Molecular imaging for assessment of mesenchymal stem cells mediated breast cancer therapy

Abstract

The tumor tropism of mesenchymal stem cells (MSCs) makes them an excellent delivery vehicle used in anticancer therapy. However, the exact mechanisms of MSCs involved in tumor microenvironment are still not well defined. Molecular imaging technologies with the versatility in monitoring the therapeutic effects, as well as basic molecular and cellular processes in real time, offer tangible options to better guide MSCs mediated cancer therapy. In this study, an in situ breast cancer model was developed with MDA-MB-231 cells carrying a reporter system encoding a double fusion (DF) reporter gene consisting of firefly luciferase (Fluc) and enhanced green fluorescent protein (eGFP). In mice breast cancer model, we injected human umbilical cord-derived MSCs (hUC-MSCs) armed with a triple fusion (TF) gene containing the herpes simplex virus truncated thymidine kinase (HSV-ttk), renilla luciferase (Rluc) and red fluorescent protein (RFP) into tumor on day 13, 18, 23 after MDA-MB-231 cells injection. Bioluminescence imaging of Fluc and Rluc provided the real time monitor of tumor cells and hUC-MSCs simultaneously. We found that tumors were significantly inhibited by hUC-MSCs administration, and this effect was enhanced by ganciclovir (GCV) application. To further demonstrate the effect of hUC-MSCs on tumor cells in vivo, we employed the near infrared (NIR) imaging and the results showed that hUC-MSCs could inhibit tumor angiogenesis and increased apoptosis to a certain degree. In conclusion, hUC-MSCs can inhibit breast cancer progression by inducing tumor cell death and suppressing angiogenesis. Moreover, molecular imaging is an invaluable tool in tracking cell delivery and tumor response to hUC-MSCs therapies as well as cellular and molecular processes in tumor.

Keywords: Bioluminescence imaging (BLI); Cancer therapy; Mesenchymal stem cells (MSCs); Molecular imaging; Near infrared (NIF) imaging.

Fig. 1

Transduction of MAD-MB-231 cells, hUC-MSCs with double fusion (DF) and triple fusion (TF) reporter genes, respectively. (A) Schema of the DF reporter gene containing Fluc and eGFP driven by an ubiquitin promoter. (B) Schema of the TF reporter gene contains Rluc-RFP-HSV-ttk driven by an EF-1α promoter. (C) Transduced MDA-MB-231 cells are strongly positive for eGFP on fluorescence microscopy. (D) RFP was expressed robustly in hUC-MSCs. (E & F) Ex vivo imaging analysis of stably transduced MDA-MB-231 cells and hUC-MSCs shows a robust correlation between cell numbers and Fluc/Rluc reporter gene activity. Scale bar = 100 μm.

Fig. 2

In vivo inhibition of tumor growth by hUC-MSCs via HSV-ttk system. (A) Schema of treatment of breast cancer with hUC-MSC-TF cells. (B) Firefly luciferase (Fluc) imaging of tumor progression (left). Quantitative analysis of Fluc signal (right). (C) Renilla luciferase (Rluc) imaging of hUC-MSCs-TF from day 0 to day 4 in the second cycle of hUC-MSCs administration (left). Quantitative analysis of Rluc signal. Bioluminescence activity is showed as photons/s/cm²/sr. #P < 0.01 compared to control.

Fig. 3

In vivo reduction of tumor angiogenesis and enhancement of cell apoptosis by hUC-MSCs. (A) Near infrared fluorescence (NIR) imaging of integrin αvβ₃ activity after two consecutive hUC-MSCs administration via injection of IntegriSense™. Angiogenesis index was indicated by relative radiance compare to the control group. (B) Representative CD31 staining of tumor sections from each group. Quantitative analysis of capillary density was significantly lower in the hUC-MSCs and hUC-MSCs/GCV groups compared with the control and GCV groups. (C) Representative images after hUC-MSCs administration by intravenous injection of Annexin-Vivo™. Apoptosis index was indicated by relative radiance compared to the control group. (D) Apoptosis was measured by the TUNEL assay. Quantitative analysis of TUNEL assay. Tumor cell apoptosis was promoted in hUC-MSCs-TF treated group and this effect can be enhanced by GVC application. Scale bar = 50 μm. *P < 0.05, ^#P < 0.01 compared to control.

Fig. 4

hUC-MSCs-TF exhibit a great sensitivity to prodrug GCV and bystander effect on MDA-MB-231 cells in vitro. (A) hUC-MSCs or hUC-MSC-TF cells were plated and treated with increasing concentrations of GCV, and viability of cells was measured 2 days later. (B) hUC-MSCs or hUC-MSC-TF cells were cultured with GCV (40 μg/ml) for 3 days, and cell viability was measured by CCK-8 assay every day. (C) Mixtures of hUC-MSC-TF and MDA-MB-231 cells in the ratios (hUC-MSC-TF: MDA-MB-231=0, 1:10, 1:5, 2:5, 4:5, 1:1) were treated with GCV (40 μg/ml) for 48 h, and the viability of MDA-MB-231 cells was indicated by the relative radiance compared to 0% group by imaging Fluc activity in MDA-MB-231 cells. (D) Mixtures of hUC-MSC-TF cells and the same number of MDA-MB-231 cells were treated with PBS or GCV (40 μg/ml) for 3 days. The cell viability of MDA-MB-231 was measured as mentioned above. More than 70% of MAD-MB-231 cells were killed in the presence of 40 μg/ml GCV at day 3, but the cell proliferation was not affected without GCV. ^#P < 0.05, *P < 0.01 compared to control. Note: MSC, hUC-MSC; MSC-TF, hUC-MSC-TF; MDA, MDA-MB-231.

Fig. 5

hUC-MSCs have no significant effect on MDA-MB-231 cell proliferation in vitro. (A) Expression of proliferation marker Ki67 and apoptosis marker cleaved caspase-3 in MDA-MB-231 cells was not affected by MSC-CM. (B) FACS analysis of MDA-MB-231 cells treated with MSC-CM was conducted by staining MAD-MB-231 with Annexin V and PI (Propidium Iodide). Quantitative analysis results revealed that the cell apoptosis of MAD-MB-231 was not affected significantly by hUC-MSCs. (C) The cell proliferation of MDA-MB-231 was inhibited MSC-CM, but no significant difference. (D) The viability of MDA-MB-231 cells was not affected by hUC-MSCs through direct contact.

Fig. 6

hUC-MSCs down-regulate the expression of angiogenic factors in MAD-MB-231 cells and inhibit endothelial cell migration. (A) Analysis of angiogenic factors expression in MDA-MB231 treated with MSC-CM. (B) Representative photographs of Transwell assay of HUVECs treated with MSC-CM. Quantitative analysis results revealed that the migration of HUVECs was significantly suppressed by hUC-MSCs. Scale bar = 100 μm. *P < 0.05, ^#P < 0.01 compared to control.

Fig. 7

Putative model outlining the tumor-suppressive effects of hUC-MSCs by inhibiting tumor angiogenesis or inducing tumor cell death through bystander effect via the HSV-ttk/GCV suicide system. EC, endothelial cell.