Utility of spheroid-seeded scaffolds as a drug screening tool. It has been previously shown that 3-D systems lead to an increased drug resistance when compared with 2-D systems. More specifically, 3-D systems utilizing spheroids only or polymeric scaffolds only have been demonstrated as useful tumor models to mimic the in vivo tumor behavior.(13,20,22) Our study has shown that combining the two techniques by seeding 3-D scaffolds with spheroids instead of dispersed, monolayer-cultured cells increases the drug resistance significantly. For our studies using doxorubicin and irinotecan, our 2-D IC50 values, 0.05–0.19 μM for doxorubicin and 16–20 μM for irinotecan, corresponded well with previous studies.(29,30) Also, cells in SS exhibited approximately 10-fold and threefold, respectively, higher drug resistance than cells in MS. Since both doxorubicin and irinotecan have been used in vivo, it was possible to compare the dose response of our in vitro system with that of the in vivo studies to assess the system’s ability to screen drugs more accurately. One study has reported the concentration of liposomal doxorubicin around a glioma tumor to be approximately 7 μM for an effective intravenous injection dose.(31) Interestingly, this compared fairly well with our IC50 of 20 μM. Another study using irinotecan and rats with implanted xenograft glioma tumor models showed that delivery of 3 mg/mL (4800 μM) of irinotecan slightly improved survival time.(32) Although the IC50 value of 798 μM is still well below the effective in vivo concentration, the chemoresistance of SS was closer to the in vivo data than that of MS. Moreover, in this analysis, two drugs of very different physiochemical properties were used. While doxorubicin and irinotecan both have very similar molecular weights (543 and 586 g/mol, respectively), their respective partition coefficients (−0.5 and 3.2; octanol-water), predicted water solubilities (1.2 and 0.1 mg/mL), and half-lives (55 and 6–12 h) are considerably different.(33) Thus, because the drug response of our system for both doxorubicin and irinotecan was the closest to in vivo data among all in vitro systems observed, it suggests that the system may be a useful in vitro screening tool for evaluating the efficacy of drugs of various properties. It should be emphasized that these comparisons do not suggest that our current system can replace any in vivo models.
We have further determined that the polymer itself in the system can play a role, albeit small, in raising the apparent chemoresistance by sequestering the drugs. Although not the focus of this study, polymer matrices could have more deterring effects on the drug screening process depending on its size and chemical properties. Moreover, studies have shown that some cell lines readily form spheroids while others do not,(34) and that for some drugs, culturing as spheroids instead of a monolayer has no observable effects on cellular drug response.(35,36) Thus, the dimensions and properties of the 3-D scaffold as well as the cell line used for spheroid generation would both be important considerations when screening drugs with different properties. We have generated and characterized the morphology of another glioma cell line, LN-229, to be similar to U251 in terms of spheroid production (Won Jin Ho, Edward A. Pham, Christopher W. Ng, Daniel T. Kamei, Benjamin M. Wu, unpublished data). This may be another candidate cell line to be incorporated into 3-D scaffolds for further studies.
One clear limitation of the current spheroid-seeded scaffold system is that the spheroids dissociate in the scaffolds by the fourth day of culture. This restricts its utility in any long-term studies. However, we have observed in additional experiments that differential modifications of the scaffold surfaces such as conjugating Arg-Gly-Asp (RGD) peptide, which is a prevalent extracellular peptide involved in cellular adhesion, can control the spheroid dissociation rate (data not shown). Since cell spreading is dependent on the density of RGD peptides on a surface,(37) the amount of RGD-conjugation could be altered in order to optimize spheroid adhesion to the scaffolds. Efforts to promote cell–cell adhesion within the spheroid, as well as attempts to prevent cell adhesion to the 3-D scaffold surface, may maintain the aggregations of spheroids for longer periods.
Understanding the basis of increased drug resistance. Previous studies have explained the higher drug resistance of 3-D systems based on a variety of mechanisms. One possible component of chemoresistance is drug penetration.(38) Cellular aggregation as well as the presence of tortuous, 3-D scaffold structures may limit the drug exposure to some cells. However, we found that drug uptake in SS was efficient and that doxorubicin was found inside the spheroids within 3 h of exposure. This was consistent with spheroid permeability data previously obtained for various drugs including doxorubicin.(23,39) In fact, one previous study has shown that even at relatively low concentrations (∼10 μM), doxorubicin rapidly traverses more than 120 μm from the periphery upon exposure.(23) Thus, coupled with the observation of intercellular spaces throughout the spheroids using 150 kD FITC-dextran, the presence of doxorubicin throughout the 300 μm diameter spheroids was not surprising. Others have shown that despite efficient drug penetration, spheroids still exhibited higher drug resistance than cells in a monolayer.(40) Therefore, our study confirms the idea presented by many others that increased drug resistance in 3-D systems cannot merely be explained by the effects on drug transport.
Another distinguishing characteristic of real-life tumors is high glycolytic activity. Although oxygen readily diffuses, it is a required metabolite that is rapidly consumed by cells. Thus, oxygen tension in a given cellular construct is a function of several factors including varying metabolic activities in the surrounding environment and the spatial arrangement of cells. Since 3-D systems are more susceptible to forming regions of hypoxia, cells compensate for the need of nutrients by increasing their dependence on the less efficient anaerobic pathways following glycolysis instead of oxidative phosphorylation. As expected, the highest level of lactate production was seen in the spheroid-seeded scaffolds. Importantly, as confirmed by our measurements of the intracellular pH, increased production of lactate leads to acidosis. Consequently, acidosis can trigger a variety of mechanisms leading to higher drug resistance. A previous study has shown that inhibiting glycolysis results in reduced drug resistance.(24) Further studies exploring the relevance of this mechanism to the observed behavior of SS, especially on a molecular level, would be valuable.
Another widely studied mechanism linked with hypoxia and tumor development is angiogenesis. Cancer cells respond to a lack of oxygen by stimulating neovascularization and inducing the expression of angiogenic factors such as VEGF(41) and bFGF.(42) Many earlier studies showed that inhibiting the activities of these growth factors leads to increased chemosensitivity, increased expression of proteins that participate in the induction of apoptosis, and thus reduced cell survival.(27,28,43,44) Although our results did not show a definitive role of angiogenic factors in the enhanced drug resistance of SS, there was a significantly higher production of VEGF in SS, which may partly be due to the role of acidosis in up-regulating VEGF.(25) These results are also suggestive of the usefulness of SS in screening VEGF-targeted therapies.