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Doxorubicin distribution in multicellular prostate cancer spheroids evaluated by confocal laser scanning microscopy
M Wartenberg1, J Hescheler, H Acker
1Department of Neurophysiology, University of Cologne, Germany. hs@physiologie.uni-koeln.de
Abstract:
Multicell-mediated drug resistance is a major impediment for the effectiveness of chemotherapeutic approaches and has been shown to be a feature of many solid tumors. We used confocal laser scanning microscopy to evaluate the depth distribution of the fluorescent cytostatic drug doxorubicin (Dox) in two size classes of multicellular cancer spheroids (MCS) (psi150+/-50 microm and 350+/-50 microm). MCS (psi150+/-50 microm) solely consist of proliferating cells, whereas in MCS (psi350+/-50 microm) peripheral proliferating cell layers are followed in the depth of the tissue by drug resistant quiescent cell areas. A technique was developed which allows noninvasively to trace fluorescence distributions down to a depth of approximately 180 microm in living MCS. This was achieved by confocal radial recordings of the mean Dox fluorescence in 600 microm2 regions of interest (ROI), equidistantly spaced (10 microm) from the center of MCS towards their periphery. The resulting fluorescence intensity profiles were subsequently corrected for absorbtion and light scattering in the depth of the tissue by a convenient algorithm. A 10 min incubation of MCS (psi150+/-50 microm) with Dox (10 microM) led to a peripheral accumulation, after 2 h Dox was homogeneously distributed within the whole MCS. In contrast, after Dox treatment of MCS (psi350+/-50 microm) for 2 h, the drug was accumulated within the peripheral proliferating cell rim of 78+/-8 microm, whereas deeper, quiescent cell layers remained unstained. When MCS were incubated with verapamil, cyclosporin A, orthovanadate, and quinidine, which are known to reverse P-glycoprotein (Pgp)-mediated multidrug resistance (MDR), Dox accumulated also in deeper cell layers. Genistein and indometacin which reverse multidrug resistance mediated by the multidrug resistance-associated protein (MRP) were without effects. The optical probe technique proved to be well suited to study MDR in a living three dimensional tissue context.
Insights
Multicell-mediated drug resistance hinders chemotherapy. Researchers developed a 3D imaging technique to track doxorubicin distribution in multicellular cancer spheroids, revealing drug penetration differences in proliferating versus quiescent cells.
Area of Science:
- Oncology
- Pharmacology
- Biophysics
Background:
- Multicell-mediated drug resistance is a significant challenge in cancer chemotherapy, particularly in solid tumors.
- Understanding drug distribution within tumor tissues is crucial for developing effective treatment strategies.
Purpose of the Study:
- To evaluate the depth distribution of doxorubicin (Dox) in multicellular cancer spheroids (MCS) using confocal laser scanning microscopy.
- To investigate the role of different cell populations (proliferating vs. quiescent) and drug resistance mechanisms in drug penetration.
Main Methods:
- Confocal laser scanning microscopy was employed to visualize doxorubicin distribution in two sizes of MCS (small: ~150 µm, large: ~350 µm).
- A novel technique allowed noninvasive tracing of drug fluorescence up to 180 µm depth in living MCS.
- Fluorescence intensity profiles were corrected for light absorption and scattering using a specialized algorithm.
Main Results:
- In small MCS (proliferating cells only), doxorubicin distribution became homogeneous within 2 hours.
- In large MCS (proliferating periphery, quiescent core), doxorubicin accumulated in the outer proliferating layer, with deeper quiescent cells remaining unstained after 2 hours.
- Inhibitors of P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) enhanced doxorubicin penetration into deeper layers, while MRP inhibitors had no effect.
Conclusions:
- The developed optical probe technique is effective for studying multidrug resistance (MDR) in a 3D tissue context.
- Quiescent cells in larger multicellular cancer spheroids exhibit significant drug resistance, limiting doxorubicin penetration.
- Targeting Pgp-mediated MDR could improve drug delivery to resistant cell populations within tumors.