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Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis.

Shen K, Luk S, Elman J, Murray R, Mukundan S, Parekkadan B - Sci Rep (2016)

Bottom Line: Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME).Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone.The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School and the Shriners Hospitals for Children, Boston, Massachusetts 02114, USA.

ABSTRACT
Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy.

No MeSH data available.


Related in: MedlinePlus

In vivo elimination of suicide gene-engineered stromal cells.(a) NOD/SCID mice were inoculated with MDA-Luc + CAF-iCasp tumors at mammary fatpads on both flanks, and half of the population was injected with CID homodimerizer at day 10 and 11. Animals were sacrificed on dash-line indicated days and tumor were stained with a human mitochondria-specific antibody. (b) Human mitochondria staining of xenograft tumor sections where MDA/Luc and CAF-iCasp cells were co-implanted into the mammary fatpads of NOD/Scid mice. Fibroblastic human CAF cells remain visible 30 days after implantation in the NTX condition, and hardly seen in the tumors from mice hosts treated with CID injection on day 10 and 11. Scale bar: 100 μm. (c) Morphology-based quantification shows dramatic reduction of human fibroblasts in tumors treated with CID drug (p = 1.4 × 10−5, Student’s t-test). Total images analyzed: NTX: n = 13; CID TX: n = 14. For in vivo study, N = 5 animals per endpoint, per condition. Error bars: standard error of the mean (SEM).
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f2: In vivo elimination of suicide gene-engineered stromal cells.(a) NOD/SCID mice were inoculated with MDA-Luc + CAF-iCasp tumors at mammary fatpads on both flanks, and half of the population was injected with CID homodimerizer at day 10 and 11. Animals were sacrificed on dash-line indicated days and tumor were stained with a human mitochondria-specific antibody. (b) Human mitochondria staining of xenograft tumor sections where MDA/Luc and CAF-iCasp cells were co-implanted into the mammary fatpads of NOD/Scid mice. Fibroblastic human CAF cells remain visible 30 days after implantation in the NTX condition, and hardly seen in the tumors from mice hosts treated with CID injection on day 10 and 11. Scale bar: 100 μm. (c) Morphology-based quantification shows dramatic reduction of human fibroblasts in tumors treated with CID drug (p = 1.4 × 10−5, Student’s t-test). Total images analyzed: NTX: n = 13; CID TX: n = 14. For in vivo study, N = 5 animals per endpoint, per condition. Error bars: standard error of the mean (SEM).

Mentions: The engineered cell line was advanced to in vivo studies to confirm whether suicide-induction can be achieved in situ in a mouse model of cancer. We focused on breast cancer based on previous evidence that phenotypic changes in the fibroblastic stroma of breast cancer patients have been a strong predictor signature to poor outcomes17. MDA-MB-231 breast cancer cells carrying a luciferase gene18 (MDA/Luc) were co-implanted with equal numbers of CAF-iCasp cells into the mammary fat pads of immune compromised female NOD/SCID mice. A cohort of the animals were randomized and treated with two doses of CID drug on day 10 and 11 to eliminate CAF-iCasp cells. Tumor-bearing mice were sacrificed on day 3, 10, 15, and 30 to characterize tumor morphology and CAF distribution (Fig. 2a). Tumor sections were immunostained for human mitochondria to specifically detect the human origin MDA/Luc and CAF-iCasp cells. The two cell types were readily distinguishable by rounded epithelial morphology (blue arrows) and extended fibroblastic morphology (red arrows), respectively (Fig. 2b), in the tumors without CID treatment at all the time points in the 30-day study. We found implanted cells generally underwent an initial cell loss through day 10 and 15, with shrunk human cell areas in the tumor sections. By day 30, cancer cells had proliferated extensively to occupy the majority of the tumor sections; yet, distinct human CAF-iCasp cells were still visible as indicated by the red arrow. In contrast, human CAF-iCasp cells were largely absent from tumor sections at day 15 and 30 in the CID treated groups (Fig. 2b, green box; Fig. 2c), confirming CAF cells were selectively targeted and eliminated from the TME in our in vivo model.


Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis.

Shen K, Luk S, Elman J, Murray R, Mukundan S, Parekkadan B - Sci Rep (2016)

In vivo elimination of suicide gene-engineered stromal cells.(a) NOD/SCID mice were inoculated with MDA-Luc + CAF-iCasp tumors at mammary fatpads on both flanks, and half of the population was injected with CID homodimerizer at day 10 and 11. Animals were sacrificed on dash-line indicated days and tumor were stained with a human mitochondria-specific antibody. (b) Human mitochondria staining of xenograft tumor sections where MDA/Luc and CAF-iCasp cells were co-implanted into the mammary fatpads of NOD/Scid mice. Fibroblastic human CAF cells remain visible 30 days after implantation in the NTX condition, and hardly seen in the tumors from mice hosts treated with CID injection on day 10 and 11. Scale bar: 100 μm. (c) Morphology-based quantification shows dramatic reduction of human fibroblasts in tumors treated with CID drug (p = 1.4 × 10−5, Student’s t-test). Total images analyzed: NTX: n = 13; CID TX: n = 14. For in vivo study, N = 5 animals per endpoint, per condition. Error bars: standard error of the mean (SEM).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4759812&req=5

f2: In vivo elimination of suicide gene-engineered stromal cells.(a) NOD/SCID mice were inoculated with MDA-Luc + CAF-iCasp tumors at mammary fatpads on both flanks, and half of the population was injected with CID homodimerizer at day 10 and 11. Animals were sacrificed on dash-line indicated days and tumor were stained with a human mitochondria-specific antibody. (b) Human mitochondria staining of xenograft tumor sections where MDA/Luc and CAF-iCasp cells were co-implanted into the mammary fatpads of NOD/Scid mice. Fibroblastic human CAF cells remain visible 30 days after implantation in the NTX condition, and hardly seen in the tumors from mice hosts treated with CID injection on day 10 and 11. Scale bar: 100 μm. (c) Morphology-based quantification shows dramatic reduction of human fibroblasts in tumors treated with CID drug (p = 1.4 × 10−5, Student’s t-test). Total images analyzed: NTX: n = 13; CID TX: n = 14. For in vivo study, N = 5 animals per endpoint, per condition. Error bars: standard error of the mean (SEM).
Mentions: The engineered cell line was advanced to in vivo studies to confirm whether suicide-induction can be achieved in situ in a mouse model of cancer. We focused on breast cancer based on previous evidence that phenotypic changes in the fibroblastic stroma of breast cancer patients have been a strong predictor signature to poor outcomes17. MDA-MB-231 breast cancer cells carrying a luciferase gene18 (MDA/Luc) were co-implanted with equal numbers of CAF-iCasp cells into the mammary fat pads of immune compromised female NOD/SCID mice. A cohort of the animals were randomized and treated with two doses of CID drug on day 10 and 11 to eliminate CAF-iCasp cells. Tumor-bearing mice were sacrificed on day 3, 10, 15, and 30 to characterize tumor morphology and CAF distribution (Fig. 2a). Tumor sections were immunostained for human mitochondria to specifically detect the human origin MDA/Luc and CAF-iCasp cells. The two cell types were readily distinguishable by rounded epithelial morphology (blue arrows) and extended fibroblastic morphology (red arrows), respectively (Fig. 2b), in the tumors without CID treatment at all the time points in the 30-day study. We found implanted cells generally underwent an initial cell loss through day 10 and 15, with shrunk human cell areas in the tumor sections. By day 30, cancer cells had proliferated extensively to occupy the majority of the tumor sections; yet, distinct human CAF-iCasp cells were still visible as indicated by the red arrow. In contrast, human CAF-iCasp cells were largely absent from tumor sections at day 15 and 30 in the CID treated groups (Fig. 2b, green box; Fig. 2c), confirming CAF cells were selectively targeted and eliminated from the TME in our in vivo model.

Bottom Line: Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME).Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone.The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School and the Shriners Hospitals for Children, Boston, Massachusetts 02114, USA.

ABSTRACT
Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy.

No MeSH data available.


Related in: MedlinePlus