Bruk av induserte pluripotente stamceller (iPSc) utviklet fra fibroblaster for kreft behandling
1 Aim
To test an innovative therapeutic strategy which is targeting the invasive cancer cells by using mesenchymal stem cells (MSC) differentiated from induced pluripotent stem cells (iPSc) generated from own patient’s derived skin and/or oral fibroblasts as a carrier to deliver drugs or functionalized nanodiamond particles (nDPs) to the cancer cells present at invasion sites. More specifically, first we will differentiate MSC in vitro from the iPSc reprogrammed from skin or oral fibroblasts (iPSc-MSC) and we will test in vivo if they infiltrate after IV injection in immunocompromised mice bearing human tumor xenografts. In the second step, we will load the iPSc-MSCs with various functionalized nanodiamond particles and perform the same 'homing' test after IV injection in immunocompromised mice bearing human tumor xenografts and then evaluate their effect on tumor progression.
2 Adverse effects
Mice will develop tumor xenografts from human oral cancer cells injected in the tongue. The ability of mice to eat will be affected by tumors when they reach a certain size. We have experience with this tumor model so we have established humane endpoints to minimize the suffering of the mice due to these xenografts. Reports on human MSCs injected into immunocompromised mice do not report side effects.
3 Expected utility
The study will provide basis for a novel oncology application of functionalized nDPs for targeted therapy of cancer with particular benefit for the bone-invasion cancer-suffering patients.
4 Number of animals and species
288 NOD / SCID ILgamma2-deffcinet mice
Step 1 – testing of MSC tropism: 144 mice
10x6 oral cancer cells (HPV+ and HPV-) will be xenotransplanted in tongues of NOD/SCID ILgamma2 deficient mice in 25 ul PBS. After 2 weeks, 10x6 normal oral fibroblasts, matched iPSc-MSCs cells, and BM-MSCs from bone marrow labeled with GFP, will be injected in the vein tail.
Mice will be sacrificed (at 1h, 1 day, 1 week, 2 weeks), tumor xenografts will be harvested and immunohistochemistry for anti-GFP will be performed to visualize MSCs present in the tumor.
Nr Mice: 6micex3types cells injected IV (Fibs/iPS-MSC/BM-MSC) = 18 mice for each xenotransplanted cancer cell line. For two cancer cell lines (HPV+ and HPV-) : 18x2 = 36 mice for each time point. For 4 time points: 36 x 4 time points = 144 mice.
Step 2 – testing of functionalized nDP for specific tumor targeting: 144 mice
10x6 oral cancer cells (HPV+/HPV-) and 10x5 CAFs will be xenotransplanted in the tongue of NOD/SCID ILgamma2 deficient mice. After 2 weeks, 10x6 iPSc-MSCs cells loaded with nDPs (ctrl, anti EGFR and anti Fibrin) will be injected in the vein tail.
Nr Mice: 6x3=18, 18x2=36, 36 at each time point x 4 time points = 144 mice
5.3R
The iPSC-MSC derived from matched oral and skin adult fibroblasts will be first tested for cancer tropism on in vitro 3D models before the in vivo models and the molecular interactions between iPSc-MSC cells and oral cancer cells and activated oral cancer-fibroblasts will be analyzed in vitro in 2D and 3D organotypic models.
To test an innovative therapeutic strategy which is targeting the invasive cancer cells by using mesenchymal stem cells (MSC) differentiated from induced pluripotent stem cells (iPSc) generated from own patient’s derived skin and/or oral fibroblasts as a carrier to deliver drugs or functionalized nanodiamond particles (nDPs) to the cancer cells present at invasion sites. More specifically, first we will differentiate MSC in vitro from the iPSc reprogrammed from skin or oral fibroblasts (iPSc-MSC) and we will test in vivo if they infiltrate after IV injection in immunocompromised mice bearing human tumor xenografts. In the second step, we will load the iPSc-MSCs with various functionalized nanodiamond particles and perform the same 'homing' test after IV injection in immunocompromised mice bearing human tumor xenografts and then evaluate their effect on tumor progression.
2 Adverse effects
Mice will develop tumor xenografts from human oral cancer cells injected in the tongue. The ability of mice to eat will be affected by tumors when they reach a certain size. We have experience with this tumor model so we have established humane endpoints to minimize the suffering of the mice due to these xenografts. Reports on human MSCs injected into immunocompromised mice do not report side effects.
3 Expected utility
The study will provide basis for a novel oncology application of functionalized nDPs for targeted therapy of cancer with particular benefit for the bone-invasion cancer-suffering patients.
4 Number of animals and species
288 NOD / SCID ILgamma2-deffcinet mice
Step 1 – testing of MSC tropism: 144 mice
10x6 oral cancer cells (HPV+ and HPV-) will be xenotransplanted in tongues of NOD/SCID ILgamma2 deficient mice in 25 ul PBS. After 2 weeks, 10x6 normal oral fibroblasts, matched iPSc-MSCs cells, and BM-MSCs from bone marrow labeled with GFP, will be injected in the vein tail.
Mice will be sacrificed (at 1h, 1 day, 1 week, 2 weeks), tumor xenografts will be harvested and immunohistochemistry for anti-GFP will be performed to visualize MSCs present in the tumor.
Nr Mice: 6micex3types cells injected IV (Fibs/iPS-MSC/BM-MSC) = 18 mice for each xenotransplanted cancer cell line. For two cancer cell lines (HPV+ and HPV-) : 18x2 = 36 mice for each time point. For 4 time points: 36 x 4 time points = 144 mice.
Step 2 – testing of functionalized nDP for specific tumor targeting: 144 mice
10x6 oral cancer cells (HPV+/HPV-) and 10x5 CAFs will be xenotransplanted in the tongue of NOD/SCID ILgamma2 deficient mice. After 2 weeks, 10x6 iPSc-MSCs cells loaded with nDPs (ctrl, anti EGFR and anti Fibrin) will be injected in the vein tail.
Nr Mice: 6x3=18, 18x2=36, 36 at each time point x 4 time points = 144 mice
5.3R
The iPSC-MSC derived from matched oral and skin adult fibroblasts will be first tested for cancer tropism on in vitro 3D models before the in vivo models and the molecular interactions between iPSc-MSC cells and oral cancer cells and activated oral cancer-fibroblasts will be analyzed in vitro in 2D and 3D organotypic models.