- LCM ACCUMULATE PREFERENTIALLY AT TUMOR SITE
- LCM ARE INTERNALIZED BY TUMOR CELLS
- LCM CAN DELIVER TAXOL TO TUMOR CELLS
- EFFECTS OF TAXOL-LCM ON C6 GLIOMA CELLS IN TISSUE CULTURE
- MECHANISM OF LCM UPTAKE BY TUMOR CELLS
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Protocols for cancer therapy include chemotherapy, immunotherapy, radiation therapy, and cavitation, singly or in combination. The approach described here focuses on chemotherapy exclusively, and summarizes the effects on tumors of a known drug, taxol, administered in a vehicle designated in the earlier literature as LCM (for "lipid-coated microbubbles", -- but can be more accurately described as a (coated-microbubble/nanoparticle) lipid nanoemulsion [tradename Filmix®]).
Brain tumors are difficult to treat because of limited agent penetration into the tumor. Although there is a leaky capillary endothelium in the tumor-blood vessel interface (Stewart et al., 1985), there does not appear to be specific accumulation of intravenously (i.v.) administered agents into the tumor. When rats bearing brain tumors were injected i.v. with LCM, the latter were found in and around the tumor (see Preliminary Data below) (Simon et al., 1992; D'Arrigo et al., 1991, 1993). This suggested to us that LCM could be used as a vehicle for anti-cancer drugs. An important additional property of LCM is that they are capable of physically carrying some drugs with them, and, as described in Preliminary Data, are internalized by tumor cells, thus delivering their content inside the cells. The mechanism for the uptake specificity appears to involve "lipoprotein receptor"-mediated endocytic pathways (D'Arrigo, 2003), -- and at least 4 different types of tumor cells do interact with LCM in that manner suggesting that LCM specificity may be for tumor cells in general. Finally, LCM have been shown to be non-toxic and safe, and are easily administered (D'Arrigo et al., 1991). LCM thus may provide a targeted delivery system, for some types of chemotherapeutic agents, that is potentially more efficient than conventional ones and which has less side effects.
Taxol is FDA-approved for clinical use in ovarian and breast cancer. It has shown cytotoxic activity against a number of leukemias, as well as Walker 256 carcinosarcomas and lung tumors (for a review see Rowinsky et al., 1990), but its development has been hampered by poor aqueous solubility. Taxol toxicology has been so far studied with the drug presented in polyoxyethylated castor oil (cremophor) (CRE). The preclinical toxicology of taxol has been studied in mice, rats and beagle dogs using taxol administered in CRE. The side effects of taxol were seen mostly in hematopoietic, lymphatic, and gastrointestinal tissues (NCI brochure, 1983). CRE itself had serious effects, particularly in dogs, such as vasodilation, labored breathing, lethargy, and hypotension (Lorenz et al., 1977). Transient asymptomatic bradycardia has been noted in patients during taxol infusions (McGuire et al., 1989). The use of LCM as a vehicle for taxol would circumvent the problems associated with CRE. Furthermore, the targeting ability of LCM may reduce the side effects of taxol on hematopoietic, lymphatic, and gastrointestinal tissues.
The targeting ability of LCM was studied by cytochemistry. LCM were labeled with a fluorescent dye (diO) prior to injection in C6 or 9L tumor-bearing rats. Injection was done in the tail vein, and the rats sacrificed 2 min. later. Vibratome sections were examined by fluorescence microscopy to localize LCM. The well circumscribed C6 tumor was intensely and uniformly fluorescent, indicating the presence of tagged LCM within the tumor (data not shown). The normal tissue adjacent to the tumor was not labeled. 9L gliosarcoma gives rise to multiple small tumors widely dispersed in the brain parenchyma. In this case, the tumors were identified with Texas Red conjugated wheat germ agglutinin which binds specifically to them. Fig. 1B (size - 93.3K) shows the distribution of diO labeled LCM (green), and Fig. lA (size - 93.3K) that of the tumor cells (red) in the same field. The scale bar represents 100 microns. It is apparent that LCM are present on or in a majority of tumor cells.
A high magnification view of the tumor revealed that LCM have been internalized by the tumor cells (Fig. lC (size - 93.3K)). The cell nucleus appears black; the microbubbles are green and immediately adjacent to the nucleus; the wheat germ agglutinin appears red and delineates the contours of the cells; overlap of microbubbles and the agglutinin shows in yellow. The scale bar is 25 microns. Serial optical sectioning confirmed the intracellular distribution of LCM (data not shown).
The uptake of LCM by C6 cells in culture is time and temperature dependent, as illustrated in Fig. 2 (size - 15.6K). It is minimal at 4 °C and maximal at 37 °C.
In order to check if the uptake is due to endocytosis, the acidic compartments (which comprise endosomes and lysosomes) of live C6 cells were labeled with DAMP, and the cells further incubated with diO-labeled LCM. At the end of the incubation period, the cells were fixed, and DAMP was visualized with a Texas Red labeled antibody. LCM (green) (Fig.3A (size - 17.9K)) and the acidic compartments (red) (Fig.3B (size - 17.9K)) were visualized simultaneously using double channel recording fluorescence microscopy. Over 80% of LCM coincided with acidic compartments, suggesting that LCM are found in endosomes and/or lysosomes. In contrast to C6 and 9L cells, primary astrocytes do not adsorb LCM at their surface and do not internalize them (data not shown).
Rats bearing C6 brain tumor were treated with taxol in CRE, or taxol in LCM. The treatment consisted of one i.v. injection of 250 ug of taxol-CRE/kg body weight, or 250 ug of taxol-LCM/kg, at 4 days after tumor inoculation. The rats were sacrificed at 4 days after treatment. The brain was dissected out and processed for histology. Fig. 4 (size - 84.8K) is a representative view of the tumor in taxol-CRE (A) and taxol-LCM (B,C) treated animals, respectively. Taxol given in CRE, at the above concentration, has no effect on the tumor morphology. Tumor cells appear healthy (A). In animals treated with the same dose of taxol but provided in LCM, the situation is dramatically different: (B) shows extensive necrosis in the core of the tumor, while (C) shows that the margins of the tumor are literally covered with lymphocytic cells.
Figure 5 (size - 88.7K) compares the morphology of cells in control culture, taxol-saline and taxol-LCM treated cultures. Overall cell morphology indicates that taxol-LCM (5C) had a more pronounced effect than taxol-saline (5B) under the conditions tested. In taxol-LCM cultures the cells were found to have retracted processes, while this process is incomplete in taxol-saline cultures after 8 hours. The cell number of the taxol-LCM treated culture is also reduced further than that of taxol-saline, as compared to control (see Table 1 (size - 17.9K)), at 8 and 24 hours.
In order to understand the mechanism of LCM uptake by tumor cells, we performed experiments on cells in culture where conditions could be manipulated. When fluorescently labeled LCM (diO labeled) were incubated at 37 °C with C6 cells grown on coverslips, they collected at the cell surface, where they remained as discrete vesicles ( Fig. 6A (size - 28.8K)). Isosurface rendering of a portion of the cell in Fig. 6A confirmed that labeled LCM are present as spherical discrete objects ( Fig. 6B (size - 28.8K)). By contrast, cells labeled with diO alone show a smooth pattern of uniform surface labeling ( Fig. 6C, D (size - 28.8K)). This is in agreement with results obtained with diO in other systems. It indicates that the punctate pattern observed with LCM can only be obtained if diO remains associated with LCM during the incubation period. During the next 30 min, LCM were taken up by the cells at the rate of 2 LCM/min. Uptake was slower at room temperature (1 LCM/min), and minimal at 4 °C.
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