation (see green arrows). The magnified images

are shown in the blue boxes. Images of 24 h and 48 h clearly demonstrated the permeability of

BMSCs. Compared with 24 h, BMSCs obviously permeated into the tumor spheroid as the green

arrows shown.

Supplementary Fig. 2. Fluorescence intensities of CM-Dil labeled BMSCs in vivo after

systemic delivery from day 1 to day 5 in pulmonary melanoma metastasis-bearing C57BL6 mice

(left) or healthy normal mice (right).

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Supplementary Fig. 3. Tumor regression effects post suicide gene treatment. The metastatic

nodules on lung post treatment with PBS group, indicating phosphate buffer solution intravenous

injection only; TK-BMSCs + GCV-so group, indicating TK-BMSCs co-administrated with GCV

aqueous solution for intravenous injection; and TK-BMSCs + GCV-lipo group, indicating TK-

BMSCs co-administrated with GCV encapsulated liposomes for intravenous injection. n = 7.

3. Supplementary Tables

Table 1. Formulation of the liposome impacts on the size and entrapment efficiency. When the

GCV concentration was 1.5 mg/mL, the mass ratio of egg phosphatidylcholine (EPC) and

cholesterol (CH) was 5:1, and the volume ratio of oil/water was 3:1, the liposomes achieved the

highest encapsulation efficiency and desired particle size (see the yellow area). : precipitate; -:

no measurement.

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Table 2. Number of the metastatic nodules and weight of the lung.

Descriptive statistics for number of the metastatic nodules and weight of the lung.

4. Other Supplementary Files

Video 1. Permeability of BMSCs into the 3D tumor spheroid 24 h post incubation.

Video 2. Permeability of BMSCs into the 3D tumor spheroid 48 h post incubation.

Video 3. Permeability of BMSCs into the 3D tumor spheroid 72 h post incubation.

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