Chemotherapeutic Drug Primed Stem Cells
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0 40 80 120 1 2 3 4 5 6 7 Cell Proliferation (%) A549 HCC827 Non–small-cell lung cancer (NSCLC) accounts for the most cancer deaths worldwide, along with a 5-year survival of only 5% to 15%. Chemotherapy remains an important modality for treatment of this tumour. However, response rates to chemotherapy are only approximately 30%, making its treatment a challenge. The major limitations include short drug half-lives, insufficient delivery, and suboptimal specificity for malignant tissue. There has been considerable interest in the use of stem cells as trophic vehicles for delivery of drugs, proteins, and other therapeutic agents specifically to tumours due to their lack of immune rejection and inherent tumour-homing capabilities. We hypothesized human adipose-derived mesenchymal stem cells (hAD-MSCs) primed in vitro with anti-cancer drugs could release drugs and inhibit tumour growth, thereby serving as vehicles for delivering effective and targeted therapy to tumours. Introduction Experimental Model Materials and Methods hAD-MSCs were isolated from peri-umbilical adipose tissue and stably transfected with green fluorescent protein (GFP); three human non-small-cell lung cancer cell lines (A549, HCC827 and H520) were cultured . Doxorubicin internalisation was analysed by fluorescence microscopy. Cell proliferation was detected by Alarma blue assay. Cell migration was assessed by Boyden chamber assay. Paclitaxel concentration was determined by UHD accurate-mass Q-TOF LC/MS. Results 24 hours 48 hours 72 hours Paclitaxel produced a strong anti-proliferative effect on three lung carcinoma cells (A549, HCC827 and H520) in a dose- and time- dependent manner whereas hAD-MSCs were highly resistant. Drug Intake Drug Release hAD-MSC Tumour cell Chemotherapeutic drugs Paclitaxel (ng/ml) Paclitaxel (ng/ml) Paclitaxel (ng/ml) After 1 hour priming, the internalisation of doxorubicin by hAD-MSCs was appreciable. The staining was intense and enriched in cytoplasm at the end of priming (24 hours). After 24 hours, doxorubicin distribution in cytoplasm decreased, suggesting a possible excretion. Scale bar: 100 μm. 1 School of Healthcare Science, Faculty of Science and Engineering, Manchester Metropolitan University 2 School of Science & The Environment, Faculty of Science and Engineering, Manchester Metropolitan University 3 School of Biomedicine, Faculty of Medicine and Human Sciences, University of Manchester Wen-Hui Fang 1 , David Smith 2 , Shant Kumar 1, 3 , Glenn Ferris 1 , Garry McDowell 1 , Mark Slevin 1 Conclusion and Future Work hAD-MSCs primed in vitro with chemotherapeu- tic drugs could release drugs and inhibit lung cancer cell growth, and the tropism of hAD- MSCs to human lung carcinomas might be exploited to therapeutic advantage as vehicles for delivering targeted therapy to tumours. Future work would focus on mechanisms of chemotherapeutic drug intake and release by hAD-MSCs, the enhanced and more specific tumour-homing, and the utilization of therapeutic modified hAD-MSCs in the treatment of cancer. hAD-MSCs were exposed to 100 μg/ml paclitaxel for 24 hours. Paclitaxel primed hAD-MSCs were further cultured and conditioned media were collected and replaced every day. The concentrations of paclitaxel in conditioned media were measured by LC-MS/MS and conditioned media were tested for anti- proliferative activity on A549 and HCC827 cells. Wild type hAD-MSCs Day 2 Day 3 Day 7 Day 21 DAPI DOX Merge GFP-hAD-MSCs Untreated 1 hour 2 hours DAPI DOX 4 hours 8 hours 12 hours 24 hours Untreated 1 hour 2 hours 4 hours 8 hours 12 hours 24 hours This work was supported by a grant of the Ministry of National Education, CNCS – UEFISCDI, project number PN-II-ID-PCE-2012-4-0133. Many thanks to Dr Valentina Ceserani, Dr Andrew Ryan and Dr John Brognard for kindly providing cell lines. Lund TC et al. Nat Rev Clin Oncol, 2015;12:163-74; Pessina A et al. J Control Release, 2014;192:262-70; Keung EZ et al. Stem Cells, 2013;31:227-35. Paclitaxel-primed hAD-MSCs steadily released paclitaxel into media over 7 days. Drug release (Day) Doxorubicin (DOX) 50 μM Doxorubicin (DOX) 50 μM GFP Merge The conditioned media from paclitaxel primed hAD-MSCs produced a potent growth inhibition on lung cancer cells. Drug release (Day) Chemotherapeutic Drug Primed Mesenchymal Stem Cells Induce Anti-cancer Effects on Human Lung Cancer Cells Chemotherapeutic Drug Primed Mesenchymal Stem Cells Induce Anti-cancer Effects on Human Lung Cancer Cells hAD-MSC A549 HCC827 0 20 40 60 80 100 0 6.25 12.5 25 50 100 200 400 800 1600 3200 6400 12800 25600 51200 102400 204800 Cell Proliferation (%) H520 0 20 40 60 80 100 0 6.25 12.5 25 50 100 200 400 800 1600 3200 6400 12800 25600 51200 102400 204800 Cell Proliferation (%) 0 20 40 60 80 100 0 6.25 12.5 25 50 100 200 400 800 1600 3200 6400 12800 25600 51200 102400 204800 Cell Proliferation (%) 0 40 80 120 1 2 3 4 5 6 7 Cell Proliferation (%) A549 HCC827 Negative control RPMI+10%FBS A549-10%RPMI Positive control Conditioned media of lung cancer cells Basal IMDM 5%IMDM+EndoPM DMEM+10%FBS HCC827-10%DMEM H520-10%RPMI Media control Acknowledgement and Key References 0 10 20 30 40 Basal IMDM Number of Migration Cells × 10 3 RPMI +10%FBS DMEM +10%FBS A549 10%RPMI H520 10%RPMI HCC827 10%DMEM 5%IMDM +EndoPM ** ** ** 0 5 10 15 20 1 2 3 4 5 6 7 Paclitaxel (ng/ml) Conditioned medium from human lung carcinomas promoted hAD-MSCs migration. Scale bar: 100 μm. 4. The oncotropism of hAD-MSCs 3. Paclitaxel-primed hAD-MSCs sustainedly released paclitaxel 2. hAD-MSCs ware resistant to paclitaxel 1. The uptake and release of doxorubicin by hAD-MSCs
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Transcript of Chemotherapeutic Drug Primed Stem Cells
0
40
80
120
1 2 3 4 5 6 7
Ce
ll P
rolif
era
tio
n (
%)
A549
HCC827
Non–small-cell lung cancer (NSCLC) accountsfor the most cancer deaths worldwide, alongwith a 5-year survival of only 5% to 15%.Chemotherapy remains an important modalityfor treatment of this tumour. However, responserates to chemotherapy are only approximately30%, making its treatment a challenge. Themajor limitations include short drug half-lives,insufficient delivery, and suboptimal specificityfor malignant tissue.
There has been considerable interest in the useof stem cells as trophic vehicles for delivery ofdrugs, proteins, and other therapeutic agentsspecifically to tumours due to their lack ofimmune rejection and inherent tumour-homingcapabilities.
We hypothesized human adipose-derivedmesenchymal stem cells (hAD-MSCs) primed invitro with anti-cancer drugs could release drugsand inhibit tumour growth, thereby serving asvehicles for delivering effective and targetedtherapy to tumours.
Introduction
Experimental Model
Materials and Methods
hAD-MSCs were isolated from peri-umbilicaladipose tissue and stably transfected withgreen fluorescent protein (GFP); three humannon-small-cell lung cancer cell lines (A549,HCC827 and H520) were cultured .
Doxorubicin internalisation was analysed byfluorescence microscopy.
Cell proliferation was detected by Alarma blueassay. Cell migration was assessed by Boydenchamber assay.
Paclitaxel concentration was determined byUHD accurate-mass Q-TOF LC/MS.
Results
24 hours 48 hours 72 hours
Paclitaxel produced a strong anti-proliferative effect on three lung carcinoma cells (A549, HCC827 andH520) in a dose- and time- dependent manner whereas hAD-MSCs were highly resistant.
Drug Intake
Drug Release
hAD-MSC
Tumour cell
Chemotherapeuticdrugs
Paclitaxel (ng/ml)Paclitaxel (ng/ml)Paclitaxel (ng/ml)
After 1 hour priming, the internalisation of doxorubicin by hAD-MSCs was appreciable. The staining wasintense and enriched in cytoplasm at the end of priming (24 hours). After 24 hours, doxorubicindistribution in cytoplasm decreased, suggesting a possible excretion. Scale bar: 100 μm.
1School of Healthcare Science, Faculty of Science and Engineering, Manchester Metropolitan University2School of Science & The Environment, Faculty of Science and Engineering, Manchester Metropolitan University
3School of Biomedicine, Faculty of Medicine and Human Sciences, University of Manchester
Wen-Hui Fang1, David Smith2, Shant Kumar1, 3, Glenn Ferris1, Garry McDowell1, Mark Slevin1
Conclusion and Future Work
hAD-MSCs primed in vitro with chemotherapeu-
tic drugs could release drugs and inhibit lung
cancer cell growth, and the tropism of hAD-
MSCs to human lung carcinomas might be
exploited to therapeutic advantage as vehicles
for delivering targeted therapy to tumours.
Future work would focus on mechanisms of
chemotherapeutic drug intake and release by
hAD-MSCs, the enhanced and more specific
tumour-homing, and the utilization of
therapeutic modified hAD-MSCs in the
treatment of cancer.
hAD-MSCs were exposed to 100 μg/ml paclitaxel for 24 hours. Paclitaxel primed hAD-MSCs were further
cultured and conditioned media were collected and replaced every day. The concentrations of paclitaxel
in conditioned media were measured by LC-MS/MS and conditioned media were tested for anti-
proliferative activity on A549 and HCC827 cells.
Wild type hAD-MSCs
Day 2 Day 3 Day 7 Day 21
DAPI
DOX
Merge
GFP-hAD-MSCs
Untreated 1 hour 2 hours
DAPI
DOX
4 hours 8 hours 12 hours 24 hours
Untreated 1 hour 2 hours 4 hours 8 hours 12 hours 24 hours
This work was supported by a grant of the Ministry of National Education, CNCS – UEFISCDI, project number PN-II-ID-PCE-2012-4-0133. Many thanks to Dr Valentina Ceserani, Dr Andrew Ryan and Dr John Brognard for kindly providing cell lines.
Lund TC et al. Nat Rev Clin Oncol, 2015;12:163-74; Pessina A et al. J Control Release, 2014;192:262-70; Keung EZ et al. Stem Cells, 2013;31:227-35.
Paclitaxel-primed hAD-MSCs steadily released
paclitaxel into media over 7 days.
Drug release (Day)
Doxorubicin (DOX) 50 μM
Doxorubicin (DOX) 50 μM
GFP
Merge
The conditioned media from paclitaxel primed
hAD-MSCs produced a potent growth inhibition
on lung cancer cells.
Drug release (Day)
Chemotherapeutic Drug Primed Mesenchymal Stem Cells Induce
Anti-cancer Effects on Human Lung Cancer Cells
Chemotherapeutic Drug Primed Mesenchymal Stem Cells Induce
Anti-cancer Effects on Human Lung Cancer Cells
hAD-MSC A549 HCC827
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Ce
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%)
A549
HCC827
Negative control
RPMI+10%FBS A549-10%RPMI
Positive control Conditioned media of lung cancer cells
Basal IMDM 5%IMDM+EndoPMDMEM+10%FBS HCC827-10%DMEMH520-10%RPMI
Media control
Acknowledgement and Key References
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Basal IMDM
Nu
mb
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s ×
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3
RPMI
+10%FBS
DMEM
+10%FBS
A549
10%RPMI
H520
10%RPMI
HCC827
10%DMEM
5%IMDM
+EndoPM
****
**
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Pa
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Conditioned medium from human lung carcinomas
promoted hAD-MSCs migration. Scale bar: 100 μm.
4. The oncotropism of hAD-MSCs
3. Paclitaxel-primed hAD-MSCs sustainedly released paclitaxel
2. hAD-MSCs ware resistant to paclitaxel
1. The uptake and release of doxorubicin by hAD-MSCs
