Mesenchymal stem cells from human fat engineered to secrete BMP4 are nononcogenic, suppress brain cancer, and prolong survival

Abstract

Purpose: Glioblastoma is the most common adult primary malignant intracranial cancer. It is associated with poor outcomes because of its invasiveness and resistance to multimodal therapies. Human adipose-derived mesenchymal stem cells (hAMSC) are a potential treatment because of their tumor tropism, ease of isolation, and ability to be engineered. In addition, bone morphogenetic protein 4 (BMP4) has tumor-suppressive effects on glioblastoma and glioblastoma brain tumor-initiating cells (BTIC), but is difficult to deliver to brain tumors. We sought to engineer BMP4-secreting hAMSCs (hAMSCs-BMP4) and evaluate their therapeutic potential on glioblastoma.

Experimental design: The reciprocal effects of hAMSCs on primary human BTIC proliferation, differentiation, and migration were evaluated in vitro. The safety of hAMSC use was evaluated in vivo by intracranial coinjections of hAMSCs and BTICs in nude mice. The therapeutic effects of hAMSCs and hAMSCs-BMP4 on the proliferation and migration of glioblastoma cells as well as the differentiation of BTICs, and survival of glioblastoma-bearing mice were evaluated by intracardiac injection of these cells into an in vivo intracranial glioblastoma murine model.

Results: hAMSCs-BMP4 targeted both the glioblastoma tumor bulk and migratory glioblastoma cells, as well as induced differentiation of BTICs, decreased proliferation, and reduced the migratory capacity of glioblastomas in vitro and in vivo. In addition, hAMSCs-BMP4 significantly prolonged survival in a murine model of glioblastoma. We also demonstrate that the use of hAMSCs in vivo is safe.

Conclusions: Both unmodified and engineered hAMSCs are nononcogenic and effective against glioblastoma, and hAMSCs-BMP4 are a promising cell-based treatment option for glioblastoma.

Figure 1. hAMSCs-BMP4 decrease migration of BTICs in vitro

(A) hAMSCs were infected with BMP4 or vector retroviruses. Western blots were performed using cell lysates and concentrated media. β-actin served as a loading control. (B) Schematic of conditioned media (CM) collection and co-culturing methods. (C) BTICs were treated with 50 ng/ml BMP4, or were cultured in hAMSC-Vector-CM, hAMSC-BMP4-CM, or control media for 24 hours and a Boyden transwell assay was performed. Results were normalized and compared to the control media condition. (D) Schematic of cells migrating on a nanopattern. (E) Nanopattern assay of BTICs cultured in hAMSC-Vector-CM, hAMSC-BMP4-CM,control media, or treated with 50 ng/ml BMP4. *p<0.05, **p<0.01, ***p<0.001.

Figure 2. hAMSCs-BMP4 decrease BTIC proliferation in vitro

BTICs were cultured in (A) hAMSC-CM or (B) BMP4 treated media (100ng/ml) for 2 weeks, and MTS assays were performed to measure BTIC proliferation. EdU assay of GFP-BTICs (C) co-cultured with hAMSCs for 5 days, (D) treated with BMP4 (50 ng/ml) for 48 hours, or (E) co-cultured with hAMSCs-BMP4 for 5 days to determine the proliferation of BTICs. Results were normalized and compared to BTICs condition.*p<0.05, **p<0.01, ***p<0.001.

Figure 3. hAMSCs-BMP4 promote BTIC differentiation in vitro

(A) BTICs were cultured in control media (stem cell media, undifferentiated BTICs), differentiation media (stem cell media+10%FBS, differentiated BTICs), hAMSC-Vector-CM, hAMSC-BMP4-CM, or BMP4 (100 ng/ml) for 2 weeks and immunofluorescence staining for Tuj1 and GFAP was performed, with magnification in the upper right insets. Scale bar, 200 μm. (B) The percentages of Tuj1+/DAPI and GFAP+/DAPI were calculated from 5 random fields for the different conditions. ***p<0.001.

Figure 4. hAMSCs remain multipotent and retain proliferation capacity when exposed to BTIC-CM or transduced with BMP4 in vitro

(A) MTS assay of hAMSCs cultured in BTICCM or control media for 2 weeks to measure proliferating hAMSCs every 3 days. (B) EdU assay of td-tomato-hAMSCs co-cultured with BTICs for 5 days to determine proliferating hAMSCs. (C) Real-time RT-PCR was performed. Markers for adipocyte, osteocyte and chondrocyte lineages were tested. GAPDH served as a control. Other groups were normalized and compared to the undifferentiated hAMSCs group. (D) hAMSCs were cultured in control media (undifferentiated hAMSCs), differentiation media (differentiated hAMSCs), or BTIC-CM for 3 weeks. Various stains were performed to assess differentiation capabilities (scale bar, 100 μm) and (E) EdU assay of hAMSCs treated with BMP4 (50 ng/ml) for different time periods (24 hours and 48 hours) to measure hAMSC proliferation. Results were normalized and compared to the 0 hour condition. (F) EdU assay comparing proliferation of hAMSCs-Vector and hAMSCs-BMP4 cells after 5 days of culturing. Results were normalized and compared to hAMSCs-Vector. (G) hAMSCs-Vector, hAMSCs-Vector treated with BMP4 (100 ng/ml), or hAMSCs-BMP4 cultured in control media or differentiation media for 3 weeks. Various lineage stains were performed as described previously. Scale bar, 100 μm. (H-I) Transwell assays: hAMSCs-Vector (H) or hAMSCs-BMP4 (I) were seeded on top chamber. BTIC-CM or control media+2% FBS was at the bottom of the chamber. Results were normalized and compared to control.*p<0.05, **p<0.01, ***p<0.001; N.S., not significant.

Figure 5. hAMSCs are not tumorigenic and do not transform into tumor associated fibroblasts (TAFs) in vitro or in vivo

(A-B) hAMSCs were cultured in BTIC-CM or control media for 1-3 weeks and (A) Western blots (β-actin served as a control) and (B) Real-time RTPCR (GAPDH served as a control) were performed to quantify TAF markers (vimentin and ACTA2). (C) Schematic of the co-injection experiment where PBS, GFP-BTICs, GFP/bioluminescent-hAMSCs (GFP-hAMSCs), or GFP-BTICs mixed with td-tomato-hAMSCs (td-hAMSCs) were injected into mice and sacrificed and sacrificed 4 weeks later. (D) Quantification of mean tumor area of the GFP-BTIC and co-injection groups using DAPI staining. The co-injection group had a smaller mean tumor area of 135,700 μm² as compared to GFP-BTIC group, with a mean tumor area of 209,800 μm² (p=0.0189). (E) Live animal imaging of the GFP-hAMSCs condition. At 14 days post GFP-hAMSCs injection, the hAMSC signal drastically decreases. Each mouse brain represents the counts of bioluminescent signal at each time point. (F) DAPI and human nuclei stain for GFP-BTICs and co-injection groups (n=5). Larger tumors were only seen in the GFP-BTICs condition compared the co-injection condition. Brain sections and tumor mass are outlined. Scale bar, 200 μm. N.S., not significant.

Figure 6. hAMSCs-BMP4 increase the median survival time of GBM bearing mice, drive differentiation, and decrease proliferation and migration of GBM cells in vivo

(A) Immunoreactivity for GFP and BMP4 to test the expression of BMP4. Scale bars, 200 μm. (B) GFP-hAMSCs-BMP4 cells were seen near satellite Nestin+ cells away from the main tumor bulk. Scale bars, 200 μm. (C) Representative pictures and quantification of GFP and Ki67 staining to test the proliferation of GBM cells. Scale bars, 200 μm. (D) Representative pictures and quantification of GFP, Nestin, GFAP, and Tuj1 staining to test the differentiation of BTICs. Arrowheads in the GFP-hAMSC-BMP4 GFAP staining correspond to magnified insets of GFP-hAMSC-BMP4 and GFAP+ cells at the tumor center, and a GFAP+ cell with mature astrocytic morphology at the tumor periphery. Magnified pictures are shown on the left. Scale bars, 200 μm. (E) Representative pictures (right hemisphere) and quantification of migratory GBM cells. The average distance of migrated GBM cells, identified as human nuclei+/DAPI+/GFP- cells outside tumor bulk, from the center of tumor mass (outlined) was measured. Scale bars, 200 μm. *p<0.05, **p<0.01, ***p<0.001. (F) Schematic of the in vivo experiment for which immunofluorescence staining was performed in panels A-E: BTIC culture 276 were intracranially injected into 6-8 week-old nude mice. At 4 weeks post-injection, GFP-hAMSCs-Vector (n=7), GFP-hAMSCs-BMP4 (n=5), or equal volumes of PBS (n=5) were injected intracardially. Mice were sacrificed 2 weeks later. (G) U87 cells were intracranially injected into 6-8 week-old nude mice. Ten days post-injection, GFP-hAMSCs-Vector (n=7), GFP-hAMSCs-BMP4 (n=5), or equal volumes of PBS (n=10) were injected intracardially. Mice were followed for 125 days to monitor survival. Kaplan-Meier survival analysis resulted in the median survival of mice treated with hAMSCs-BMP4 (undefined) was significantly greater than that of mice treated with hAMSCs-Vector (p=0.01) (76 days) and control mice (p=0.002) (52 days), with no significant difference between the PBS and hAMSCs-Vector group (p=0.09).