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. 2012 Sep;15(3):273-82.
doi: 10.4048/jbc.2012.15.3.273. Epub 2012 Sep 28.

Stem Cell Implants for Cancer Therapy: TRAIL-Expressing Mesenchymal Stem Cells Target Cancer Cells In Situ

Michaela R Reagan  1 , F Philipp Seib, Douglas W McMillin, Elizabeth K Sage, Constantine S Mitsiades, Sam M Janes, Irene M Ghobrial, David L Kaplan
Affiliations

Stem Cell Implants for Cancer Therapy: TRAIL-Expressing Mesenchymal Stem Cells Target Cancer Cells In Situ

Michaela R Reagan et al. J Breast Cancer. 2012 Sep.

Abstract

Purpose: Tumor-specific delivery of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), an apoptosis-inducing peptide, at effective doses remains challenging. Herein we demonstrate the utility of a scaffold-based delivery system for sustained therapeutic cell release that capitalizes on the tumor-homing properties of mesenchymal stem cells (MSCs) and their ability to express genetically-introduced therapeutic genes.

Methods: Implants were formed from porous, biocompatible silk scaffolds seeded with full length TRAIL-expressing MSCs (FLT-MSCs). under a doxycycline inducible promoter. In vitro studies with FLT-MSCs demonstrated TRAIL expression and antitumor effects on breast cancer cells. Next, FLT-MSCs were administered to mice using three administration routes (mammary fat pad co-injections, tail vein injections, and subcutaneous implantation on scaffolds).

Results: In vitro cell-specific bioluminescent imaging measured tumor cell specific growth in the presence of stromal cells and demonstrated FLT-MSC inhibition of breast cancer growth. FLT-MSC implants successfully decreased bone and lung metastasis, whereas liver metastasis decreased only with tail vein and co-injection administration routes. Average tumor burden was decreased when doxycycline was used to induce TRAIL expression for co-injection and scaffold groups, as compared to controls with no induced TRAIL expression.

Conclusion: This implant-based therapeutic delivery system is an effective and completely novel method of anticancer therapy and holds great potential for clinical applications.

Keywords: Breast neoplasms; Mesenchymal stem cells; TNF-related apoptosis-inducing ligand; Tissue engineering; Tissue therapy.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
In vitro characterization of full length TRAIL-expressing MSCs (FLT-MSCs) and their impact on breast cancer cells. (A) TRAIL protein enzyme-linked immunosorbent assay (ELISA) of FLT-MSCs exposed to doxycycline (denoted as "dox") for denoted times. Cell specific-bioluminescence assay at 24 hours for MDA-MB-231 cell proliferation in cultures of cancer cells alone, cancer cells with FLT-MSCs, and co-culture with FLT-MSCs and doxycycline. Data is graphed as mean±standard error of the mean (SEM), n=3. (B) For direct co-culture imaging of FLT-MSCs and MDA-MB-231 breast cancer cells, breast cancer cells were grown alone, or with FLT-MSCs with and without doxycycline. After 48 hours of co-culture, MDA-MB-231 cells showed significant decreases in cell numbers in the FLT-MSC doxycycline group compared to the FLT-MSC no doxycycline group or the no MSC group. Statistics were done using an ANOVA and a Tukey's multiple comparison test. *p<0.001 (mean±SEM, n=4). TRAIL=tumor necrosis factor-related apoptosis-inducing ligand; MSC=mesenchymal stem cell; NS=not significant.
Figure 2
Figure 2
Scaffold design and characterization. (A) Timeline of in vitro scaffold screening experiment. (B) Representative live-dead confocal imaging of water-based (WB) or solvent-based (HFIP) scaffolds. Samples were pre-differentiated for 0, 3 or 4 weeks, (denoted as 0 wk, 3 wk, or 4 wk, respectively), re-seeded with DiD mesenchymal stem cells (MSCs), and imaged after 1 week. Cyan, DiD cells; Green, live cells; Red; dead cells and scaffold. All scale bars represent 300 µm. (C) Flow cytometric analysis of CD73 and (D) CD90 expression on DiD MSCs removed from array of scaffolds. No significant differences were found between groups using an ANOVA (mean±SEM, n=4).
Figure 3
Figure 3
Silk scaffold mesenchymal stem cell (MSC) retention after in vitro and in vivo culture. Live-dead confocal images of scaffolds (A) 5 days and (B) 16 days post-seeding demonstrating good viability in vitro of MSCs on scaffolds. Green, live cells; Red, dead cells and silk scaffold. Frozen section imaged on confocal microscope of full length TRAIL-expressing MSC (FLT-MSC)-seeded HFIP scaffolds removed (C) 2 weeks post-implantation of (D) 8 weeks post-implantation demonstrating retention of DiD FLT-MSCs in vivo. Red, DiD; Green, scaffold autofluorescence. Images made from z-stacked maximum or average projections. All scale bars equal in size and represent 300 µm. TRAIL=tumor necrosis factor-related apoptosis-inducing ligand.
Figure 4
Figure 4
DiD full length TRAIL-expressing MSCs (FLT-MSCs) fluorescence at 6 weeks after tumor inoculation for in vivo groups with doxycycline. (A) Co-injection group, (B) tail vein injection group, (C) implant group, and (D) breast cancer cells alone.
Figure 5
Figure 5
In vivo tumor growth and metastasis. (A) In vivo tumor growth assessed at weeks 1, 3, and 6 for the following mouse groups with and without doxycycline (Dox): breast cancer cells alone (BCCs), co-injection groups, tail vein injection groups, and Implant groups for mice. Statistics were done using a one-way ANOVA and a post-hoc Dunnett's test: *p<0.001; †0.001<p<0.01; ‡0.05< p<0.01. (B) Metastatic frequency (number of metastases out of total samples) in mouse femurs, liver, and lungs for mice treated with doxycycline. (C) Representative Images of (B): organs positive (left) and negative (right) for metastasis in brain, lung, liver, and bone using unvarying settings for imaging.
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