Cultivation of PMSCs
Human PMSCs were donated by the Stem Cell Research Institute of the General Hospital of Ningxia Medical University (Yinchuan, China). PMSCs were cultured and concentration was adjusted to 3 × 106 cells/mL, 100 uL cell suspension was added to Eppendorf (EP) tube, and then, 2 uL primary antibodies (CD29, CD105, CD73, CD11b, CD14, CD45, and IgG1) was added, incubated at 4 ℃ for 30 min, centrifuged, washed, and the supernatant was removed. Afterwards, 2uL FITC-conjugated secondary antibody was added to incubate in an ice box in the dark for 30 min, which then centrifuged, washed with phosphate-buffered saline (PBS), and finally the positive expression rate of cell surface markers was detected.
Prepared cells were seeded into a six-well plate with a density of 2 × 104 cells/ml. After the cells adhere to the wall, add 1.5 mL culture medium, and then incubated. Next, USPIOs and poly-L-lysine (PLL) were mixed at a ratio of 1:0.03, placed in a vortex mixer at 2200 r/min, and shaken for 30 min to form PLL-USPIO complex, then add to cultere medium at a final concentration of 10uL/mL. After incubation for 24 h, it was fixed with 4% paraformaldehyde, and stained with Perls' Prussian blue. The number of Prussian blue-stained and unstained cells were counted using hemocytometer (× 100) under an inverted microscope and labeling efficiency was determined by percentage of Prussian blue-stained positive cells over total cells.
Comparing biological characteristics of PMSCs
The Trypan Blue dye exclusion test: This test was carried out to determine the number of viable cells present in a cell suspension. Take an equal amount of PMSCs suspension and 0.4% Trypan blue solution to mix and count, due to incomplete cell membrane, dead cells can be stained blue and living cells are not stained. Besides, cell counting kit-8 (CCK-8) was used to assess cell proliferation; for this purpose, 2 × 103 PMSCs were seeded into 96-well plates, and we attempted to consider the labelled cells as experimental group, the unlabelled cells as control group, and the medium without cells was Blank group (there were 5 duplicate wells in each group). On the 1st, 3rd, 5th, and 7th days after labeling, 10 uL CCK-8 solution was added to 100 uL complete medium, incubated for 2 h to detect optical density (OD) value of each well at a wavelength of 450 nm, then, the OD value of different groups were compared, and the cell proliferation curve was plotted. Additionally, Annexin V-FITC/PI cell apoptosis detection kit was utilized to detect the rate of apoptosis rate; to this end, labelled and unlabelled PMSCs were collected, and the cell density was adjusted to 1 × 105 cells/ml. Furthermore, 500 µL of 1 × Annexin V Binding Buffer was added to re-suspend the cells. Next, the cells were gently vortexed, and incubated at room temperature for 15 min in the dark.
Establishment of animal models
Herein, 20 healthy female BALB/c nude mice (body weight, 18–22 g; age, 4–6 week-old) were tested, and fed at Experimental Animal Center of Ningxia Medical University (Yinchuan, China). Moreover, 20 nude mice were randomly divided into two groups (n = 10 mice for each group). The concentration of HT-29 cells was adjusted to 107 cells/ml, and subcutaneously inoculated into the right groin of nude mice. The experiment was performed on the day 14 when the tumor diameter was about 5 mm. In the experimental group, 5 × 106 cells/mL of USPIOs-labelled PMSCs were injected into tumor-bearing mice, while no injection was undertaken in the control group. The growth of xenograft tumors of nude mice was assessed, then, diameter of each transplanted tumor was measured every 3–4 days, and the growth curve was plotted to calculate the volume of tumor.
MRI scan in vivo
4% isoflurane to induce anesthesia, when the mice under deep anesthesia adjusted the concentration to 1.5% for continuing anesthesia. Scanning parameters: T2WI fast spin echo sequence TR 2000 ms, TE 140 ms, echo chain length 15, flip angle = 150°, FOV 35 mm × 35 mm, acquisition matrix 176 × 176; T2 mapping sequence: TR 2000 ms, TE n * 13 ms, six echoes of 13, 26, 39, 52, 65, 78 ms, flip angle = 90°, FOV 50 mm × 50 mm, acquisition matrix 124 × 124; T2 * mapping sequence: TR 28 ms, TE n * 3 ms are six echoes of 3, 8, 12, 16, 20, 24 ms, flip angle = 20°, FOV 30 mm × 30 mm, matrix 100 × 96. Transverse scanning, layer thickness 2 mm, layer spacing 0.2 mm,and scanned before the PMSCs injection, on the 1st, 2nd, 3rd, 7th, 10th, and 14th days, analyzed the signal change rates (△SI, △T2, △T2 *) and plotted time signal value curve, compared the SNR, CNR and C values of each sequence. △SI = [(SIlabeled-SItumor) / SItumor] × 100%, △T2 = (T2labeled-T2tumor) / T2tumor] × 100%, △T2 * = (T2 * labeled-T2 * tumor) / T2 * tumor] × 100%, SNR = SItumor / SDnoise, CNR = SItumor-SIlabeled / SDnoise, C = SItumor / SIlabeled (SIlabeled, SItumoris is the signal strength of labeled cells and tumor, SDnoise is the standard deviation of background signal strength). Avoided bleeding and necrosis to measure tumor tissue, and choosed the contralateral thigh's largest section to measure muscle, The region of interest(ROI) size of tumor and image background was 3 cm2 and the labeled area was 0.5 cm2, three ROIs were selected and averaged.
Herein, 2 mice were selected from both the experimental group and the control group respectively to be sacrificed on the 2nd day of PMSCs injection for Perls' Prussian blue staining, and the remaining mice were sacrificed after MRI on the 14th day of PMSCs injection. The inhibition rate of tumor growth was calculated using the following formula: inhibition rate (%) = (mean tumor weight in control group – mean tumor weight in experimental group)/ mean tumor weight in control group × 100%. Moreover, hematoxylin and eosin (H&E), Perls' Prussian blue,and CD31, CD34, and ki67 staining methods were performed, and microvascular density (MVD) was calculated by modifying the Weidner method.
The data were statistically analyzed by SPSS 23.0 software (IBM, Armonk, NY, USA), and all experimental data were expressed as mean ± standard deviation (x ± s). The rates of survival, apoptosis, proliferation, and tumor inhibition of labelled and unlabelled cells were compared by the independent t-test. The volume of transplanted tumors was analyzed by repeated measures analysis of variance (ANOVA). The values of signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) for each sequence were compared by ANOVA. P < 0.05 was considered statistically significant.